Increased circulating follicular helper T cells with decreased programmed death-1 in chronic renal allograft rejection

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Increased circulating follicular helper T cells with decreased programmed death-1 in chronic renal allograft rejection

Jian Shi1, Fengbao Luo1, Qianqian Shi1, Xianlin Xu1, Xiaozhou He1* and Ying Xia2*

Author Affiliations

1 Third Clinical College of Soochow University, Changzhou, Jiangsu, China

2 The University of Texas Medical School at Houston, Houston, TX, USA

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BMC Nephrology 2015, 16:182  doi:10.1186/s12882-015-0172-8

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2369/16/182

Received: 26 May 2015
Accepted: 19 October 2015
Published: 3 November 2015

© 2015 Shi et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

Chronic antibody-mediated rejection is a major issue that affects long-term renal allograft survival. Since follicular helper T (Tfh) cells promote the development of antigen-specific B cells in alloimmune responses, we investigated the potential roles of Tfh cells, B cells and their alloimmune-regulating molecules in the pathogenesis of chronic renal allograft rejection in this study.

Methods

The frequency of Tfh, B cells and the levels of their alloimmune-regulating molecules including chemokine receptor type 5 (CXCR5), inducible T cell co-stimulator (ICOS), programmed death-1 (PD-1), ICOSL, PDL-1 and interleukin-21 (IL-21), of peripheral blood were comparatively measured in 42 primary renal allograft recipients within 1–3 years after transplantation. Among them, 24 patients had definite chronic rejection, while other 18 patients had normal renal function.

Results

Tfh-cell ratio was significantly increased with PD-1 down-regulation in the patients with chronic renal allograft rejection, while B cells and the alloimmune-regulating molecules studied did not show any appreciable change in parallel.

Conclusions

The patients with chronic renal allograft rejection have a characteristic increase in circulating Tfh cells with a decrease in PD-1 expression. These pathological changes may be a therapeutic target for the treatment of chronic renal allograft rejection and can be useful as a clinical index for monitoring conditions of renal transplant.

Keywords:

Chronic renal allograft rejection; Tfh cells; PD-1

Background

Renal transplantation remains an effective treatment for end-stage renal dysfunction [1], facilitating a return to normal health and prolonging life. However, antibody-mediated rejection is a major issue that affects long-term renal allograft survival. Despite the rapid development of new immunosuppressive drugs for attenuating acute rejection, improving the long-term survival of grafts is still a challenge mainly because of chronic allograft rejection. The features of chronic renal allograft rejection are hypertension, proteinuria, progressive deterioration of graft function, peritubular capillary C4d deposition, presence of donor-specific antibodies (DSA) and morphological changes with transplant vasculopathy, glomerulopathy, fibrosis and lymphocyte infiltration. However, the causes leading to chronic rejection are complex and not well understood yet [2].

Allograft rejection is characterized by an increase in activated CD4+ T-lymphocytes, especially regulatory and cytotoxic T cells, leading to an imbalance of immune responses in the transplant recipients [3], [4]. Functionally, CD4+ T helper cells that interact with antigen-specific B cells are required for the production of alloantibodies [5]. Among them, Tfh cells, a recently defined subset of CD4+ T cells, play a particular role in mediating B cell-driven allogeneic responses. Tfh cells can migrate into germinal centers and promote B-cell activation and differentiation into immunoglobulin-producing plasmablasts or plasma cells [5]. They can express PD-1, CXCR5, ICOS, IL-21 and the transcription factor B-cell lymphoma 6 (Bcl-6) [6], [7], thereby displaying their regulatory functions.

Circulating Tfh cells, peripheral counterparts of conventional Tfh cells, express PD-1, CXCR5, ICOS and IL-21, but not Bcl-6 [5]–[7]. They play an important role in human humoral immunity through these functional molecules. Their abnormal activities are critically involved in the onset of several human diseases such as autoimmune disorders, cancer and infective diseases [7]–[10]. Therefore, an alteration in circulating Tfh cells may be correlated with disease conditions and might be used as a biomarker of certain diseases [11]–[13]. Moreover, recent clinical studies have shown that peripheral Tfh cells in the kidney transplant recipients with acute rejection can regulate B-cell alloreactivity and the number of these Tfh cells alters the immunization status and DSA levels [14]. However, their function and relevance to chronic renal allograft rejection are not known yet.

This study was conducted to explore the potential association between circulating Tfh cells and chronic rejection in kidney transplant recipients. The outcome results may provide a useful hint for clinical prediction of renal status after transplantation and for a potential new therapy for chronic allograft rejection.

Methods

This study was approved by the Institutional Ethics Committee of Third Affiliated Hospital of Soochow University, Jiangsu Province, China. Written-informed consent was obtained from all participants of the study.

Subjects

The patients with primary renal transplantation for 1–3 years were enrolled from October 2013 to December 2014. Totally 42 recipients were studied in this work, including 24 patients with chronic rejection (CR group) and 18 patients with normal renal function as the normal control (NC group). All of them received the treatment with cyclosporine A, methylprednisolone and mycophenolate mofetil or azathioprine after the renal transplantation. The diagnosis for chronic allograft rejection was confirmed by renal biopsy, biochemical measurements and immunological assays, including DSA as described in other studies [15]–[18]. In specific,the diagnostic criteria included 1) clinical evidence of slowly deteriorating graft function; 2) biopsy evidence and diffuse deposition of C4d; and 3) the presence of circulating DSA at the time of biopsy. Their peripheral blood samples were collected in a standard way by the clinical laboratory of the hospital. All the subjects had no infective disease when sampling blood.

Surface staining and flow cytometry analysis

The whole blood was subjected to flow cytometry detection by a BD bioscience FACSCantoII cytometer with FACSDiva software for measuring the frequency of circulating Tfh cells and B cells as well as the expression of their surface markers. The following conjugated monoclonal antibodies were used to stain the cells: CD4-FITC, CXCR5-APC, ICOS-PE, PD-1-PE, CD19-PE-Cy5.5, ICOSL-PE and PDL-1-APC. The cells were incubated with the antibodies for 30 min at room temperature in the dark. Totally 50,000 lymphocytes were acquired in each sample. Data were analyzed using Flow Jo software 7.6.1.

Enzyme-linked immunosorbent assay (ELISA)

The levels of serum IL-21 were quantified by using the human IL-21 ELISA kit (eBioscience) according to the manufacturer’s instructions. The concentration in each individual sample was calculated according to the standard curve.

Statistical analyses

All experimental data were analyzed by Graph Prism version 5.0. The results were expressed as mean ± SD and subjected to t test for statistical comparisons between the NC and CR groups. If a p-value was found to be less than 0.05, the result would be considered statistically significant.

Results

Demographics

The general information of the renal transplant recipients was summarized in Table 1. Gender and age were similar between CR and NC groups. The mean serum creatinine (sCr) and blood urea nitrogen (BUN) were almost three-fold higher in the CR patients than those of NC group (p < 0.001).

Table 1. General information of the renal transplant patients

Increased circulating Tfh cells in the CR patients

To determine if chronic rejection was associated with an alteration in circulating Tfh cells in the renal transplantation recipients, we first evaluated the frequency of CD4 + CXCR5+ Tfh cells through flow cytometry. As shown in Fig. 1, the percentage of CD4 + CXCR5+ Tfh cells among total CD4+ T cells was significantly increased in the CR group as compared to that of the NC group (35.3 ± 8.5 % vs.19.0 ± 5.0, P < 0.001).

thumbnailFig. 1. Frequency of Tfh cells in the patients with renal allograft. a, representative contour plots of the ratio of Tfh cells in the NC and CR groups, b, mean values of the frequency of Tfh cells in the two groups. ***, P < 0.001. Note a significant increase in Tfh cells in the CR group compared to that of the control group

Differential changes in PD-1, CXCR5, ICOS, and IL-21 of Tfh cells in the CR patients

We further detected the changes of PD-1, CXCR5 and ICOS in the high frequency of Tfh cells and found that PD-1 was significantly down-regulated in these Tfh cells (Fig. 2a). In sharp contrast, there were no significant changes in CXCR5 and ICOS in the CR group as compared to the NC group (Fig. 2b and c).

thumbnailFig. 2. Comparative changes in PD-1, CXCR5 and ICOS expression on Tfh-cell surfaces. a,PD-1. b, CXCR5. c, ICOS. * P < 0.05. Note a significant decrease in PD-1 expression with no appreciable changes in CXCR5 and ICOS expression in the CR patients as compared to the NC group

IL-21 is a biological hallmark of Tfh cells because this immune cytokine is produced by Tfh cells and is involved in mediating Tfh-B-cell interaction [6]. Therefore, we further measured the concentration of IL-21 in the serum of the CR patients (Fig. 3). Similarly as the changes in CXCR5 and ICOS, serum IL-21 did not show any significant change in the CR patients as compared to that of the control group (406.9 ± 123.9 pg/ml vs. 449.1 ± 101.7 pg/ml, P > 0.05).

thumbnailFig. 3. The level of serum IL-21 in renal allograft patients. Note that there was no statistic difference in the IL-21 level between the CR and NC groups

No changes in circulating B cells, PDL-1 and ICOSL in the CR patients

Since B cells are key players in the graft rejection [19], we further determined the frequency of total B cells and compared their change with that of Tfh cells. The ratio of B lymphocytes was not significantly different between CR and NC groups (Fig. 4), unlike Tfh cells. Because PDL-1-expressing B cells interact with PD-1+ Tfh cells to regulate the maturation and survival of B cells [20], we next detected the expression of PDL-1 in B cells. Unlike the change in the PD-1 of Tfh cells, PDL-1 did not decrease in B cells at all in the CR group (Fig. 5a). Also, ICOSL had no significant change in the CR patients (Fig. 5b).

thumbnailFig. 4. The frequency of B cells. Note that there was no significant difference between two groups

thumbnailFig. 5. The expression of PDL-1 and ICOSL in B cells. Note that there were no appreciable differences in both PDL-1 (a) and ICOSL (b) between two groups

All these results suggest that the increase in circulating Tfh cells with PD-1 down-regulation is a specific and characteristic change in the CR patients.

Discussion

We have made a novel finding in this work, i.e., a major increase in circulating Tfh cells with a significant decrease in PD-1 in the patients with chronic renal allograft rejection. In sharp contrast, B cells and the alloimmune-regulating molecules such as CXCR5, ICOS, ICOSL, PDL-1 and IL-21 did not show any appreciable change in parallel.

Tfh cells display multiple features for their helper functions in secondary lymphoid organs or tissues with inflammation [21]. They migrate into B cell follicles of germinal centers [22] and thereby help B cells generate antibodies for humoral immunity [3], [6], [23]. In fact, Tfh cells, as an immune regulator, are critically involved in the pathological processes of many immune diseases [8]. In renal allograft rejection, the germinal center reactions are dependent on Tfh, while B cells are indispensable for the immune attack to the newly transplanted kidney [24].

Tfh cells express PD-1 [6], while B cells produce PDL-1, an endogenous ligand of PD-1 [19]. The PD-1/PDL-1 signaling has been shown to play an important role in regulating immune functions and affecting the activation of regulatory T cells, cytotoxic T cells and Dendritic cells [25]. Such signaling also influences the generation and differentiation of Tfh and B cells themselves [26]. Recent evidence suggests that blocking the PD-1 signaling induces an up-regulation of Tfh generation and differentiation, which may directly lead to autoimmune encephalomyelitis [27]. In contrast, stimulating this pathway can prolong the survival of the patients after cardiac allograft transplantation [28]. More recently, PD-1 ligands are found to protect the kidneys from ischemia reperfusion injury [29]. In fact, PDL-1, the endogenous ligand of PD-1, has been demonstrated as a required factor for peripheral transplantation tolerance and protection aganist chronic allograft rejection [30].

Taken together, PD-1 signaling is a key regulator for attenuating the Tfh cells and down-regulating the overreaction of humoral immunity against the transplanted kidneys. Therefore, the novel finding of the present study strongly suggests that the deficiency of PD-1 expression causes the increase in Tfh cells, thereby leading to an overreaction of humoral immunity against the allergenic organ, which may be a major reason for chronic allograft rejection.

Tfh and B cells also express many other immune-regulating molecules such as CXCR5, ICOS, ICOSL, and IL-21 [6], [19], [26]. All these molecules are actively involved in the regulation of immune function [26]. For example, an increase in the expression of CXCR5 can enable Tfh cells to migrate into germinal centers [21]. On the other hand, ICOS, another surface receptor like PD-1, also mediate the generation, development and function of Tfh cells by activating ICOS/ICOSL signaling [31], [32]. Moreover, IL-21,a pro-inflammatory cytokine secreted by Tfh cells,has an important role in Tfh cell differentiation, B cell proliferation [33], and the expression of PD-1 [34] and CXCR5 [26]. However, all of these immune regulators did not shown any change in the patients with chronic allograft rejection. We are therefore confident that the deficient PD-1 expression with increased circulating Tfh cells is a specific and characteristic change in chronic allograft rejection.

In the lymph nodes, CD4+ CXCR5+ Tfh cells are more effective in helping B cells than their peripheral counterparts [7]. Humoral response in the lymph nodes can be suppressed by anti-CD40 mAb via regulating Tfh cells [23]. In the transplanted kidneys with acute rejection, infiltrated Tfh cells have been found to participate in the antibody-mediated rejection [14]. However, little is known about the role of these special Tfh cells in the transplanted kidneys with chronic rejection. We speculate that the increase in circulating Tfh cells with a decrease in PD-1 expression might, at least partially, contributes to the genesis of the renal chronic rejection by migrating and infiltrating into germinal centers of renal allografts and lymphoid organs. We will further clarify this issue in our future work.

In addition, pre-existent DSA storing before renal transplantation and de-novo DSA developing after renal transplantation are associated with antibody-mediated rejection and allograft failure [35]. However, recent studies have shown that despite the numbers of circulating Tfh cells were higher in the patients with pre-existent DSA than those without pre-existent DSA, the levels of circulating Tfh cells were not different among the patients with or without de-novo DSA [35], [36]. Therefore, the relationship between the frequency of circulating Tfh cells and the level of DSA in renal allograft rejection is not clear yet and needs more investigations.

Conclusions

Our first data show that decreased PD-1 expression may contribute to the increase in circulating Tfh cells in the patients with chronic renal allograft rejection. This finding provides a potential hint for a new target for the treatment of chronic rejection. Moreover, a dynamic change in the expression of PD-1 and the number of circulating Tfh cells may be used as an index for monitoring chronic allograft rejection after kidney transplantation as.

Abbreviations

Tfh: Follicular helper T cells

CXCR5: Chemokine receptor type 5

ICOS: Inducible T cell co-stimulator

PD-1: Programmed death-1

ICOSL: Inducible T cell co-stimulator ligand

PDL-1: Programmed death-1 ligand

IL-21: Interleukin-21

Bcl-6: Transcription factor B-cell lymphoma 6

CR: Chronic rejection

NC: Normal control

ELISA: Enzyme-linked immunosorbent assay

sCr: Serum creatinine

BUN: Blood urea nitrogen

DSA: Donor-specific antibodies.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

JS, XX, XH and YX conceived and designed the experiments. JS, FL and QS performed the experiments. JS analyzed the data. XH provided research reagents. JS and YX wrote the paper. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No.81273267) . YX was partially supported by NIH (AT-004422).

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Quantitative analysis of a Māori and Pacific admission process on first-year health study

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Quantitative analysis of a Māori and Pacific admission process on first-year health study

Elana Curtis1*, Erena Wikaire1, Yannan Jiang2, Louise McMillan2, Robert Loto1, Airini3 and Papaarangi Reid1

Author Affiliations

1 Te Kupenga Hauora Māori, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92015, Auckland, New Zealand

2 Department of Statistics, Faculty of Science, University of Auckland, Private Bag 92015, Auckland, New Zealand

3 Faculty of Human, Social and Educational Development, Thompson Rivers University, Thompson, BC, Canada

For all author emails, please log on.

BMC Medical Education 2015, 15:196  doi:10.1186/s12909-015-0470-7

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1472-6920/15/196

Received: 15 December 2014
Accepted: 20 October 2015
Published: 3 November 2015

© 2015 Curtis et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

Universities should provide flexible and inclusive selection and admission policies to increase equity in access and outcomes for indigenous and ethnic minority students. This study investigates an equity-targeted admissions process, involving a Multiple Mini Interview and objective testing, advising Māori and Pacific students on their best starting point for academic success towards a career in medicine, nursing, health sciences and pharmacy.

Methods

All Māori and Pacific Admission Scheme (MAPAS) interviewees enrolled in bridging/foundation or degree-level programmes at the University of Auckland were identified (2009 to 2012). Generalised linear regression models estimated the predicted effects of admission variables (e.g. MAPAS Maths Test; National Certificate in Educational Achievement (NCEA) Rank Score; Any 2 Sciences; Followed MAPAS Advice) on first year academic outcomes (i.e. Grade Point Average (GPA) and Passes All Courses) adjusting for MAPAS interview year, gender, ancestry and school decile.

Results

368 First Year Tertiary (bridging/foundation or degree-level) and 242 First Year Bachelor (degree-level only) students were investigated. NCEA Rank Score (estimate 0.26, CI: 0.18-0.34, p< 0.0001); MAPAS Advice Followed (1.26, CI: 0.18-1.34, p = 0.0002); Exposure to Any 2 Sciences (0.651, CI: 0.15-1.15, p = 0.012); and MAPAS Mathematics Test (0.14, CI: 0.02-0.26, p = 0.0186) variables were strongly associated with an increase in First Year Tertiary GPA. The odds of passing all courses in First Year Tertiary study was 5.4 times higher for students who Followed MAPAS Advice (CI: 2.35-12.39; p< 0.0001) and 2.3 times higher with Exposure to Any Two Sciences (CI: 1.15-4.60; p = 0.0186). First Year Bachelor students who Followed MAPAS Advice had an average GPA that was 1.1 points higher for all eight (CI: 0.45-1.73; p = 0.0009) and Core 4 courses (CI: 0.60-2.04; p = 0.0004).

Conclusions

The MAPAS admissions process was strongly associated with positive academic outcomes in the first year of tertiary study. Universities should invest in a comprehensive admissions process that includes alternative entry pathways for indigenous and ethnic minority applicants.

Keywords:

Admission; Selection; Indigenous; Ethnic minority; Health professional; Higher education; Widening participation; Workforce development; Māori; Pacific

Background

Worldwide, tertiary institutions are attempting to widen participation to historically underserved populations including indigenous and ethnic minority students [1] Often driven by social inclusion and social accountability policies, universities have devised a number of strategies to increase diversity. Within an indigenous and ethnic minority health workforce context, a pipeline approach is recommended to address well-known barriers to accessing and succeeding in university-level studies. A pipeline approach often includes early exposure interventions aimed at raising aspirations and academic preparation for a career in health [2]–[4]; addressing educational disadvantage via the provision of bridging/foundation programmes [5], [6] and improving student performance by providing comprehensive support programmes [7]–[9]. Given the highly competitive context of health professional programme selection, it is also recommended that universities provide more flexible and inclusive selection and admission policies for students from underserved populations [1], [10].

Universities have a choice of selection tools that can be used to inform student admission including prior academic performance, interview scores and results from aptitude tests. Both cognitive and non-cognitive tools are used by universities when selecting students; however it is arguable that prior academic performance remains a dominant tool for medical selection in many universities [11]. Given this reality, indigenous and ethnic minority students are required to aim to achieve a high level of academic performance within the pathways used for future selection into medical or health professional programmes of study [12]. Unfortunately, students from underserved populations are less likely to receive access to science-rich subjects and are more likely to leave high school with lower qualifications than their peers [5], [10], [13]. Providing an admissions process that can determine whether indigenous and ethnic minority applicants are academically (and socially) ready to achieve success in pre-medical degree pathways and the provision of alternative entry pathways is recommended for tertiary institutions committed to widening participation [14], [15].

An extensive body of research identifies the tertiary conditions and factors that impact on academic success within the first year of study at university [16]–[20]. Indicators of prior academic performance such as: secondary school grade point averages [21]; secondary school factors including markers of socio-geographic status (e.g. school decile) [22]; and student characteristics (e.g. autonomy, confidence, motivation, control) [17], [23] have been identified as important factors impacting on academic performance in the first year of study. In addition, factors associated with the environment of the tertiary institution also impact on student engagement; such factors include: opportunities for teachers and students to engage with each other [18]; levels of institutional support to provide environments conducive to learning [20]; and the provision of academic, social and personal support [16].

To date, few studies have explored the effect of equity-targeted admission processes on the academic performance of indigenous and ethnic minority students in their first year of tertiary study. As a result, tertiary institutions have little empirical evidence to understand the effect of equity-targeted selection processes and whether such initiatives are likely to support a widening participation agenda.

This article explores the predictive effect of admission variables associated with an equity-targeted admission process on academic outcomes for Māori (the indigenous peoples of Aotearoa New Zealand) and Pacific (a heterogeneous composite of peoples with Pacific nation ancestry born and/or living in New Zealand) applicants applying under the Māori and Pacific Admission Scheme (MAPAS) to the Faculty of Medical and Health Sciences (FMHS) at the University of Auckland (UoA).

Methods

FMHS entry pathways

Admission into FMHS health professional programmes is generally via direct entry into First Year Bachelor level undergraduate study for those applicants who meet the necessary entry requirements [24]. The FMHS also offers a one-year, MAPAS-specific bridging/foundation programme, the Certificate in Health Sciences (CertHSc) through which Māori and Pacific students who achieve a CertHSc GPA above B+ can gain alternative entry into First Year Bachelor undergraduate study. Hence, Māori and Pacific First Year Tertiary students within FMHS could either be enrolled in the CertHSc bridging foundation programme, or, the first year of bachelor level study (Table 1). The first year of bachelor level study also acts as a ‘pre-medical’ year prior to admission into the FMHS Medical programme in year 2. Table 1 provides definitions of the Certificate in Health Sciences, First Year Tertiary, and First Year Bachelor terms used within this study (Table 1).

Table 1. Definition of terms used within the FMHS context

Māori and Pacific Admission Scheme (MAPAS)

MAPAS operates an equity-targeted admissions process for applicants with indigenous Māori and Pacific ancestry. The process aims to gather a broad range of information about Māori and Pacific applicant preparation for tertiary health study. The December interview process involves a Multiple Mini Interview (MMI), an English test and a mathematics test.

The MMI is an alternative form of admission interview that aims to reduce interviewer bias by consisting of a number of short interview stations with multiple interviewers. The MMI has been shown to be reliable, acceptable and feasible in a variety of tertiary health study contexts [25]. In building on the original pilot of the MMI [26], other studies have taken advantage of the intended benefit of the flexibility of station development in their own contexts [27], [28]. Whilst the original authors aimed to assess suitability of applicants as health professionals, the MAPAS MMI aims to assess Māori and Pacific applicant preparation for and potential to succeed in FMHS programmes. In the MAPAS context, the MMI has been redeveloped to include four 8-min stations assessing career aspirations; academic preparation; family support and student information. The MAPAS mathematics and English testing are used in addition to the MAPAS MMI to objectively assess academic numeracy and literacy skills. Using MMI and testing information, two assessments are made about: 1) potential to succeed within the CertHSc, and 2) potential to succeed within the Bachelor of: Health Sciences; Science (Biomedicine)1 ; Nursing; or Pharmacy. Potential to succeed is assessed as: pass, borderline or fail (objective testing) for the English and mathematics testing and few, some, or major concerns (subjective testing) for each MMI station. A MAPAS Recommendations Team reviews the combination of results and provides a provisional MAPAS recommendation (advice regarding the applicant’s recommended best starting point given their intended health career) for applicants (and families) on the day of their interview. Recommended starting points are reflected within three categories: (1) Bachelor i.e. start at degree-level; (2) CertHSc i.e. start at bridging/foundation; or (3) Not FMHS i.e. start in a pathway not provided by FMHS (likely to need further academic preparation not offered by the FMHS). Following the release of secondary school results in January, all information is re-reviewed and a final MAPAS recommendation is provided. MAPAS recommendations are not binding if an applicant has met guaranteed entry criteria for any FMHS programme. In this context, the applicant can choose to follow MAPAS advice (or not)2 .

Methodology

This study used a Kaupapa Māori Research (KMR) approach, broadly defined and responsive to Pacific research methodologies [29], [30]. This approach recognises that issues associated with power, privilege and agency within society are hypothesised to act similarly on both Māori and Pacific students [31], [32]. In this instance KMR aims to: ensure research outputs are positive for Māori and Pacific students; explicitly challenge ‘victim blame’ or ‘cultural deficit’ analyses that may blame Māori or Pacific students for educational failure; and provide a structural analysis to promote institutional change targeting Māori and Pacific student success [14], [33]. This research was led by senior Māori and Pacific researchers with input from a FMHS advisory group.

Study design

The predictive effect of MAPAS admission process variables on academic outcomes in the first year of tertiary study was explored. Applicant data were obtained from the MAPAS admissions database and the university’s centralised student data management system for all MAPAS interviewees (2008 – 2011) who subsequently enrolled in relevant tertiary health programmes (2009 – 2012) within the FMHS at the UoA. Approval to complete this research was granted by the University of Auckland Human Participant Ethics Committee (Ref 8110). As per ethics protocols, written informed consent was not required for this research project due to the use of secondary administrative data sources. All secondary data obtained from these datasets were de-identified by an independent research member with no student contact or teaching responsibilities and data analysis occurred via a coding system. Two student cohorts are identified: First Year Tertiary Students i.e. students enrolled in either the CertHSc or the first year of a bachelor programme in the year following their MAPAS interview; and First Year Bachelor Students i.e. students enrolled in a bachelor programme in either the first or second year following their MAPAS interview (may include CertHSc graduates).

Variables

Demographic variables include: Year of Admission (2009–2012); Gender (Female, Male); Ancestry (Māori, Pacific, Both) and School Decile (High, Medium and Low). Secondary schools with a mid-low decile rating have been linked to higher levels of deprivation associated with reduced access to, and outcomes from, tertiary education [34] (Table 2).

Table 2. Descriptive summary of first year tertiary and first year bachelor student demographic and outcome variables

Admission predictor variables include: MAPAS Testing results (%); MMI Station results (Some or Major Concerns (SMC) versus Few Concerns (FC)); Provisional December Recommendation (CertHSc, Bachelor, Not FMHS); secondary school results including New Zealand’s NCEA Rank Score3 (out of 320); Level 3 NCEA Subject Credits (number of credits achieved in English, biology, chemistry, physics, mathematics); Exposure to Any 2 Sciences of senior biology, chemistry or physics (yes, no)4 ; Followed MAPAS Advice (yes, no); and Final January Recommendation made in January (CertHSc, Bachelor, Not FMHS).

Academic outcome variables include: Grade Point Average (GPA) Eight Courses, 09 (i.e. GPA achieved across a total of eight courses over the year); GPA Core 4 Courses, 09 (i.e. GPA achieved across four core courses5 taken in the first year of bachelor study that are specifically assessed for selection into second year medicine at the UoA); Passes All Courses, yes/no (i.e. across total of eight courses); Passes All Core 4 Courses, yes/no (i.e. across the four core courses).

Statistical analysis

All downloaded data were recorded in Microsoft Office Excel spread sheets. Statistical analyses were performed using SAS version 9.3 (SAS Institute, Cary, NC, USA). Continuous variables were presented as mean and standard deviation (SD); categorical variables as frequencies (n) and percentages (%) (Tables 2 and 3). Generalised linear and logistic regression models were used to estimate the predicted effects of individual admission variables on academic outcomes (i.e. GPA and Passes All); adjusting for pre-defined demographic variables (i.e. MAPAS interview year, gender, ancestry and school decile) (Tables 4, 5, 6 and 7). Admission variables that showed significant single predictive effect (i.e. MAPAS Maths Test, NCEA Rank Score, Any 2 Sciences and Followed MAPAS Advice) were included in the multiple regression analyses to determine their joint effects on the academic outcomes of interest (Tables 8 and 9). All statistical tests were two-sided at 5 % significance level.

Table 3. Descriptive summary of first year tertiary and first year bachelor student predictor variables

Table 4. Univariate regression analysis results – GPA eight courses

Table 5. Univariate regression analysis results – GPA core 4 courses

Table 6. Univariate regression analysis results – passes all eight courses

Table 7. Univariate regression analysis results: passes all core 4 courses

Table 8. Multiple regression analysis results – linear regression a

Table 9. Multiple regression analysis results – logistic regression a

Results

Descriptive variables

A total of 368 students were identified in the First Year Tertiary cohort. Of these, 37 % were Māori, 57 % Pacific and 6 % had Both Māori and Pacific ancestry. Two thirds were female (67 %), the mean age was 19.2 years (SD 4.2 %) and 70 % or more came from a secondary school with a medium or low school decile (representing more deprived communities). The First Year Bachelor cohort had a total of 242 students with a similar demographic profile to First Year Tertiary students (Table 2).

Predictor variables

Mathematics and english testing

The First Year Tertiary cohort had a mean percentage mark for the mathematics test of 79.0 % (SD 18.3 %) and 68.4 % (SD 13.6 %) for the English test. This represents a borderline-fail result for bachelor-level study and a pass result for CertHSc-level study as the best starting point of entry across both assessments. The First Year Bachelor cohort had a slightly higher mean mark for both the mathematics (80.4 %, SD 18.3 %) and English tests (70.6 %, SD 12.8 %) (Table 3).

MMI

Over 80 % of all students from both cohorts were assessed as having few concerns for CertHSc-level entry across the four MMI stations. Forty-four percent of all First Year Tertiary students were assessed as having some or major concerns for bachelor-level entry at the Academic Preparation and Student Information MMI stations. For First Year Bachelor students, the stations with the highest proportion of some or major concerns for bachelor-level entry were Career Aspirations (48 %) and Student Information (39 %) (Table 3).

School results

The average NCEA rank score (out of a total of 320) was 190.5 (SD 51.3) for First Year Tertiary and 201.8 (SD 52.7) for First Year Bachelor students. Both averages fall below requirements for guaranteed entry within FMHS (set at a rank score between 210 – 250 depending on the programme). The average number of subject credits for both cohorts were 0.3–3.4 credits below requirements for guaranteed entry (i.e. 16 – 18 subject credits depending on programme) (Table 3). At least two thirds of all students admitted into either the CertHSc or bachelor programmes had taken two or more science subjects in their final year of secondary school (Table 3).

MAPAS recommendations

For First Year Tertiary students, MAPAS recommended CertHSc to 72 % of all students, followed by Bachelor (26 %) and Not FMHS (2 %). For First Year Bachelor students, 58 % were recommended to start at the CertHSc level, followed by 39 % Bachelor and 3 % Not FMHS (Table 3).

Followed MAPAS advice

Over 83 % of all students followed MAPAS advice regarding the best starting point for success with only 12 – 17 % of students from each cohort not following their final MAPAS recommendation (Table 3).

Outcome variables

GPA All eight courses and core 4 courses

The average GPA for all eight courses (out of a total of 9) was 4.3 (SD 2.0) for First Year Tertiary and 4.1 (SD 2.1) for First Year Bachelor students. The average GPA achieved for the Core 4 Courses was 3.8 (SD 2.4) for First Year Bachelor students.

Passes All eight courses and passes All core 4 courses

Seventy-five percent of First Year Tertiary students and 60 % of First Year Bachelor students passed all eight courses. Sixty-four percent of First Year Bachelor students passed all Core 4 Courses (Table 2).

Multiple regression analysis

First year tertiary – GPA

As shown in Table 8, all predictors had a statistically significant effect on First Year Tertiary GPA, with the most significant predictor being NCEA Rank Score, then MAPAS Advice Followed, Any 2 Sciences and MAPAS Mathematics Test results. First year Tertiary GPA increased by an average of 0.3 (out of a total 9) for every 20 point increase in NCEA Rank Score (CI: 0.18-0.34; p < 0.0001). Students who followed MAPAS advice had on average a GPA that was 1.2 points higher (out of a total 9) than students who did not (CI: 0.57-1.78; p = 0.0002).

First year tertiary – passes All courses

The odds of passing all eight courses was 5.4 times higher for those students who followed MAPAS advice versus those students who did not (CI: 2.36-12.39; p < 0.0001) (Table 8). The odds of passing all eight courses was 2.3 times higher for those students who had exposure to Any 2 Sciences versus those students who did not (CI: 1.15-4.61; p = 0.019) (Table 8).

First year bachelor – GPA

For every 20 point increase in NCEA Rank Score, the GPA achieved by First Year Bachelor students increased by an average of 0.4 for all 8 courses (CI: 0.30-0.50; p < 0.0001) and for Core 4 courses (CI: 0.26-0.50; p < 0.0001) (Table 7). Students who followed MAPAS advice had on average a GPA that was 1.1 points higher than students who did not follow MAPAS advice for all eight courses (CI: 0.45-1.73; p = 0.0009) and Core 4 courses (CI: 0.60-2.04; p = 0.0004) (Table 8).

First year bachelor – passes All courses

A 20 point increase in NCEA Rank Score increased the odds of passing all first year bachelor courses by a factor of 1.5 (CI: 1.24-1.74; p < 0.0001), with similar results for passing all Core 4 courses (Table 8). The odds of passing all first year bachelor courses (CI: 1.45-7.69; p = 0.005) and all Core 4 courses (CI: 1.39-7.69; p = 0.007) was 3.3 times higher for those students who followed MAPAS advice versus those students who did not (Table 9).

Discussion

Our findings confirm that the MAPAS admissions process is strongly associated with positive academic outcomes in the first year of tertiary study. Our results reinforce the evidence-base showing a strong association between secondary school performance via NCEA rank score (a marker of the quality of grades achieved) and positive tertiary academic outcomes [35]. The existing literature base has also been extended, given our identification of a strong association between exposure to two or more senior science subjects (a marker of breadth of knowledge) and first year academic outcomes. Similar to other studies, our findings show that the number of credits achieved within NCEA subjects appear to be less strongly correlated with tertiary outcomes [35].

Overall, our findings suggest that there is value in providing a comprehensive admissions process for indigenous and ethnic minority students applying under equity targeted admission programmes. Students admitted into tertiary institutions under targeted admission programmes have been shown to experience peer/educator stigma and ‘everyday racism’. Demonstrating the effectiveness of targeted admission programmes may assist some indigenous and ethnic minority students to override this societal (and potentially internalised) stigma to receive the benefits that targeted admission programmes have to offer.

Increasing the odds of passing all first year courses has relevance for all students. This is important for applicants pursuing medicine as even small increments in first year bachelor GPA, particularly within the Core 4 courses used for medical selection, may have a profound impact on potential selection [12], [19]. A student’s progress towards completion of total point requirements within their degree has been shown to improve student retention and increase the likelihood of degree completion [36]. Aligning MAPAS admission to a comprehensive process focussed on achieving equity in access and performance is likely to have contributed to the recent increase in numbers and improved performance observed for Māori and Pacific students within the FMHS [5], [37].

Our data suggests secondary schooling is yet to demonstrate the ability to prepare Māori and Pacific students adequately for tertiary health professional study. Both teaching and subject selection are critical factors. Māori and Pacific students and their families are not to blame for the observed inequities in secondary education. Rather, Māori and Pacific students and their families should receive greater support to navigate NCEA subject selection and ensure that students achieve the right number and quality of credits [38]. This is consistent with international evidence showing that indigenous and ethnic minority students are less likely to receive high-quality careers or university advice [38], [39] and in some instances may be actively discouraged from pursuing a health professional career [2].

Based on our findings, it appears that the secondary education sector is failing to ensure that indigenous and ethnic minority students are ‘university-ready’ for health-professional study. Unfortunately, this is not a new issue [5], [14], [40], [41] and nor is it unique to New Zealand [3], [42]. Action by secondary schools and educators to address their own role in the creation and maintenance of ethnic inequities in academic outcomes is recommended [43]. Likewise, tertiary institutions are expected to be part of the solution [44]. Pechenkina & Anderson (2011) call for “more effective institutional response to the lack of adequate preparation of indigenous students…via greater investment in the pipeline and provision of transitioning programmes” (p. 5-6). Our findings further support the delivery of bridging/foundation programmes targeting indigenous and ethnic minority students.

Strengths

This study explores a unique application of the MMI within an equity-targeted context [14], [26]. Although we identified varied associations between individual MMI stations and academic outcomes, we believe that our overall findings support maintaining the MMI within the MAPAS admissions process. This reflects the strong association observed between following MAPAS advice (a predictor variable that is determined by the combined assessment of all results) and higher academic outcomes.

Using both cognitive (e.g. NCEA school results, MAPAS Maths and English test) and non-cognitive (e.g. MMI results) tools for student selection within the total MAPAS admission process supports a widening participation agenda and is consistent with recommendations to use more inclusive selection tools [10], [45]-[47]. This is particularly important when assessing the potential of alternative admission or older applicants who may possess maturity shown to be positively associated with tertiary programme completion [36], [48].

Limitations

This study has a number of limitations. The analysis relied on secondary data and is therefore limited by the quality of data sources. However, combining central university and MAPAS datasets has reduced the potential for data misclassification by using verified ancestry and increased the admission variables available for analysis [49], [50]. Our research was limited to first-year outcomes due to resource and time constraints. Ideally, the effect of predictor variables on long-term outcomes across all FMHS programmes should be examined. Comparing academic outcomes across all ethnic groups may also highlight issues of disadvantage and privilege [51]. This research is in progress and is drawing on the methods developed within this study. We acknowledge that combining Māori and Pacific data is not ideal from an indigenous rights or Pacific-centric perspective. However, this is consistent with our methodological approach as it maximises statistical power (to aid student success) and supports a structural critique of the effect of ‘society’ on ‘ancestry’ [14]. As the quantum of Māori and Pacific data increases, further research should investigate Māori-specific and Pacific-specific predictors of academic success.

Conclusion

Tertiary institutions committed to widening participation should prioritise the funding and delivery of a comprehensive, flexible and inclusive admissions process that includes alternative entry pathways for indigenous and ethnic minority applicants [10], [52], [53].

Ethical approval

This project was approved by the University of Auckland Human Participants Ethics Committee, Ref 8110.

Abbreviations

CertHSc: Certificate in Health Sciences (Hikitia Te Ora)

CIE: Cambridge International Exam

FMHS: Faculty of Medical and Health Sciences

GPA: Grade Point Average

IB: International Baccalaureate

KMR: Kaupapa Māori Research

MAPAS: Māori and Pacific Admission Scheme

NCEA: National Certificate of Educational Achievement

UoA: University of Auckland

Competing interest

The authors declare that they have no competing interest.

Authors’ contributions

EC led the study design, methodological approach, interpretation of the data analysis, and drafted the manuscript. EW contributed to study design and provided research assistance to obtain and clean data variables. She contributed to drafting and revising the manuscript and was responsible for producing the data tables. YJ provided senior statistical expertise for data analysis. She contributed to drafting and revising the manuscript. LM provided junior statistical expertise and contributed to drafting and revising the manuscript. RL contributed to the study design and provided Pacific research methodological expertise in the drafting and revising of the manuscript. A provided senior Pacific educational and research expertise and contributed to drafting and revising the manuscript. PR provided senior Māori educational, institutional and KMR expertise and contributed to drafting and revising the manuscript. All authors read and approved the final manuscript for submission. All authors agreed to be accountable for all aspects of the work

Authors’ information

EC (Te Arawa, FNZCPHM, MPH (Distinc), MBChB) is a specialist in public health medicine who has experience in research and policy concerned with eliminating ethnic and indigenous inequalities in health. Elana is a Senior Lecturer and the Director Vision 20:20 at Te Kupenga Hauora Māori, The University of Auckland. She is a postgraduate Doctor of Medicine (MD) candidate (exploring indigenous and ethnic minority health workforce development) and has ongoing research interests in ethnic inequities in service utilisation and health outcomes.

EW (Ngāti Hine, PGDipPH (Distinc), BHSc) is a Māori Physiotherapist who has experience in research concerned with Māori and Indigenous health workforce development, cultural competence, and psycho-oncology in Māori and Indigenous populations. Erena is currently completing a Masters in Public Health whilst working as Researcher at Te Kupenga Hauora Māori, University of Auckland. Ongoing research interests include Māori health workforce development and addressing ethnic inequalities in health.

YJ (Chinese, PhD) is a Senior Research Fellow at the Department of Statistics and Senior Statistical Consultant at the Statistical Consulting Centre (SCC), Faculty of Science, University of Auckland, New Zealand. Ongoing research interests include: randomised controlled trial design and analysis, national surveys, longitudinal and case-control studies with response-selective sampling and missing data problems.

LM (Pākehā, MSc, MMath) is an Assistant Analyst at the Statistical Consulting Centre (SCC), Faculty of Science, The University of Auckland, New Zealand. She is a PhD candidate in the Department of Mathematics and Statistics.

RL (Samoa, PGDipPsych-Community, MSocS-Hons) proudly hails from the villages of Fagamalo and Avao (Savai’i) where he was raised as a young child. Rob is a Professional Teaching Fellow within Hikitia Te Ora – Certificate in Health Sciences programme at Te Kupenga Hauora Māori, FMHS, UoA. Rob is a Registered Community Psychologist and his aspirations are firmly rooted in the wellbeing and development of Māori and Pacific communities in regards to identity and health.

A (PhD, MEd (Distinc), MBA, BA, DipTchg, CertARM) specialises in higher education research, with a particular focus on Pasifika, indigenous and under-served students. Airini has Samoan ancestry, has a national and international record in Pasifika education research and recognised expertise in Pasifika methodologies. Airini is Dean, Faculty of Human, Social and Educational Development, Thompson Rivers University, BC, Canada. With a view to informing further education system reform in New Zealand and internationally, as a Fulbright Scholar based in Washington DC Airini investigated how to convert education policy into better results for under-served students.

PR (Te Rarawa, DipComH, BSc, MBChB, DipObst, FNZCPHM) is Tumuaki and Head of Department of Māori Health at the Faculty of Medical and Health Sciences, University of Auckland, New Zealand. She is a specialist in public health medicine and her research interests include analysing disparities between indigenous and non-indigenous citizens as a means of monitoring government commitment to indigenous rights.

Acknowledgements

The authors would like to thank members of the Te Hā Advisory Group: Dr Teuila Percival; Dr Vili Nosa; Dr Malakai Ofanoa; Associate Professor Mark Barrow; Lynley Pritchard; James Clark and Carolyn (Shaoxun) Huang. Andrew Sporle and Joanna Stewart are acknowledged for providing input into the early stages of project design from a statistical perspective. Dr Elana Curtis was supported by Te Kete Hauora, Ministry of Health (New Zealand) to conduct this research via the provision of a Research Fellowship (Contract 414953/337535/00). We also thank Ngā Pae o Te Māramatanga for their support for Erena Wikaire to attend and present these research findings at the Leaders in Indigenous Medical Education (LIME) Connection V conference in Darwin, Australia 2013.

End notes

    1. Completion of the first year of study within either the Bachelor of Health Sciences or the Bachelor of Science (Biomedicine) programme is required for an undergraduate application to the medical programme at the UoA

    1. For additional information, see previous publications 5. Curtis E, Reid P. Indigenous health workforce development: Challenges and successes of the Vision 20: 20 programme. Australian & New Zealand Journal of Surgery. 2013;83(2013):49-54, 13. Curtis E, Reid P, Jones R. Decolonising the Academy: The process of re-presenting indigenous health in tertiary teaching and learning. In: Cram F, Phillips H, Sauni P, Tuagalu C, editors. Māori and Pasifika Higher Education Horizons. Bingley, U.K.: Emerald Group Publishing Limited; 2014. p. 147-66, 14. Curtis, E., Wikaire, E., Jiang, Y., McMillan, L., Loto, R., Airini, & Reid, P. (2015). A tertiary approach to improving equity in health: Quantitative analysis of the Māori and Pacific admission scheme (MAPAS) process, 2008-2012. International Journal for Equity in Health, 14(7). 10.1186/s12939-015-0133-7. or https://www.fmhs.auckland.ac.nz/en/faculty/for/future-undergraduates/maori-and-pacific-admission-scheme.html

    1. The National Certificate of Educational Achievement (NCEA) is the major assessment method used in New Zealand secondary schools. The NCEA Rank Score reflects the best 80 credits at Level 3 or higher, over a maximum of five approved subjects. It reflects a system of Grade Point Average and is used by the UoA to assist with admission to limited entry programmes 23. Shulruf B, Hattie J, Tumen S. New Zealand’s standard-based assessment for secondary schools (NCEA): implications for policy makers. Asia Pacific Journal of Education. 2010;30(2).

    1. Exposure to a minimum of two final year secondary school science subjects is recommended for success within the CertHSc (alongside English and mathematics rich subjects). This variable includes secondary school results from NCEA, International Baccalaureate (IB) and Cambridge International Examinations (CIE).

  1. The Core 4 courses include: CHEM110 (Chemistry of the living world), POPLHLTH 111 (Population Health), MEDSCI 142 (Biology for Biomedicine Science: Organ Systems) and BIOSCI 107 (Biology for Biomedicine Science: Cellular Processes and Development).

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An epidemiological investigation to reconstruct a probable human immunodeficiency virus -1 transmission network: a case report

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An epidemiological investigation to reconstruct a probable human immunodeficiency virus -1 transmission network: a case report

Sara Serafino1, Eleonora Cella12, Claudia Montagna3, Eugenio Nelson Cavallari1, Pietro Vittozzi1, Alessandra Lo Presti2, Marta Giovanetti24, Laura Mazzuti3, Ombretta Turriziani3, Giancarlo Ceccarelli1, Gabriella d’Ettorre1, Vincenzo Vullo1 and Massimo Ciccozzi256*

Author Affiliations

1 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy

2 Department of Infectious Parasitic and Immunomediated Diseases, Reference Centre on Phylogeny, Molecular Epidemiology and Microbial Evolution (FEMEM)/Epidemiology Unit, National Institute of Health, Rome, Italy

3 Department of Molecular Medicine, Laboratory of Virology, Sapienza University of Rome, Rome, Italy

4 Department of Biology, University of Rome Tor Vergata, Rome, Italy

5 University of Biomedical Campus, Rome, Italy

6 Epidemiology Unit, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità- V.le Regina Elena, Roma, 299 – 00161, Italy

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Journal of Medical Case Reports 2015, 9:253  doi:10.1186/s13256-015-0717-2

The electronic version of this article is the complete one and can be found online at: http://www.jmedicalcasereports.com/content/9/1/253

Received: 31 October 2014
Accepted: 28 September 2015
Published: 3 November 2015

© 2015 Serafino et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

Recently published studies have highlighted the importance of phylogenetic and phylodynamic analyses in supporting epidemiological investigations to reconstruct the transmission network of human immunodeficiency virus. Here, we report a case of sexual transmission of human immunodeficiency virus type 1 between a man and a woman that marks once more the importance of a tightened collaboration between phylogeny and epidemiology.

Case presentation

We describe a case of human immunodeficiency virus type 1 subtype B transmission in a stable Caucasian heterosexual couple. The man was 30 years old and the woman was 21 years old at the time of their presentation to the Department of Public Health and Infectious Diseases of the University of Rome “Sapienza”. The couple reported a history of drug abuse.

Conclusion

Phylogenetic analysis is a powerful technique that if properly used can prove valuable in research investigations. In the case presented here, a phylogenetic analysis alongside epidemiological evidence allowed us to determine the most probable source of the human immunodeficiency virus infection. The dated tree allowed us to date the transmission event, the time point, and the direction of transmission based on the phylogeny, which agreed with the presumptive time of infection determined from clinical history-taking.

Keywords:

Case report; HIV-1; Phylogeny

Background

The human immunodeficiency virus (HIV) is characterized by great genetic heterogeneity driven by several factors, such as the lack of proofreading ability of the reverse transcriptase [1], [2], the rapid turnover of HIV-1 in vivo[3], host selective immune pressures [4], and recombination events during replication [5].

The majority of HIV-1 strains cluster within a large group called M (for Main), which includes nine subtypes (A–D, F–H, J, and K) with distinct phylogeny. Subtypes A and F can be further divided into sub-subtypes A1–A4 and F1 and F2, respectively. A number of inter-subtype recombinant viruses have also been observed [6]–[8].

HIV-1 group M subtypes are responsible for most of the HIV infections worldwide. In Italy the estimated percentage of non-B subtype infections has been reported to range from 2.4 to 19.4 %, thus confirming a significant increase in non-B subtypes prevalence [9]–[15], but, in this country, the first phase of the HIV epidemic was mainly confined to the intravenous drug users risk group, with an absolute predominance of HIV-1 B clade, as other Western Countries [16].

Phylogeny is a branch of molecular biology that infers knowledge about taxonomy and the evolution of species [17]. It is a powerful tool widely used in the study of rapidly evolving RNA viruses that cause chronic infections. The present case report underlines the importance of phylogenetic analysis to support epidemiological investigations into the reconstruction of transmission networks.

We present a case of HIV-1 subtype B transmission in a stable heterosexual couple living together was described to mark once more the importance of the “tightened collaboration” between phylogeny and epidemiology.

Case presentation

We describe a case of HIV-1 subtype B transmission in a stable Caucasian heterosexual couple. The man was 30 years old and the woman was 21 years old at the time of their presentation to the Department of Public Health and Infectious Diseases of the University of Rome “Sapienza”. He became addicted to injected drugs in 1996 at the age of 14 and entered a rehabilitation community after 14 years of drug abuse, during which he practiced needle exchange. She was addicted to injected drugs from the age of 12 until the age of 19 and also practiced an unsafe needle exchange. They met after she joined his rehabilitation community.

Our epidemiological investigation was conducted in two different phases. At the beginning of April 2013, he came to our attention because of a suspected infection with hepatitis C virus (HCV). He reported a virulent and recent episode of shingles on his right hemi-thorax. During a physical examination we noticed the presence of several genital lesions, suggestive of condylomas. On the basis of his epidemiological history and these clinical findings, we proposed our patient be tested for HIV infection. He was found to be positive for HCV IgG (Anti HCV Advia Centaur Immunoassay System, Siemens Healthcare Diagnostic, Tarrytown, NY, USA) and negative for HCV RNA (Versant HCV RNA 1.0 assay (kPCR), Siemens Healthcare Diagnostics). He was HIV-Ag/Ab-positive (Advia Centaur Systems HIV Ag/Ab Combo assay, Siemens Healthcare Diagnostics) with an HIV RNA count of 102,900 copies/mL (Versant HIV 1.0 RNA assay (kPCR), Siemens Healthcare Diagnostics) and a CD4+ T cell count of 20 cells/μL (1.55 %). A genotypic resistance test (Trugene HIV-1 genotyping assay, Siemens Healthcare Diagnostics) showed a wild-type virus. Our patient started combination antiretroviral therapy (cART) with tenofovir, emtricitabine, atazanavir, and ritonavir. Owing to these findings, we tested our patient’s partner for HIV and HCV infections. She was HIV-Ag/Ab-negative, HCV IgG-positive, and HCV RNA-negative.

After two weeks, the woman presented to the emergency department of an urban hospital with an elevated temperature and a skin rash on every part of her body. Results of a blood test showed a white blood cell count of 1200 cell/mL. She was discharged with a diagnosis of a viral infection and instructed to present for ambulatory care to an infectious diseases clinic.

The day after this discharge, our patient arrived at our center and reported that her menses was two days late. She tested positive for beta-human chorionic gonadotropin. The test for HIV-Ag/Ab was repeated, again with negative results, but we tested her anyway for HIV RNA with a result of 4161 copies/mL. As stated by the US Department of Health and Human Services guidelines, in consideration of <10,000 copies/mL of HIV RNA with a negative HIV-Ag/Ab test, we repeated the HIV RNA test using a different specimen from the same patient, and found an HIV RNA count of 1,302,000 copies/mL. At this time, an HIV-Ag/Ab test had “undetermined” results, with a single gp41 positive band on a confirmatory western blot test. cART was initiated with tenofovir, emtricitabine, and raltegravir. As was the case with her partner, a genotypic resistance test revealed a wild-type virus. A genotyping test revealed coincident viral tropism within the couple, with CXCR4 tropism as predicted by a geno2pheno algorithm set at a false-positive rate of 10 % [18]. At the beginning of May, our patient had a spontaneous abortion due to acute retroviral syndrome.

To support the epidemiological investigation, we reconstructed the transmission network within a calendar timescale on the basis of a recently described phylogenetic–statistical framework, using the env viral sequences from our two patients [19], [20]. For virologic and phylogenetic analysis, we performed peripheral blood mononuclear cell isolation and DNA extraction as previously described [21]. The env region was amplified by a nested polymerase chain reaction and the following primers were used for the sequencing reaction: 5′-CTGTTAAATGGCAGTCTAGC-3′, 5′-GCAATGTATGCCCCTCCCATC-3′, and 5′GCTCCATGTTTTTCCAGGTC-3′. Sequence analyses were performed by Sequencher and Bioedit software packages. The subtypes of the two sequences was determined by uploading the sequences individually into the REGA HIV-1 automated Subtyping Tool v2.0 [22].

We built two different datasets: one with both the male and female sequences, and one without the female sequence to date the male infection. Nucleotide sequences were aligned with 35 reference HIV-1 subtype B sequences of known provenance and date (26 from Italy and six from other countries) using CLUSTAL W software and edited manually according to their codon-reading frame by BioEdit [23], [24]. These reference sequences were obtained with the Blast similarity search.

We performed a hierarchical likelihood ratio test using ModelTest 3.7 implemented in the PAUP* 4.0 software [23], [24], and identified the evolutionary model as the best-fitting nucleotide substitution model.

Dated phylogenies were obtained by simultaneously inferring the evolutionary rate and population and model parameters using a Bayesian Markov Chain Monte Carlo (MCMC) method implemented in the BEAST package version 1.8 [18], [25]. Statistical support for specific clades was obtained by calculating the posterior probability of each monophyletic clade. The trees were generated using the HKY +I+G model of substitution, chosen by ModelTest.

The MCMC was run for 50×10 6 generations, under both strict and relaxed clock conditions, until convergence was achieved on the basis of the effective sampling size (ESS). Only ESS values of >250 were accepted. As coalescent priors, we compared three parametric models (constant, exponential, expansion growth) and a Bayesian Skyline plot non-parametric model.

For the first dataset, the expansion growth model under a relaxed (uncorrelated log normal) clock was selected, whereas for the second dataset, the exponential growth model under a relaxed (uncorrelated log normal) clock was selected.

REGA subtyping analysis classified the two sequences as subtype B. Our patients’ isolates formed a significant monophyletic cluster (posterior probability = 1) (Fig. 1a), showing a strong relationship and affirming infection by the same virus.

thumbnailFig. 1. Bayesian time-scaled tree of the HIV-1 B sequences. The asterisks (*) along a branch represent significant statistical support for the clade subtending that branch (posterior probability = 100 %). The numbers at the internal node represent the estimated date of the origin and the uncertainty indicated by 95% highest posterior density intervals. a Couple (male and female) tree. b Dated male viral phylogeny

In the male dated phylogeny (Fig. 1b), the male sequence was related to a sequence from the USA, and the time to most recent common ancestor was estimated to be 1998 (95 % highest posterior density, 1991 to 2004).

The Bayesian analysis confirmed the transmission network and allowed us to date the transmission event with a probability of 99 %. Moreover, the existence of an epidemiological relationship between the two patients confirmed the phylogenetic analysis and agreed with the presumptive date of infection on the basis of clinical history-taking. The Bayesian analysis also confirmed that our male patient probably acquired the infection about two years after starting illicit drug use and before having a relationship with our female patient.

Conclusions

HIV-1 and HIV-2 transmission networks are already described in nosocomial and non-hospital-acquired infections [19], [20], [26]. A report of healthcare workers infection with HIV-1 by a needle stick injury was recently reported and published in 2010 [23]. A connection between epidemiological investigations and phylogenetic analyses was also recently demonstrated in case report analyses and population studies [24], [26]. Prospective surveillance studies conducted throughout the world report that the risk of HIV transmission ranges from 0.09 to 0.3 % [27].

A phylodynamic reconstruction, created using Bayesian methods, of the transmission network within a calendar timescale, that agreed with the epidemiological data, provided a well-documented transmission framework to significantly improve the investigation in our case.

Phylogenetic analysis is a powerful technique that if properly used can prove valuable in research investigations. In our case, we found it remarkable how the phylogenetic analysis and epidemiological evidence aligned to allow us to determine the most probable source of HIV infection. Our findings in these cases has strengthened the evidence that Bayesian phylogenetic analysis can be an important way of tracing epidemiological relationships.

Consent

Written informed consent was obtained from the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Abbreviations

cART: combined antiretroviral therapy

ESS: effective sampling size

HCV: hepatitis C virus

HIV: human immunodeficiency virus

MCMC: Markov Chain Monte Carlo

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SS, EC, ALP and MG performed the phylogenetic analysis and contributed to manuscript writing. ENC, GC, GdE and PV collected the clinical data and revised the manuscript; CM, LM, OT and SS performed sequencing and data analysis. VV supervised and coordinated the study. MC supervised the phylogenetic analysis and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors wish to thank Dr Valerio Ciccozzi for the English revision of the manuscript.

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Update on mesenchymal stem cell-based therapy in lupus and scleroderma

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Update on mesenchymal stem cell-based therapy in lupus and scleroderma

Audrey Cras12*, Dominique Farge3*, Thierry Carmoi4, Jean-Jacques Lataillade5, Dan Dan Wang6 and Lingyun Sun6

Author Affiliations

1 Assistance Publique-Hôpitaux de Paris, Saint-Louis Hospital, Cell Therapy Unit, Cord blood Bank and CIC-BT501, 1 avenue Claude Vellefaux, Paris, 75010, France

2 INSERM UMRS 1140, Paris Descartes, Faculté de Pharmacie, 4 avenue de l’observatoire, Paris, 75004, France

3 Assistance Publique-Hôpitaux de Paris, Saint-Louis Hospital, Internal Medicine and Vascular Disease Unit, CIC-BT501, INSERM UMRS 1160, Paris 7 Diderot University, Sorbonne Paris Cité, 1 avenue Claude Vellefaux, Paris, 75010, France

4 Hôpital du Val de Grace, Internal Medecine Unit, 74 boulevard de Port Royal, Paris, 75005, France

5 Percy Military Hospital, Department of Research and Cell Therapy, 101 Avenue Henri Barbusse, Clamart, 92140, France

6 Department of Immunology, The affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhong Shan Road, Nanjing 210008, China

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Arthritis Research & Therapy 2015, 17:301  doi:10.1186/s13075-015-0819-7
Audrey Cras, Dominique Farge, Dan Dan Wang and Lingyun Sun contributed equally to this work.

The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/17/1/301

Published: 3 November 2015

© 2015 Cras et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Current systemic therapies are rarely curative for patients with severe life-threatening forms of autoimmune diseases (ADs). During the past 15 years, autologous hematopoietic stem cell transplantation has been demonstrated to cure some patients with severe AD refractory to all other available therapies. As a consequence, ADs such as lupus and scleroderma have become an emerging indication for cell therapy. Multipotent mesenchymal stem cells (MSCs), isolated from bone marrow and other sites, display specific immunomodulation and anti-inflammatory properties and appear as ideal tools to treat such diseases. The present update aims at summarizing recent knowledge acquired in the field of MSC-based therapies for lupus and scleroderma.

Introduction

Autoimmune diseases (ADs) are a group of heterogeneous conditions characterized by aberrant activation of the immune system with failure of the immune regulation to maintain adapted tolerance. They are traditionally classified as “organ-specific AD”, where the consequences of organ failure can be improved by a replacement opotherapy or an organ transplantation, and as “diffuse or systemic AD”, notably including systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). However, progressive identification of the genetic background of each AD type [1] and elucidation of the mechanisms associated with self-directed tissue inflammation, unrelated to T- or B-cell abnormalities, revealed the important differences between autoimmunity and autoinflammation [2]. SLE, type 1 diabetes, and autoimmune thyroiditis are polygenic ADs with a predominant autoimmune component, whereas other polygenic ADs, such as Crohn’s disease, are characterized by a predominant autoinflammatory component. Therefore, the optimal treatment of AD should be discussed in light of this specific pathological continuum between autoimmunity and autoinflammation, which variably interacts in each AD phenotypic expression. Indeed, chronic immunosuppression is responsible for high treatment-related morbidity and still is associated with significant disease- and treatment-related mortality, notably in patients with severe inflammatory SLE or refractory SSc and with kidney, heart-lung, or brain damage. With a view to developing innovative therapies for AD, mesenchymal stem cell (MSC)-based therapies theoretically appear as ideal tools to target the respective autoinflammatory and autoimmune components of such diseases, and this update aims at summarizing recent knowledge acquired in the field.

A need for innovative stem cell therapies in severe or refractory forms of systemic lupus erythematosus and systemic sclerosis

SLE, with a prevalence of 40 to 50 out of 100,000 people, is a heterogeneous chronic multisystemic autoimmune inflammatory disorder whose original flare can be controlled by conventional immunosuppressive therapy. However, definitive cure is rarely achieved by this therapy and life-long immunosuppression is often required. Response rates vary from 20 to 100 % at 6 months according to the definition of response or improvement, the extent of visceral damage, the ethnic origin, and the socioeconomic profile. First-line validated standard therapies used to induce remission within the first 6 to 9 months of disease flare are the corticosteroids in combination with either (a) cyclophosphamide (CY), using the classic National Institutes of Health regimen or lower doses for shorter duration over the course of 3 months with a similar efficacy, according to the Eurolupus regimen [3], [4], or (b) mycophenolate mofetil, with good efficacy and tolerability [5], [6]. Other monoclonal antibodies against the T- or B-cell receptors, such as rituximab as an anti-CD20, or against the adhesion molecules involved in the T- or B-cell interaction and their co-stimulatory signals, have been used despite the paucity of validated therapeutic targets and the failure to demonstrate the efficacy of rituximab in renal and extra-renal manifestations of SLE [7]. In 2011, a monoclonal antibody against B cell-activating factor of the tumor necrosis factor family (BAFF), belimumab anti-Blys, was the first targeted therapy to demonstrate its efficacy in mild to moderate SLE by a randomized clinical trial [8]. Despite early diagnosis and treatment with immunosuppressive agents as well as a tight control of hypertension and infections, there is still a subgroup of patients with SLE that does not respond to the treatment and that has 10-year mortality of 10 % [9]. In addition, early death from rapidly progressive atherosclerosis in SLE suggests that, despite apparent reasonable disease control, subclinical inflammatory disease promotes endothelial damage and plaque formation and that prolonged exposure to corticosteroids and immunosuppressive drugs leads to further damage beyond the SLE itself.

SSc, which has a prevalence of 5 to 50 per 100,000, is a rare AD characterized by early vascular endothelium damage with consequent activation of the immune response and enhanced collagen synthesis, leading to progressive fibrosis of the skin and internal organs. Both antigen stimulation and genetic susceptibility may contribute to autoimmunity, with consequent early T-cell infiltration as well as B-cell and fibroblast activation, by pro-fibrotic cytokines, mainly transforming growth factor-beta (TGF-β) and connective tissue growth factor. Most patients progress, and the overall 10-year survival is only 66 %, and there is significant morbidity and altered quality of life among survivors. In rapidly progressive SSc, mortality rates reach 30 to 50 % in the first 5 years after disease onset, according to the extent of skin, cardiopulmonary, and renal involvement [10]. No treatment has ever shown any benefit in this severe disease, except autologous hematopoietic stem cell transplantation (HSCT), whose efficacy was recently established by a unique international multi-center, open-label phase III, ASTIS (Autologous Stem cell Transplantation International Scleroderma) trial [11] that enrolled 156 patients over the course of 10 years with early diffuse cutaneous SSc, showing that HSCT confers a significantly better long-term survival rate than 12 monthly intravenous pulses of CY.

In this context, new therapeutic approaches with fewer long-term side effects are warranted. Bone marrow (BM) stromal cells or MSCs, which can also be obtained from other human tissues, have recently enlarged the therapeutic tool set for SLE and SSc. Because MSCs display specific immunomodulation and immunosuppressive properties as well as regenerative potential, there is a strong rationale for MSC-based therapy in SLE and SSc to treat their respective autoimmune and autoinflammatory components at a certain time point of each disease evolution.

Biology of mesenchymal stem cells

Definition, isolation, and characterization of mesenchymal stem cells

MSCs were originally identified in BM by Friedenstein in 1976 as a fibroblast-like cellular population capable of generating osteogenic precursors [12]. Since then, these cells have been extensively investigated and given various names, until 1991 when Caplan proposed the definition ‘mesenchymal stem cells’ (MSCs) [13], which after consensus of the Mesenchymal and Tissue Stem Cell Committee of International Society for Cellular Therapy (ISCT) was changed to “multipotent mesenchymal stromal cells”. ISCT has provided three minimal criteria to define MSCs [14]: (a) plastic adherence in standard culture conditions; (b) differentiation into osteoblasts, adipocytes, and chondroblasts under specific conditions in vitro; and (c) expression of nonspecific markers CD105, CD90, and CD73 along with the lack of expression of hematopoietic markers such as CD34, CD45, CD14 or CD11b, CD79a, or CD19. MSCs show intermediate levels of major histocompatibility complex (MHC) class I molecules on their cell surface and have no detectable levels of MHC class II, mainly HLA-DR, and co-stimulatory molecules (CD40, CD80, and CD86), which allow their transplantation across MHC barriers [15]. Therefore, their privileged immunological phenotype makes them an appropriate stem cell source for allogeneic transplantation. They can also synthesize trophic mediators, such as growth factors and cytokines—macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-11, IL-15, stem cell factor, and vascular endothelial growth factor—involved in hematopoiesis regulation, cell signaling, and modulation of the immune response [16].

BM-MSCs were discovered first, and the BM was considered the main source of MSCs. BM-MSCs are classically expanded in vitro by consecutive passages in fibroblast growth factor-supplemented cell culture medium from the plastic-adherent BM cell population. Subsequently, MSCs, facilitated by their ability to adhere to plastic, have been isolated from various other sources such as skeletal muscle, adipose tissue, dental tissue, synovial membranes, placenta, cord blood, and Wharton’s jelly by using enzymatic tissue digestion and density gradient centrifugation methods [17]. These alternative sources are very attractive because BM harvesting is rather invasive and painful and is associated with potential donor-site morbidity. Moreover, because of the rarity of MSCs in the BM, where they represent 1 in 10,000 nucleated cells, tissues such as umbilical cord (UC) or adipose tissue (AT) represent promising sources. Indeed, MSCs can be more easily isolated from these tissues and considerably larger amounts of UC- or AT-derived MSCs can be obtained, compared with the BM. MSCs from these different sources share many biological features, although studies reported some differences in their immunophenotype, proliferative capacity, differentiation potential, or gene expression profile [18], [19].

Immunomodulatory properties of mesenchymal stem cells: evidence from in vitro data

Compared with other stem cell sources, such as hematopoietic stem cells (HSCs), MSCs appear as a promising source for overcoming autoimmunity because of their immunosuppressive properties [20]. MSCs modulate the immunological activity of different cell populations as shown by in vitro data. Their most important effects are T-cell proliferation and dendritic cell (DC) differentiation inhibition, which are key activating factors of autoimmune disorders. MSCs are effective in inhibiting proliferation of CD4 and CD8 T cells as well as memory and naïve T cells [21]. This mechanism relies on both cell–cell contact and several specific mediators, produced by MSCs, such as TGF-β1, prostaglandin E 2 , and indoleamine 2,3 deoxygenase [22]. The ability to suppress T-cell responses to mitogenic and antigenic signals is explained by a complex mechanism of induction of “division arrest anergy”, responsible for maintaining T lymphocytes in a quiescent state. Thus, the MSCs trigger the inhibition of cyclin D2 expression, thus arresting cells in the G 0 /G 1 phase of the cell cycle [23]. MSCs also inhibit the production of interferon-gamma (IFN-γ) and increase the production of IL-4 by T helper 2 cells. This indicates a shift in T cells from a pro-inflammatory state to an anti-inflammatory state [24], [25]. MSCs also stimulate the production of CD4 + CD25 + regulatory T cells, which inhibit lymphocyte proliferation in allogeneic transplantation [26]. In addition, MSCs inhibit B-cell proliferation through arrest at the G 0 /G 1 phase of the cell cycle and production of IgM, IgA, and IgG as well as their chemotactic abilities [27], [28]. A recent study demonstrated that this effect of MSCs on B cells is mediated by T cells [29]. However, some contradictory data showed that, in some culture conditions, IgG secretion and B-cell proliferation can be induced and B-cell survival sustained, and this effect does not depend on the presence of IFN-γ in the culture [30], [31]. MSCs have been demonstrated to interfere with DC differentiation, maturation, and function [32]–[34]. MSCs obtained from healthy human donors can indirectly reduce T-cell activation by inhibiting DC differentiation (mainly DC type I) from monocytes [35].

Although the majority of data dealing with the immunomodulatory effects of MSCs are derived from BM-MSCs, some of these effects have also been described for MSCs from other sources. Results from studies comparing the immunomodulatory effects of various tissue-derived MSCs are controversial. Some studies concluded that BM- and UC-MSCs show similar effects, whereas others demonstrated that UC-MSCs have a higher capacity of inhibiting T-cell proliferation than adult MSCs [36], [37]. Some studies also indicate that AT-MSCs can be more effective suppressors of immune response compared with BM-MSCs. Indeed, AT-MSCs modulate mitogen-stimulated B-cell immunoglobulin production in vitro to a much greater extent than BM-MSCs. Also, in comparison with BM-MSCs, they inhibit, significantly more, the differentiation of blood monocytes into DCs and the expression of functionally important co-stimulatory molecules on the surface of mature monocyte-derived DCs [38], [39]. It may be postulated that MSCs express a different set of molecules depending of their tissue of origin, resulting in different immunosuppressive activities. Taken together, these in vitro data demonstrate that MSCs modulate the action of the various cells that are involved in immune response and preferentially inhibit T-cell proliferation and differentiation of DCs. However, it would be important to further investigate the molecular mechanisms that underlie the immunomodulatory properties of various tissue-derived MSCs since these differences may have functional relevance to the therapeutic use of these cells.

Mesenchymal stem cell-based therapy in animal models

Animal models of AD can be divided into two categories. The hereditary and spontaneous AD models, such as murine (BXSB) lupus, are characterized by autoimmune manifestations that affect the majority of the animals of a susceptible line and by a strong genetic predisposition displayed by the HSCs and manifested by anomalies of thymic development and/or function of lymphocytes B or T or antigen-presenting cells such as macrophages. Other experimental models, such as arthritis adjuvant and experimental acute encephalomyelitis [40], use active immunization by exposure to a foreign antigen to induce the AD. The rationale for using MSCs for the treatment of autoimmunity was first demonstrated in experimental acute encephalomyelitis, a model for multiple sclerosis [25]. Subsequently, several preclinical studies evaluating MSC injection in a collagen-induced arthritis model [41] or in an autoimmune type 1 diabetes model [42] provided support for the potential therapy of other ADs, including SLE and SSc.

Animal models of systemic lupus erythematosus

Both Fas mutated MRL/lpr mice and NZB/W F1 mice are widely used as genetically prone lupus models, which demonstrate progressive nephritis, elevated serum autoimmune antibodies, and immune abnormalities. The role of BM-MSC transplantation in SLE and its efficacy compared with conventional CY treatment has been investigated in MRL/lpr mice as an SLE mouse model [43], [44]. MSC injection resulted in a significant reduction in serum levels of anti-double-stranded DNA (anti-dsDNA) antibodies IgG and IgM, ANA, and immunoglobulins IgG1, IgG2a, IgG2b, and IgM as well as an increased serum albumin level. When compared with MSCs, conventional CY treatment partially reduced the levels of serum autoantibodies and immunoglobulin IgG2a, restored albumin level, and failed to reduce circulating immunoglobulins IgG1, IgG2b, and IgM. MSC treatment improved renal disorders, specifically restoring kidney glomerular structure and reducing C3 and glomerular IgG deposition. Although CY treatment could reduce glomerular IgG deposition, it did not restore the glomerular structure and C3 accumulation. MSC treatment, but not CY treatment, was able to completely restore renal function, shown as normalization of serum and urine creatinine levels in MRL/lpr mice, in comparison with disease-free control mice. In their study, Ma et al. determined that murine BM-MSC transplantation improved nephritis in MRL/lpr mice by suppressing the excessive activation of B cells via inhibition of BAFF production [45]. Nevertheless, in a similar study conducted in a different SLE mouse model (NZB/W), systemic MSC administration did not provide any beneficial effect and in fact worsened the disease [46], [47]. To resolve these conflicting results, Gu et al. assessed the differential effects of allogeneic versus syngeneic MSC transplantation on lupus-like disease in both mice models [48]. They showed that, in MRL/lpr and NZB/W mice, both normal MSCs and lupus MSCs from young mice ameliorated SLE-like disease and reduced splenic T and B lymphocyte levels. However, lupus MSCs from older NZB/W mice did not significantly reduce spleen weights, glomerular IgG deposits, renal pathology, interstitial inflammation, or T or B lymphocyte levels. This study suggests that allogeneic MSCs may be preferred over syngeneic lupus-derived MSCs given the decreased overall effectiveness of post-lupus-derived MSCs, which is partly triggered by the disease and is not exclusively an intrinsic defect of the MSCs themselves. The same group reported that human lupus BM-MSCs are not as effective as human healthy BM-MSCs and umbilical cord-derived MSCs (UC-MSCs) in ameliorating disease in MRL/lpr mice [49]. Moreover, in vitro assessments of immunomodulatory functions detected a reduced capacity of lupus BM-MSCs to inhibit IFN-γ production and CD19 + B-cell proliferation, although inhibition of CD3 + proliferation and IFN-γ licensing results were indicative of immune activity by lupus BM-MSCs. Although these studies showed that lupus MSCs are not yet a suitable source of MSCs for cell therapy, it is important to continue to define differences in MSCs because it appears that donors and the origin of the MSCs impact their function.

Some studies evaluated the effectiveness of MSCs derived from sources other than BM. Sun’s team had showed that UC-MSCs alleviated lupus nephritis in MRL/lpr mice in a dose-dependent manner [50]. Both single and multiple treatments with UC-MSCs were able to decrease the levels of 24-h proteinuria, serum creatinine, anti-dsDNA antibodies, and the extent of renal injury, such as crescent formation. Further studies dealing with the underlying mechanisms showed that UC-MSC treatment inhibited renal expression of monocyte chemotactic protein 1 and high-mobility group box 1 expression but that it upregulated Foxp3 + regulatory T cells. Moreover, carboxyfluorescein diacetate succinimidyl ester-labeled UC-MSCs could be found in the lungs and kidneys after infusion [50]. Using NZB/W F1 mice, Chang et al. showed that human UC-MSC transplantation significantly delayed the onset of proteinuria, decreased anti-dsDNA, alleviated renal injury, and prolonged the life span [51]. Subsequent studies looking at the mechanisms showed that the treatment effect was not due to a direct engraftment and differentiation into renal tissue but rather to the inhibition of lymphocytes, the induced polarization of T helper 2 cytokines, and the inhibition of the synthesis of pro-inflammatory cytokines. Choi et al. showed that long-term repeated administration of human AT-MSCs ameliorated SLE in NZB/W F1 mice [52]. Compared with the control group, the AT-MSC-treated group had a higher survival rate, decreased histological and serological abnormalities, improved immunological function, and a decreased incidence of proteinuria. Transplantation of AT-MSCs led, on the one hand, to significant decreased levels of antibodies targeting dsDNA and blood urea nitrogen levels. On the other hand, it significantly increased serum levels of granulocyte-macrophage colony-stimulating factor, IL-4, and IL-10. A significant increase in the proportion of CD4 + FoxP3 + cells with a marked restoration of their capacity to produce cytokines was observed in spleens from the AT-MSC-treated group.

Animal models of systemic sclerosis

Among the various experimental models aiming at reproducing the SSc (genetic models, such as tight skin (TSK) Tsk1 and Tsk2 mice, UCD-200 chicken, Fra-2 mice, TGFβRIIΔκ, or inducible models using injections of bleomycin or vinyl chloride or graft-versus-host disease (GVHD) mice), none displayed exactly the three components of scleroderma in humans [53]. Indeed, two forms of SSc are defined in humans. The first one is characterized by extensive skin fibrosis (proximal and distal), common pulmonary fibrosis, and the presence of antibody directed against DNA topoisomerase 1. In regard to the second form, referred to as the “limited cutaneous” form, the skin disease is limited to the distal limbs and lung symptoms are rare. The autoantibodies detected in this second form are against centromere (the main target being the centromeric protein CENP-B) and not against DNA topoisomerase 1. The TSK mouse model is characterized mainly by skin lesions, which do not reach the dermis; others use the mismatch transplant BM or spleen cells in mice sublethally irradiated. A scleroderma-like syndrome associated with chronic GVHD was induced with skin and lung fibrosis and was associated with signs of autoimmunity. Finally, induction of fibrosis by bleomycin injection could be used. But none reproduced a true picture of scleroderma. The role of free radicals in the development of SSc was studied and this helped to develop a mouse model of scleroderma, based on repeated injection of hypochlorous acid [54]. This model mimics the diffuse form of the human disease (cutaneous sclerosis, pulmonary fibrosis, renal disease, and anti-topoisomerase antibodies) and is a more satisfactory way to test new therapeutic approaches than other models. Despite the lack of perfectly reproducible models of SSc, the effect of MSCs on fibrosis is known and has been studied in the model of fibrosis induced by bleomycin [55]–[57]. Injection of MSCs allowed investigators to limit the pro-inflammatory and pro-fibrotic bleomycin effect through a mechanism involving IL-1RA [58]. Even though this model only partially reproduces SSc disease, all of the in vitro and in vivo data suggest that MSCs may have a beneficial effect in SSc.

Characteristics of mesenchymal stem cells derived from patients with systemic lupus erythematosus and systemic sclerosis

Because the majority of pathogenic autoreactive cells are the progeny of HSCs, it is conceivable that HSCs are involved in the AD process. BM-MSCs are key components of the hematopoietic microenvironment and provide support to hematopoiesis and modulate the immune system. Little is known about how MSCs are involved in immunological disorders. However, evidence has suggested that BM-MSCs from animal models and from patients with SLE and SSc exhibited impaired capacities of proliferation, differentiation, secretion of cytokines, and immune modulation. These alterations might be the consequence of the disease or play a fundamental role in the pathogenesis of SLE and SSc.

Mesenchymal stem cells derived from patients with systemic lupus erythematosus

BM-MSCs from patients with SLE have impaired hematopoietic function [59] and show significantly decreased bone-forming capacity and impaired reconstruction of BM osteoblastic niche in vivo [43]. Moreover, BM-MSCs from patients with SLE seem larger and flatter in appearance during in vitro culture and grow progressively slower compared with those from controls, thus demonstrating early signs of senescence [60], [61]. This senescent state is associated with differences in gene expression profile of BM-MSCs between SLE patients and controls, resulting in abnormalities in actin cytoskeleton, cell cycling regulation, BMP/TGF-β, and MAPK signaling pathways in BM-MSCs from patients with SLE [62]. In their study, Gu et al. found that senescent BM-MSCs from patients with SLE display reduced ability to upregulate regulatory T cells [63]. An increased p16INK4A expression plays a major role in this cellular senescence process by regulating cytokine secretion as well as the ERK1/2 signaling pathway. Wnt/b-catenin signaling also plays a critical role in the senescence of SLE BM-MSCs through the p53/p21 pathway [64]. Finally, SLE BM-MSCs exhibit an increased apoptosis rate, as reflected by downregulation of Bcl-2 and upregulation of cytochrome C in the cytoplasm, and display an enhanced aging process as shown by the overproduction of intracellular reactive oxygen species, which might be linked with the upregulation of p-FoxO3 and its upstream gene AKT [65].

Mesenchymal stem cells derived from patients with systemic sclerosis

Studies on BM-MSCs from patients with SSc are more limited. In patients with SSc, the osteogenic and adipogenic differentiation potentials of MSCs appear to be altered when they are isolated from the BM by direct selection of nerve growth factor receptor (CD271)-positive cells and not by the conventional technique of adhesion [66]. In these patients, the ability of MSCs to differentiate into endothelial progenitor cells appear reduced, and the endothelial progenitor cells obtained have a reduced ability to migrate and a lower pro-angiogenic potential [67]. Cipriani et al. showed that although BM-MSCs from SSc patients undergo premature senescence, they maintain considerable immunosuppressive functions and a normal ability to generate functional regulatory T cells [68]. In our study, we showed that the SSc BM-MSCs have fibroblast colony-forming units ability with a phenotype and a frequency similar to those of MSCs derived from healthy donors [69]. They differentiate into adipose and osteogenic cells with variabilities similar to those observed within the BM-MSCs from healthy controls. In regard to the immunoregulatory activity of MSCs in SSc, we reported that MSCs from patients were capable of supporting normal hematopoiesis and retained their immunosuppressive properties on T cells, thus confirming the data published by Bocelli-Tyndall et al. [69], [70]. We have recently shown a significant increase of the level of receptor type II TGF-β in MSCs from SSc patients compared with MSCs from healthy donors, associated with an activation of the TGF-β signaling pathway, leading to an increase in the synthesis of target genes, including the gene encoding collagen type 1 [71]. This activation of MSCs in response to stimulation by TGF-β, known for its major role in the pathogenesis of the disease, obviously limits their clinical use and justifies the use of allogeneic MSCs in these patients.

All of these findings suggest that BM-MSCs from patients with SLE or SSc are defective in regard to certain functions. Therefore, we can speculate that an allogeneic rather than an autologous MSC-based therapy might be preferable for treatment. Even though some data bring their early senescence to light, MSCs maintain some immunosuppressive properties that support the potential autologous clinical application. These data emphasize the necessity for a better understanding of the MSC involvement in the pathogenesis and the underlying MSC-immunomodulatory mechanisms.

Hematopoietic stem cell-based and mesenchymal stem cell-based therapy in patients with systemic lupus erythematosus and systemic sclerosis

Use of hematopoietic stem cell transplantation in systemic lupus erythematosus or systemic sclerosis

The use of HSCT in patients with AD to induce tolerance by resetting the immune responses is supported by both experimental data and clinical evidence. The direct relationship between the hematopoietic system and AD was evidenced in 1985 by Ikehara et al., who first demonstrated that AD originated from defects in the HSCs [72]. Thereafter, data from genetically prone and immunized animal models of AD treated with allogeneic, syngeneic, and autologous BM transplantation (BMT) showed that allogeneic BMT (but not syngeneic or autologous) could be used to treat AD-prone mice [73]. Conversely, the AD transfer was possible in normal mice after allograft from a mouse lupus nephritis showing that it was in fact a stem cell disorder. Consensus indications for the use of transplantation of BM-derived or peripheral HSCs to treat severe ADs were first elaborated in 1997 [74] and were updated in 2012 [75]. Today, more than 3500 patients worldwide have received an HSCT for an AD alone; approximately 200 autologous HSCTs were for refractory SLE and 500 were for severe SSc. This allowed a sustained and prolonged remission with qualitative immunological changes not seen with any other forms of treatment. In SLE, these beneficial effects were limited by the increased short-term mortality underlying the need to develop new strategies. In severe SSc, adequate prospective trials allowed investigators to ensure the safety of non-myeloablative autologous HSCT for SSc when careful patient selection, follow-up, and center effect are considered, to avoid misleading use of CY when it is unlikely to be clinically meaningfully effective. In case of allogeneic transplantation, more data suggest preclinical and clinical evidence for a graft versus autoimmunity effect in replacement of a dysfunctional immune system by allogeneic HSCT, which also represents an attractive prospect. In this setting, analysis of the regenerating adaptive immune system showed normalization of the restricted T-cell repertoire, with sustained shifts in T- and B-cell subpopulations from memory to naïve cell dominance supporting a thymic reprocessing and re-education of the reconstituting immune system [76], [77]. Disappearance of circulating plasmablasts and restoration of normal or raised levels of CD4+ and CD8 + FoxP3+ regulatory T cells were shown in SLE following autologous HSCT. This normalization was accompanied by complete inhibition of pathogenic T-cell response to autoepitopes from histones in nucleosomes [78], [79]. This has never been shown previously after the use of conventional immunosuppressive therapies. Such clinical and immunological results allowed investigators to take into account the non-specific immunosuppressive changes, which can be observed both in blood and in tissues after cytotoxic therapy [76], [80], and immune re-educative changes supporting immune tolerance [81]. Therefore, for the first time in AD treatment, the interruption of the vicious circle of autoimmunity allowed the emergence of normal regulatory mechanisms and the eradication of the last auto-reactive T cell, which is one of the proposed mechanisms for using HSCs in the treatment of SLE and SSc.

Mesenchymal stem cell-based therapy in systemic lupus erythematosus and systemic sclerosis

Discovery and identification of MSCs within the BM content and of their therapeutic properties have led us and others to use MSCs derived from various tissues to treat AD. Indeed, the supportive function for HSCs in the BM niche and the immunomodulatory capacities of MSCs suggest their potential use for cell therapy. Allogeneic donor-derived BM-MSCs have already been used in several phase I and II and very few phase III clinical trials for the treatment of acute GVHD following allogeneic HSCT for leukemia or hematological malignancies [82]. With a better understanding of the combined components of autoimmunity and autoinflammmation in each AD, there is a rationale to propose combined therapies with different tools.

BM-MSCs and UC-MSCs have been transplanted in patients with severe SLE, who were not responsive to conventional therapies. The 4-year follow-up demonstrated that about 50 % of the patients entered clinical remission after transplantation, although 23 % of the patients relapsed [83]. MSC infusion induced disease remission for lupus nephritis [84], diffuse alveolar hemorrhage [85], and refractory cytopenia [86]. The multi-center clinical study showed that 32.5 % of patients achieved major clinical response (13 out of 40) and 27.5 % of patients achieved partial clinical response (11 out of 40) during a 12-month follow-up, respectively. However, 7 (17.5 %) out of 40 patients experienced a disease relapse after 6 months of follow-up, after a prior clinical response, which indicated that another MSC infusion would be necessary after 6 months [87].

Few data are available about MSC-based therapy in patients with SSc. A patient with severe refractory SSc received an intravenous injection of allogeneic MSCs [88]. Three months after injection of MSCs, a significant decrease in the number of digital ulcers was observed. At 6 months, blood flow to the hands and fingers seemed significantly improved, and transcutaneous partial pressure of oxygen was increased. Rodnan skin score dropped from 25 to 11. The titer of anti-Scl-70 antibody, however, remained high, and enumeration of lymphocytes T, B, and natural killer cells did not change. This first observations were supplemented by four other cases reported by the same German team using allogeneic MSCs to treat severe forms of SSc, without major side effects or specific abnormalities observed after respective follow-ups of 44, 24, 6, 23, and 18 months [89]. The first two patients received fresh MSCs, whereas the three others received cryopreserved allogeneic MSCs. No conclusion about the efficacy of the MSC transplantation can be drawn from these clinical cases, although skin improvement was noted in three out of five cases and these patients did not have a detailed immunomonitoring.

Although further studies are necessary, preclinical and clinical data underline the therapeutic potential of MSCs in patients with SLE and SSc. Now it is important to design a controlled study to further investigate the clinical efficacy of MSC transplantation, compared with conventional immunosuppressive therapies, or the efficacy of MSC transplantation combined with immunosuppressive drug treatment compared with drugs alone. Careful patient selection and performance are crucial for the proper use of this therapy.

Note

This article is part of a thematic series on Biology and clinical applications of stem cells for autoimmune and musculoskeletal disorders, edited by Christian Jorgensen and Anthony Hollander. Other articles in this series can be found at http://www.biomedcentral.com/series/MSC

Abbreviations

AD: Autoimmune disease

AT: Adipose tissue

BAFF: B-cell-activating factor of the tumor necrosis factor family

BM: Bone marrow

BM-MSC: Bone marrow-derived mesenchymal stem cell

BMT: Bone marrow transplantation

CY: Cyclophosphamide

DC: Dendritic cell

dsDNA: Double-stranded DNA

GVHD: Graft-versus-host disease

HSC: Hematopoietic stem cell

HSCT: Hematopoietic stem cell transplantation

IFN-γ: Interferon-gamma

IL: Interleukin

ISCT: International Society for Cellular Therapy

MHC: Major histocompatibility complex

MSC: Mesenchymal stem cell

SLE: Systemic lupus erythematosus

SSc: Systemic sclerosis

TGF-β: Transforming growth factor-beta

TSK: Tight skin

UC: Umbilical cord

UC-MSC: Umbilical cord-derived mesenchymal stem cell

Competing interests

The authors declare that they have no competing interests.

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Autologous immuno magnetically selected CD133+ stem cells in the treatment of no-option critical limb ischemia: clinical and contrast enhanced ultrasound assessed results in eight patients

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Autologous immuno magnetically selected CD133+ stem cells in the treatment of no-option critical limb ischemia: clinical and contrast enhanced ultrasound assessed results in eight patients

Vittorio Arici1, Cesare Perotti2, Calliada Fabrizio3, Claudia Del Fante2, Franco Ragni1, Francesco Alessandrino3, Gianluca Viarengo2, Michele Pagani4, Alessia Moia1, Carmine Tinelli5 and Antonio Bozzani1*

Author Affiliations

1 Vascular Surgery Unit, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Piazzale Golgi 19, Pavia, 27100, Italy

2 Haemotransfusional Service, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

3 Radiology Service, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

4 Anesthesiology and Intensive Care Unit 2, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

5 Statistics and Epidemiology Service, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy

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Journal of Translational Medicine 2015, 13:342  doi:10.1186/s12967-015-0697-4

The electronic version of this article is the complete one and can be found online at: http://www.translational-medicine.com/content/13/1/342

Received: 5 April 2015
Accepted: 14 October 2015
Published: 3 November 2015

© 2015 Arici et al.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Objectives

Demonstrate the safety and effectiveness of highly purified CD133+ autologous stem cells in critical limb ischemia (CLI).

Design

Prospective single-center not randomized. Clinicaltrials.gov identifier: NCT01595776

Methods

Eight patients with a history of stable CLI were enrolled in a period of 2 years. After bone marrow stimulation and single leukapheresis collection, CD133+ immunomagnetic cell selection was performed. CD133+ cells in buffer phosphate suspension was administered intramuscularly. Muscular and arterial contrast enhanced ultra sound (CEUS), lesion evolution and pain management were assessed preoperatively and 3, 6 and 12 months after the implant.

Results

No patient had early or late complications related to the procedure. Two patients (25 %) didn’t get any relief from the treatment and underwent major amputation. Six patients (75 %) had a complete healing of the wounds, rest pain cessation and walking recovery. An increase in CEUS values was shown in all eight patients at 6 months and in the six clinical healed patients at 12 months and had statistical relevance.

Conclusions

Highly purified autologous CD133+ cells can stimulate neo-angiogenesis, as based on clinical and CEUS data.

Keywords:

Peripheral arterial disease; Critical limb ischemia; Stem cell therapy; Contrast enhanced ultrasound

Background

Symptomatic peripheral arterial disease (PAD) has a prevalence of 3 % in a population aged 40 years and above and of 6 % in patients over 60. Critical Limb Ischemia (CLI) is the worst and terminal clinical picture of PAD often preceding gangrene and amputation: typical symptoms are rest pain refractory to analgesics lasting more than 2 weeks and ischemic lesions (Fontaine classification stage 3–4 and Rutherford classification stage 4–6). CLI diagnosis is confirmed instrumentally by calf arterial pressure <50 mmHg, Ankle/Brachial Index (ABI) <0.5 and Transcutaneous PO 2 (TcPO 2 ) <30 mmHg. The Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II) [1] stresses the absolute indication to revascularization for these patients either by surgical or endovascular treatment. Nevertheless in some cases, mostly due to lack of distal run off, revascularization is not feasible or shows very low success rates. For the above mentioned reasons, the prognosis of these CLI patients is poor, with a major amputation rate of about 50 % at 1 year.

Many non-interventional treatments have been proposed for these “no-options” patients: spinal cord stimulation, prostanoids and prostaglandins administration, hyperbaric oxygen therapy and so on. More recently some authors have focused the attention on the local administration of stem cells, and particularly Endothelial Progenitor Cells (EPCs). EPCs local administration effectiveness in post-ischemic myocardial damage has been demonstrated in animal models and in humans [2]–[5]. Many observations lead to argue that the EPCs play a basic role in re-endothelization and neo-vascularization processes: actually the EPCs number may be reduced in peripheral blood in cardiovascular diseases, diabetes and rheumatoid arthritis and under the action of exogenous (as smoke habit) or endogenous (as high C-reactive protein levels) factors. Otherwise EPCs number may be increased by factors as physical exercise, estrogens, erythropoietin, statins and several cytokines or secreting proangiogenic factors like hepatic growth factor (HGF), insulin like growth factor (IGF-1) and the vascular endothelial growth factor (VEGF). These observations persuaded some authors to apply this cell therapy approach to CLI [6].

Our starting experience in the treatment of no options CLI with EPCs in three patients has been previously reported [7]. The bench marks of our study are the use of a selected EPCs population to investigate more precisely the cellular mechanisms, and to assess whether patients with PAD treated with EPCs show variation in muscle perfusion as displayed by contrast enhanced ultrasound (CEUS).

According to the current guidelines, reliable diagnostic tools for the management of patients with PAD, are ankle-brachial index (ABI), duplex ultrasound, transcutaneous oxygen pressure (TcPO 2 ), magnetic resonance (MR), contrast computer tomography (CT scan) and conventional angiography (CA). Among imaging modalities under development, micro bubble—based CEUS is a real-time, high spatial-resolution imaging technique that can easily be applied. Its high sensitivity, which provides detection of extremely low concentrations of micro bubbles, combined with the true blood pool distribution of contrast agent, gives CEUS the potential to help visualize and quantify the vasculature in vivo. CEUS was recently proposed as a valuable method to detect perfusion deficit and collateralization in patients with PAD [8], [9]. This method has been also validated for the detection of impaired microcirculation in patients with diabetes mellitus [10].

We present our experience in eight no-options CLI patients transplanted with peripheral immune-selected apheresis-derived autologous CD133+ cells and followed up with CEUS for 12 months.

Patients and methods

Type of the study

Prospective single centre not randomized.

Clinicaltrials.gov identifier: NCT01595776

Aim of the study

To assess the safety, feasibility and efficacy of local intramuscular administration of autologous immuno-selected CD133+ cells in patients suffering from CLI. The protocol started at our institution in July 2011 with the local Ethical Committee (EC) approval (number CHVAS-01-08-10/03/08). The investigation conforms with the principles outlined in the Declaration of Helsinki.

Enrolment criteria

All the patients enrolled were suffering from clinical stable CLI according to the TASC 2 definitions [1] and had no revascularization option, on the basis of contrast CT scan, RM or angiography imaging and the evaluation of our vascular and endovascular team. A detailed informed consent, approved by our EC, had been required.

Exclusion criteria

Patients under 18 and over 70 years of age. Elderly patients have been excluded because of expected bone marrow low responsiveness. Clinical unsteadiness of CLI (such as gangrene requiring major amputation) and poor life expectancy are exclusion criteria for supposed latency of the EPCs action. Severe systemic illness was judged to increase the risk of bone marrow stimulation. Complete inclusion and exclusion criteria are depicted in Table 1.

Table 1. Inclusion and exclusion criteria

Patients

Between September 2011 and September 2013 we enrolled eight patients with a history of Rutherford stage 4 (rest pain) or 5 (small ischemic lesions) PAD. All patients had previous vascular imaging (contrast CT or MR or angiography) excluding revascularization options, either endovascular and surgical and encountered inclusion criteria. Every patient underwent routine physical and instrumental examination including electrocardiogram, chest X-ray and blood sample analysis. The patients’ features are summarized in Table 2. The median age was 46.8 (SD 11.8, range 37–69 years), with a M:F ratio of 3:1. Younger patients met Buerger disease criteria (n = 4, 50 %), whereas others had pure atherosclerotic lesions. Only one patient (ID 7, female) had diabetes mellitus. Six patients out of eight had ischemic lesions on the forefoot (Rutherford stage 5) with poor healing and a long history of wound treatment. All patients complained moderate/severe pain and took high doses of analgesics (slow release opiates in two cases).

Table 2. Patients baseline

CEUS imaging protocol

Two operators who were blinded to treatment performed CEUS for all patients. The imaging criteria were: (1) reduced transmit power, at approximately 7–10 frames per second and one focus well below the level of the target to ensure a more uniform pressure field. (2) dual-mode presentation of a grayscale image side-by-side with the contrast image facilitating real-time identification of anatomic structures and region of interest (ROI) selection. (3) Image loops of approximately 60 s. (4) Uniform gain across the image and avoid gain saturation. (5) The time gain compensation (TGC) set such that before contrast arrival a uniform black image was shown.

A vial of contrast agent (SonoVue BR1; Bracco, Milan, Italy) was prepared at a concentration of about 2 × 10 8 sulfur hexafluoride—filled micro bubbles per milliliter, according to the manufacturer’s recommendations. The position of the probe was recorded for each patient in order to maintain the same position during follow up. The injection was made with the patient supine and after 10 min of rest to avoid exercise related micro-vascular dilatation. The radiologist maintained a constant image plane with the aid of the tissue (fundamental image) of the “Contrast Side/Side” imaging mode.

Image analysis

The main image analysis tasks were: (1) identification of anterior tibialis artery (ATA) area, (2) selection of a representative region of normal anterior tibialis muscle (ATM) and (3) formulation of time-intensity curves (TIC). Two manually defined ROI, 2 and 4 cm sided-squares, were placed, respectively, over the ATM with no evidence of arterial branches, over ATA and over a small tibialis arterial branch. The ROIs were placed in the same anatomical position for each patient to avoid unwanted differences during follow up examinations. One TIC was obtained for each ROI. The image loops were transferred to a personal computer for further analysis. From the analysis of TIC, we computed regional blood flow (RBF) and regional blood volume (RBV). TIC were extracted using commercial quantification software (QontraXt v.3.60, AMID, Rome, Italy). This software allows manual ROI selection, measurement of the selected ROI area and provides linear data for the TIC. For the ROI in the normal ATM, effort was made to place the region in an area without large vessels. The ATA ROI was a 2 cm square area and the ATM ROI was a 4 cm square area. TICs were obtained by computing the mean intensity of pixels comprised within the ROI at each time point. For each image loop were calculated:

RBV which consists in the total amount of contrast media within the selected ROI, in a period of time. Due to the characteristics of US contrast media, it reflects the quantity of blood in a defined region. It is directly related to the area under the curve (AUC).

RBF consists in the contrast media flow (related to the blood flow) in a selected ROI. It is related to the mean transit time.

Bone marrow stimulation

Human recombinant granulocyte colony-stimulating factor (rhGCSF) was administered subcutaneously for 4–5 consecutive days at a dosage of 10 µg/kg daily, split in two doses. Starting from the third day of stem cells mobilization, the CD34+/133+ cells count was monitored daily by cytofluorimetric analysis. The minimum CD34+/133+ cells count acceptable for leukapheresis collection was 20 and 10/µl, respectively. Patients were monitored for any G-CSF related side effects.

Leukapheresis (LKF) collection

A single LKF collection was planned for each patient using a third generation cell separator device (Spectra Cobe, Lakewood, CO, USA), processing at least 2.5 blood volumes according to our internal protocol for stem cell collection. Immediately after the LKF collection, a sample from patient’s peripheral blood was taken for haemocytometric analysis to evaluate platelet count and haemoglobin levels. Each LKF collection was diluted with 10 % acid citrate dextrose (ACD-A) and maintained overnight at 4 °C degrees before immuno-magnetic cell selection.

Immunomagnetic cell selection

CD133+ immunomagnetic cell selection (ICS) was performed the day after LKF collection using the Clini-MACS (Miltenyi Biotec) device according to the manufacturer’s standard protocol.

Quality controls

A sample taken from the CD133 cell positive fraction was seeded for short term (14 days) clonogenic assays to evaluate the quality of immunoselected stem cells in terms of proliferative capacity. A standard mixture of methylcellulose plus recombinant human growth factors was employed (Stem Cell Technologies, Vancouver, BC, Canada; MACS Media, Miltenyi Biotec GmbH, Bergisch Gladbach,Germany). Microbial cultures on the waste bag containing the negative fraction were carried out to detect aerobic-anaerobic bacteria and fungal contamination. A sample of 10 ml was inoculated in the culture medium (Bact/Alert FA and BacT/Alert FN, Organon Teknika Corp., Durham, NC) and incubated for 10 days at 37 °C.

Cytofluorimetric analysis

Samples obtained from peripheral blood before mobilization with G-CSF, at time of LKF and after immunomagnetic cell selection were analyzed by flow cytometry to evaluate the expression of specific stem cell and endothelial antigens. Becton–Dickinson FACSCanto was employed for all flow cytometric analysis with a lyse no-wash technique, using the following monoclonal antibodies: anti-CD45 fluorescein isothiocyanate (FITC) (Becton–Dickinson, San Jose, CA, USA), anti-CD34 Peridinin-chlorophyll-protein complex (PerCP) (8G12 clone, Becton–Dickinson), anti-CD133 phycoerythrin (PE) (AC133 clone, Miltenyi Biotec) and anti-VEGF-R2 allophycocyanin (APC) (R&D systems), following the manufacturer instructions.

Each sample was acquired with BD FACSCanto recording 100.000 events inside the lymphocyte plus monocyte gate. Data files were analyzed with FACS Diva 6.1 software. Viability was assessed using 7-amino-actinomycin D (7-AAD) (Molecular Probes, Eugene, OR, USA).

Implant procedure

After loco-regional anesthesia and below the knee cutaneous disinfection, 45–48 ml of autologous CD133+ cell in buffer phosphate (Miltenyi Biotec) suspension was administered intramuscularly with 1 ml deep injections through a 18G needle. The injections were so allocated: 10 ml in the anterior compartment of leg, 10 ml in the superficial posterior compartment, 10 ml in the deep posterior compartment, 10 ml in the lateral compartment and the remaining part in the foot (Additional file 1: Figure S1).

Baseline assessment and follow up

Pain assessment was carried out with a personal scale of 3 degrees (mild, moderate and severe) and the pain killing drugs use monitored. Ischemic lesions were treated weekly by a wound management skilled nurse. CEUS, lesion evolution and pain management were assessed at baseline and 3, 6 and 12 months after the implant.

Statistical analysis

All quantitative variables were normally distributed (Shapiro–Wilk test) and so the results were expressed as mean values and standard deviation (SD); qualitative variables were summarized as counts and percentages. Pearson’s r coefficient was used to test correlation between two study variables. Linear regression models for repeated measure were used to assess the increase over time of the CEUS parameters. Data analysis was performed with STATA statistical package (release 11.1, 2010, Stata Corporation, College Station, TX, USA).

Results

Patient’s mobilization and LKF

No relevant side effects related to G-CSF administration were registered. A single LKF collection per patient was performed. No side effects were registered. Patients did not require any red blood cells or platelet transfusion after LKF procedures. Total nucleated cells (TNC) content and viability in peripheral blood of the eight patients enrolled are depicted in Table 3.

Table 3. Cytofluorimetric analysis

Immunomagnetic cell selection

The immunomagnetic cell selection was carried out the day after LKF collection. Almost all cells expressing CD133 antigen also expressed CD34 antigen. The mean CD133+ cell recovery was 46.1 % (SD 21.5, range 9.9–81.7). The mean purity was 85.4 % (SD 19.6, range, 37.2–96.5). TNC viability was always >90 %. Results of cytofluorimetric analysis performed on samples obtained from the positive fraction are detailed in Table 3. The mean number of CD133+ infused cell per limb kilogram was 25.2 × 10 6 (SD 13.8; range 2.6–42.2). Clonogenic assays demonstrated the maintained proliferative capacity of immunoselected stem cells. The result of microbial cultures was always negative.

Clinical results

No patient had early or late complications related to the procedure. Two patients (number 3 and 7, 25 %) didn’t get any relief from the treatment and underwent major amputation. Patient number 3 had lesion and pain worsening and was amputated below the knee after 5 months. Patient number 7 had pain worsening and final gangrene of the foot and underwent above the knee amputation after 7 months; this patient had diabetes and a heavy smoking habit (40 cigarettes/day) persisting in spite of physician’s indication. Five patients (number 1, 2, 4, 5 and 6, 62.5 %) had a complete healing of the wounds, complete rest pain cessation and walking recovery or increased pain free walking distance. One patient (number 8, 12.5 %) had rest pain cessation, and a mild improvement in pain free walking distance. None statistical correlation has been found between the number of infused CD133+ cells and the clinical results.

CEUS

An increase in RBF and RBV was shown in all eight patients at 3 and 6 months and in the six clinical healed patients at 12 months and has statistical relevance: at 12 months mean increasing for TAM-RBV was 48.8 % (p = 0.018), for TAM-RBF 59.4 % (p = 0.0016), for ATA-RBV 52.8 % (p = 0.017) and for ATA-RBF 48.6 (p = 0.007). The trends in increasing values at 3, 6 and 12 months for RBV and RBF, both in artery and in muscle, are depicted in Fig. 1. No statistical correlation between RBF and RBV value, and CD133+/CD34+ infused cells was found at 6 and at 12 months. Additional file 2: Figure S2 shows the correlation between clinical healing and CEUS values improvement in patient 1. Table 4 shows a synopsis of the results.

thumbnailFig. 1. The diagrams show the percentage increase of both RBV and RBF both in muscle and in anterior tibial artery. RBF regional blood flow, RBV regional blood volume. In the x axis are months of follow-up. In the y-axis is the percentage of increase of the values of RBV and RBF compared to the baseline. Dotted lines represent single patient values. Continue line represents the mean percentage increase with the statistical significance. P values compare the value of mean increase to the baseline

Table 4. Results

Discussion

CLI is a manifestation of PAD that includes patients with typical chronic ischemic pain at rest or patients with ischemic skin lesions, either ulcers or gangrene. The term CLI should only be used in relation to patients with chronic ischemic disease, defined as symptoms lasting more than 2 weeks. The diagnosis of CLI should be confirmed by ABI, toe systolic pressure or transcutaneous oxygen tension. Ischemic rest pain most commonly occurs below an ankle pressure of 50 mmHg or a toe pressure less than 30 mmHg [1].

The first large report on the use of bone marrow-derived mononuclear cells (BM-MNC) in limb ischemia was the therapeutic angiogenesis by cell transplantation (TACT) study by Tateishi-Yuyama et al. At 4 weeks, ankle-brachial index (ABI) was significantly improved in legs injected with BM-MNC and similar improvements were seen for transcutaneous oxygen pressure. They concluded that autologous implantation of BM-MNC could be safe and effective for achievement of therapeutic angiogenesis, because of the natural ability of marrow cells to supply endothelial progenitor cells and to secrete various angiogenic factors or cytokines [11]. Since then, several trials were published, both using BM-MNC, and mobilized peripheral blood mononuclear cells (PBMNC) with intra-arterial or intra muscular administration, well reviewed by Lawall et al. [12].

In their review, Lawall et al. sustain that the role of “EPCs” in human angiogenesis in the setting of peripheral vascular obstruction remains doubtful, and the translation of a truly “EPC” based endothelial repair into clinic practice has not been achieved so far. The well substantiated concept of arteriogenesis strengthens the importance of several different bone marrow cell types, however all sharing a monocytic phenotype. They migrate to the perivascular space of sprouting collaterals and induce collateral artery growth by the release of angiogenic growth factors. A growing body of evidence strongly suggests that these secreted molecules mediate a number of protective mechanisms including cell survival, neo-vascularization, remodeling, and proliferation. The regulatory system governing paracrine factor release appears to be complex and dependent on spatiotemporal parameters. [13].

Based on available data, cell therapies in PAD based on the application of whole BM-MNC or on whole stimulated PBMNCs are more successful than methods which use subfractionated cell preparations [12], e.g. CD 133+ [14] or highly purified CD 34+ cells from peripheral blood after granulocyte- colony stimulating factor (G-CSF) mobilisation only [15]. Nevertheless available data are very scarce about efficacy of a specific subset cells population versus the entire pool of mononuclear cells, because great majority of the previous and ongoing studies employ BM-MNC or PBMNCs, due to both lower costs and relative simplicity of the method. Except the case report of Canizo et al., the only previous study employing autologous selected CD 133+ cells is by Burt et al.: they treated nine patients with positive results in 7 [16].

Previous EPCs in CLI studies considered only data measured with ABI and TcPO 2 . As a matter of fact both ABI and TCpO2 have an intra and inter patient variability due to detection method, operator experience, vasodilatation state and emotional stress, that make standardization difficult. Moreover, in case of very low velocity flow, as in peripheral blood circulation in CLI, ABI detection variability increases [17]. CEUS offers a reliable method to measure peripheral blood flow, and is a valid alternative: it’s equally a non-invasive method, because the medium contrast hasn’t got any contraindication, except hypersensitivity, and the assessment method is not operator, but dedicated software related. It has only a minimal intra patient variability, when the detection method is accurate.

The aims of our study are (1) to demonstrate that a highly purified autologous stem cells population can induce neo-angiogenesis in a safe, feasible and effective way and (2) to assess neo-angiogenesis with a non-invasive method as most objective and reproducible as possible. In consideration of the end stage disease character of CLI, we didn’t considered ethical randomizing eligible patients. The possible advantages in studying a specific cells population in order to understand the mechanisms of neo-angiogenesis are to avoid the overlapping effects of entire MNC populations infused and as second step to better identify the cytokines pattern, derived from a single cell type, given the hypothesis of a paracrine mechanism.

Stem cells mobilization with G-CSF administration induce a high white blood cells count (WBCc) and a subsequent theoretical blood hyper viscosity. In patients with CLI blood hyper viscosity can be an issue. In our series the mean WBC count at the 4th day after mobilization was 48.1 × 10 6 /ml (range 26.6–77). These values are similar to these observed in healthy donors mobilized for allogeneic hematopoietic stem cell transplant. Even in the patient (ID = 7) with the highest WBCc (77.0 × 10 6 /ml) we didn’t observe any significant side effect related to blood hyper viscosity. However, we administered a prophylactic dose of low molecular weight heparin (3800/4000 UI/daily) considering the pro thrombotic risk related to G-CSF administration and patient immobilization. Remarkably also the CD34+ and CD133+ mobilization in these patients is comparable to healthy donors showing that their stem cell reservoir is not depleted. Stem cell collection was performed following our internal protocol, processing 2.5 blood volumes without any relevant side effects. On the whole we can argue that patients with compromised peripheral circulation can tolerate very well both mobilization and LKF. The wide range of CD133+ cell recovery after the ICS may be related to the different antigen expression on the stem cell surface. All the immunoselected CD133+ cell samples showed an high in vitro clonogenic potential (similarly to hematologic field) demonstrating the good quality of the product infused. Nevertheless the purity was always very high (≥90 %), except in one case (ID = 5), showing that every patient, but one, was treated with a single cell population. From this point of view, we obtained results comparable with the study of Burt et al. [16].

Altogether, 6 out of 8 patients (75 %) had clinical improvement, with ulcer healing, cessation of rest pain, increased walk pain free distance and above all, avoided amputation and maintained their improvement for a long period of time. Indeed they had the longest follow up so far in literature, 12 months with CEUS and at least 18 months from a clinical point of view. The graphics in Fig. 1 show the increase (in percentage) from baseline of RBV and RBF, both for ATM and ATA: mean percentage increase (continuous line) is always positive and statistically significant (see also Table 4 for p values). Clinical improvement is consistent with instrumental data showing an increased blood flow in the limb, reasonably related to an induced neo-angiogenesis. Nevertheless other important local factors may be involved both in initiating and maintaining angiogenesis, like resident cells and chemokines and cytokines environment. Data demonstrate that the resident progenitor cells could differentiate into a variety of cell types in response to different culture conditions. However, collective data were obtained mostly from in vitro culture assays and phenotypic marker studies. There are many unanswered questions concerning the mechanism of cell differentiation and the functional role of these cells in vascular repair and the pathogenesis of vascular disease [18].

Patient 3, despite the maximum values of cell dose infused (42.2 × 10 6 /limb kg) showed a complete lack of responsiveness both clinical, and instrumental (Table 4). Conversely, Patient 5 received the lowest dose of CD133+ cells with the lowest purity (Table 3): however she showed a satisfactory clinical response (ulcer healing, rest pain relief and increased pain free walk distance) and TAM-RBV/RBF increasing (+28.6 and +42.9, respectively). We can speculate that these opposite results are related not exclusively to the cell dose infused, but also to responsiveness of the resident stem cells. Indeed we didn’t find any statistical correlation between the number of infused CD133+ cells (10 6 per limb kg) and the clinical and CEUS results.

Patient 7 showed apparently conflicting results: at 6 months TAM-RBV/RBF were increased around 77 % with a slightly improvement of clinical condition. Unfortunately the following month she developed a sudden worsening in the limb ischemia, with subsequent above the knee amputation. It’s crucial to emphasize that the patient had insulin dependent diabetes mellitus (IDDM) and a heavy smoking habit (around 40 cigarettes daily). We can suppose a negative role of IDDM and a precipitating role of the smoke habit during the transient neo-angiogenesis process, as assessed by CEUS values increasing. However impaired angiogenesis in diabetes has been already demonstrated both in animal models and in humans [19], [20].

In conclusion, our study shows interesting perspective and issues. Highly purified autologous CD133+ cells, routinely employed in transplant for oncohematologic diseases, can stimulate neo-angiogenesis, either directly or through a paracrine effect, as based on clinical (ulcer healing, rest pain cessation, increasing pain free walk distance and limb salvage) and CEUS data. Our study gives an instrumental demonstration of neo-angiogenesis. The limits of the study are the lack of randomization, which we judged unethical for this kind of end-stage disease, and the low number of patients, mainly due to the restrictive inclusion and exclusion criteria. Goals for future studies are to enrol a major number of patients including also less advanced stages of PAD, and to consider other powerful stem cell sources as cord blood derived or mesenchymal stem cells.

Additional files

. Additional file 1: Figure S1. The procedure of implant through multiple intramuscular 1 ml injection of CD133+ cells suspension.

Format: TIFF Size: 2.9MB Download fileOpen Data

. Additional file 2: Figure S2. The complete healing of a deep and painful ischemic lesion with bone exposure in patient 1. Beside the corresponding change in RBV and RBF measurements.

Format: TIFF Size: 3.4MB Download fileOpen Data

Authors’ contributions

Concept and design: CP, VA, CDF, AB. Supervision: VA, CP. CEUS: FC, FA. Cell management: CP, CDF, GV. Patient management: VA, AB, AM, MP, FR. Data collection: VA, CP, AB, FR, FC, FA,GV, MP, AM. Manuscript draft: all the authors. Manuscript revision and approbation: all the authors. Statistical analysis: CT.

Funding

This study was entirely supported by “Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy”.

Competing interests

The authors declare that they have no competing interests.

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