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Awareness and Perception of Plastic Surgery among Healthcare Professionals in Pune, India: Do They Really Know What We Do?

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Copyright © 2012 Nikhil Panse et al.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This article has been cited by other articles in PMC.

Abstract

Purpose. The aim of this study is to understand the level of awareness and knowledge of plastic surgery in healthcare professionals in a tertiary health care facility in Pune, India. This study also aims to highlight the perception of the medical professionals about plastic surgery and what they think a plastic surgeon does. Materials and Methods. A questionnaire-based survey was done at B.J Medical College and Sassoon Hospital, Pune in 2011. Feedback evaluation forms from hundred resident doctors and faculty were evaluated and analyzed. Results. There is not much awareness about plastic surgery as a specialty amongst health care providers. Plastic surgery is mostly perceived as cosmetic surgery, and the other spectrum of the patients we cater to goes largely unnoticed. Of all the clinical conditions given to the participants, there was not a single clinical condition where the respondents favored unanimously for plastic surgeons. Conclusion. Plastic surgery as a specialty is poorly understood by our medical colleagues, and the onus of creating and improving the awareness and perception of our specialty lies on us. Herculean unified efforts at individual as well as global level will help us achieve this goal.

1. Introduction

People in today’s world are more health conscious and are aware of the different medical specialties. Despite the tremendous advancements in the field of plastic surgery, there seems to be a limited knowledge among the general public and also among medical professionals regarding the spectrum of plastic surgery. As a medical specialty, plastic surgery is poorly understood by both the general public and some medical professionals as well.

This study is an attempt to establish the knowledge of the medical community on the specialty of plastic surgery and the spectrum of patients they cater to.

Unlike in western countries, the public literacy level and awareness in India are very disproportionate. Therefore we did not include the general public in the study. Instead, we included the medical professionals in our study. These people contribute considerably to the health care of the public. We felt that understanding the attitude and perception of our medical colleagues will be more vital than assessing the knowledge of the public. We wanted to know whom should we educate first—the doctors or the public.

2. Materials and Methods

We conducted a questionnaire-based study among a selected group of healthcare professionals to assess their attitude, knowledge, and perception of plastic surgery. A well-structured questionnaire was administered to this group of select individuals, and responses were sought and analysed. The questionnaire was handed over to a total of 100 residents and faculty of nonsurgical specialties. Doctors from general surgery and surgical super specialties, orthopedics, ENT, and ophthalmology were excluded from the study. Dermatology department was also excluded from the study because of the overlapping spectrum of plastic surgery and dermatology. Only M.B.B.S and higher qualification doctors were included in the study.

This survey was conducted at B.J Medical College and Sassoon Hospital, Pune. B.J Medical College and Sassoon Hospital is the largest government tertiary care teaching hospital in Pune. Sassoon General Hospital is 1200 plus bedded with almost 100% bed occupancy round the year. There is a functioning plastic surgery unit in the college.

A questionnaire was designed in 2 parts. In the first part, the participants were asked specific questions pertaining to plastic surgery with a multiple choice option for marking the answers. see Table 1.

In the second part, the participants were given a list of clinical situations including trauma, pathology, reconstructive, and cosmetic surgery and were asked to indicate which specialty they think would be the best to treat the clinical situation mentioned. The options of treating surgeons were ENT surgeon, plastic surgeon, ophthalmic surgeon, neurosurgeon, general surgeon, orthopedic surgeon, pediatric surgeon, uro surgeon, oral and maxillofacial surgeon, dermatologist and others (Table 2).

Table 2

Which surgeon would you expect to treat the following conditions?

3. Results

The responses of the participants are shown in Tables ​Tables33 and ​and4.4. Analysis of part 1 of questionnaire showed that 96% of the participants were aware of what training is required to be a plastic surgeon. 12% of participants felt that plastic surgery and cosmetic surgery are the same, and 80% felt that cosmetic surgery is a part of plastic surgery. Of the 100 participants, 83% did not know why plastic surgery is called plastic surgery, 5% felt that it is called plastic surgery because it involves use of plastic, and 4% felt that it is called plastic surgery because face looks shiny like plastic after the surgery. 8% of participants gave various answers of which the nearest relevant one mentioned that plastic was moldable, and in plastic surgery face is moldable, so it is called plastic surgery. A whopping 74% of the participants felt that there are no scar marks left after plastic surgical procedure. 37% of the participants felt that plastic surgery is a very expensive affair and meant for the rich and famous. 87% of participants felt that the risk involved with plastic and cosmetic surgery is similar to other surgeries.

Table 4

Which surgeon would you expect to treat the following conditions?

Analysis of part 2 of the questionnaire showed that 93% of respondents preferred an ophthalmic surgeon to suture an eyelid injury, and 92% of respondents preferred a plastic surgeon to suture a cut over the face. When treating a maxillofacial fracture, 82% preferred an oral and maxillofacial surgeon, and 12% preferred a plastic surgeon (Figure 1).

Most of the respondents (87%) preferred general surgeons over plastic surgeons in management of bed sores. 100% of the participants preferred an orthopedic surgeon to manage a hand fracture. 65% of the participants felt that injuries to nerves of hand and legs must be managed by orthopedic surgeon, 26% felt that they must be managed by a plastic surgeon, and a surprising 6% felt that nerves are best dealt by a neurosurgeon (Figure 2).

Hypospadias was divided into general surgeons (10%), pediatric surgeon (38%), urosurgeon (26%), and plastic surgeon (26%). Of the other congenital anomalies, 55% felt that plastic surgeons were best to manage a cleft, and 58% felt plastic surgeons were the best to manage congenital ear anomalies. 86% respondents preferred a plastic surgeon for managing burns, and 97% preferred a plastic surgeon for management of postburn deformities. Only 13% of participants preferred plastic surgeon as compared to 78% (orthopedic surgeon) in management of tendon injuries of the hand. The aesthetic surgery procedures have a favorable response towards plastic surgery. 99% participants favored a plastic surgeon doing a liposuction and breast reduction or augmentation procedure. 7% favored a general surgeon doing an abdominoplasty as compared to 93% favoring a plastic surgeon.

Majority of participants responded for plastic surgeon (61%) as compared to E.N.T surgeon (37%) for doing a rhinoplasty. Hair transplantation procedure had 67% participants responding for dermatologist as compared to 33% for plastic surgeons (Figure 3). Nonsurgical aesthetic procedures like Botox had 52% respondents favoring dermatologist as compared to 41% for plastic surgeons

The detailed analysis of the responses is shown in Table 4.

Although this survey was done in Pune city in Western India, we believe that the scenario in any part of India and most of the developing world would not be different. Most of our colleagues of other specialties are not really aware of what we are doing. Apart from a few aesthetic surgery procedures, where majority of the respondents favored plastic surgeons, the results for other procedures are disappointing. It was even sad to note that there was not a single clinical condition which participants thought is the exclusive domain of the plastic surgeon. Our specialty is still searching for its identity rather among the healthcare providers than among the general public.

4. Discussion

Plastic surgery is a unique specialty that defies definition, has no organ system of its own, and is based on principles rather than specific procedures.

Unlike other medical disciplines, plastic surgery is not defined by an anatomic area (OB/Gyn, ENT, thoracic surgery), organ system (gastroenterology, urology), or patient age group (pediatrics, adolescent medicine, geriatrics). It deals with everything from head to toe but is associated with the prefix plastic which symbolizes nothing to the common man and medical community at large.

There have been surveys on awareness of facial plastic surgery in general population [1], but to our knowledge survey of knowledge and perception of plastic surgery in healthcare professionals is unheard of in our part of the globe.

After analyzing the results of the survey conducted by us, we found that the findings were alarming. It was amply clear that there is not much understanding of our specialty in members of the medical community, and plastic surgery is poorly understood. We feel that if our colleagues from other specialties are not aware of the spectrum of work we do, we ourselves are to be blamed for it. We lag behind in the field of advertisement and awareness creation of our specialty.

At our unit, we have been regularly presenting our work at college-level conferences. We have also been organizing multidisciplinary symposiums of topics like wounds and burns at the local level. In spite of that, the awareness levels of our specialty were very low. To address this problem at our level, we made an activity report comprising the clinical and academic work we have done in the past two years with colour clinical photographs and distributed it amongst all the departments of our college. E-mail addresses were collected of the entire staff and as many residents and students as possible, and soft copies of the activity report were mailed to them all for wider coverage. Copies were also mailed to staff of other medical colleges in Pune.

People have a short memory, and we need to keep on reminding them of our existence. Keeping this in mind, we plan to make brochures of various subspecialties of plastic surgery like pediatric plastic surgery, hand and microsurgery, maxillofacial surgery, burns, and so forth and distribute it for awareness purpose.

We all understand the role the media plays in informing and educating the public on issues of the day, but it seems that when it comes to plastic surgery, the press has a one-track mind that leads them to cosmetic surgery. Plastic surgeons are often portrayed in the media as glamorous beings who give people a new lease of life through a “nip and a tuck”. However, the reconstructive side of our work goes largely unreported. As Reid and Malone [2] highlight, of 1191 articles published in British newspapers in 2006, 89% used the term “plastic surgery” in the context of cosmetic surgery, with only 10% referring to reconstructive work. There has been a dramatic increase in the number of people having cosmetic surgery in India over the last few years. The International Society of Aesthetic Plastic Surgery (ISAPS) has produced the ISAPS Biennial Global Survey (TM) of plastic surgeons and procedures in the top 25 countries and regions [3]. They say this ISAPS survey marks the first time reliable international plastic surgery data which has been obtained and analyzed by independent statistical specialists. India ranks fourth in the list and is emerging as a major centre of cosmetic surgery [3].

This in itself means that cosmetic surgery is a popular topic of discussion, so it is little wonder that the media want to cover it. However, no part of reconstructive aspect of plastic surgery is covered by the media. For all of us as plastic surgeons, plastic surgery is not just cosmetic and we need to educate and inform the public and nonplastic surgical specialties as to what it is that plastic surgeons actually do. Park et al. [4] looked at perception and knowledge around the work of plastic surgeons. They found that 23.7% of the local lay population could not think of five conditions treated by plastic surgeons, while 27% felt that the majority of the work plastic surgeons did was cosmetic in nature. It is not just the general public who has this perception. Journalists were interviewed across the national press, from The Times, The Daily Mail, and BBC News Online and it was found that journalists catering for a consumer audience feel that plastic surgery is synonymous with cosmetic surgery [5].

There are numerous facets to it and lot of attempts need to be made at an individual, local, regional, national, and global level to bring about this change effectively.

After analysis of the results, we realized that the name of our specialty itself is not understood by many. Unlike other specialties, where the name itself indicates the work being done by that particular specialty, plastic surgery lags behind on this front. In India, it is not uncommon for us to encounter patients who after completion of all the procedures will ask when plastic surgery will be done and plastic will be used ! We need to give serious thoughts about changing the name of our specialty to plastic, reconstructive and aesthetic surgery and name of our respective associations from Association of Plastic Surgeons to Association of Plastic, Reconstructive, and Aesthetic Surgeons. Similarly, journal names of respective bodies can be changed to journal of plastic, reconstructive, and aesthetic surgery of the respective associations. It would be an important first step in trying to improve understanding of what plastic surgeons do. It would help to change the mindset of peers and members of the public.

A similar effort was done by the British Association of Plastic Surgeons (BAPS), who changed the name of the organization to British Association of Plastic, Reconstructive and Aesthetic Surgeons (BAPRAS) in July 2006 [5].

We feel that one of the important reasons for poor knowledge and perception of plastic surgery in our survey is inadequate training and exposure of plastic surgery at undergraduate level. There has been much discussion in the western literature regarding if there is a place for plastic surgery in the undergraduate curriculum. As outlined by Wade et al. [6] many undergraduates are in favor of having plastic surgery teaching even though many may not necessarily want to pursue a career in the specialty. The portrayal of plastic surgery in the media is frequently that of purely elective cosmetic operations and this is often the understanding that undergraduates have of the Specialty until they do further research and discover the main workload carried out by a plastic surgeon. Undergraduate exposure is the single most influential factor for subsequent career interest in plastic surgery [7] with the duration of specialty exposure directly proportional to subsequent career intentions [8]. Given the inherent difficulties of changing our university curriculum, a simple but effective solution to increase plastic surgery exposure to all our medical students is required. A career in plastic surgery day has been held at the Royal College of Surgeons in London with talks from surgeons in different subspecialties and on careers in the profession, in addition to surgical skills practical sessions under the supervision of trainees in the profession [9]. An attempt to introduce a similar module can be made at other places as well. These conferences can give students a good insight into the specialty. Teaching undergraduate plastic surgery has potential benefits to all future doctors and ultimately patients, irrespective of career intentions. Exposure to plastic surgery may not lead to the development of a career interest in plastic surgery [9]. However, this is still an important reason to encourage undergraduate teaching, as a negative decision allows students to choose more suitable career paths. Given the multidisciplinary nature of plastic surgery and the abundant team working with other medical and surgical specialties, it is important that nonplastic surgery colleagues are aware of the work carried out by plastic surgeons. Referrals will therefore be more accurate, resulting in a more efficient and higher quality of service delivered to patients [9].

An attempt should be made by all of us to include in the undergraduate teaching, a topic on the spectrum of plastic surgery with clinical photographs to impress upon the young minds, as to what plastic surgery is all about. We want to attract the best and brightest of medical students into our specialty, so as a group, plastic surgeons can continue to be at the forefront in medical advances. This can be done only by creating awareness of the breadth of the plastic surgeons role and the contribution of our specialty to patient care.

Telemedicine can be an important tool for educating the medical practitioners at remote areas. Teleeducation of the doctors at remote areas about the spectrum of plastic surgery will also result in more accurate and direct referrals for our specialty.

For wider coverage and public awareness, help of media must be sought. It is necessary that we as plastic surgeons give ample information to the media regarding the reconstructive aspects of plastic surgery. Professional communication services can be hired if needed to improvise on communication and awareness. Celebrities can be requested or hired to focus on the reconstructive part of plastic surgery. Innovative ways of reaching the public must be sought and implemented.

Individuals and national-level organizations should make effective efforts for campaigns to educate the health care consumers and providers and for projects to publicize the specialty of plastic surgery. Awareness of our medical colleagues could be improved by arranging more interdisciplinary sessions and interdepartmental discussions. We feel that national-level organizations are after all a conglomeration of individuals of the specialty. Efforts from each and every individual will make a difference.

To conclude, plastic surgery as a specialty is poorly understood by our medical colleagues, and the onus of creating and improving the awareness and perception of our specialty lies on us. Herculean unified efforts at individual as well as global level will help us achieve this goal. The future of plastic, reconstructive, and aesthetic surgery is bright, and we can make it even brighter by our collective efforts in the years to follow.

References

1. Adeyemo WL, Mofikoya BO, Bamgbose BO. Knowledge and perceptions of facial plastic surgery among a selected group of professionals in Lagos, Nigeria. Journal of Plastic, Reconstructive and Aesthetic Surgery. 2010;63(4):578–582. [PubMed]
2. Reid AJ, Malone PSC. Plastic Surgery in the Press. Journal of Plastic, Reconstructive and Aesthetic Surgery. 2008;61(8):866–869. [PubMed]
3. International society of aesthetic plastic surgery; Bienel global survey http://www.isaps.org/
4. Park AJ, Scerri GV, Benamore R, McDiarmid JGM, Lamberty BGH. What do plastic surgeons do? Journal of the Royal College of Surgeons of Edinburgh. 1998;43(3):189–193. [PubMed]
5. Sachedina S, Laing H. Plastic surgery in the press: is it all about nip and tuck? Journal of Plastic, Reconstructive and Aesthetic Surgery. 2008;61(8):864–865. [PubMed]
6. Wade RG, Moses MA, Henderson J. Teaching plastic surgery to undergraduates. Journal of Plastic, Reconstructive and Aesthetic Surgery. 2009;62(2):p. 267. [PubMed]
7. Greene AK, May JW. Applying to plastic surgery residency: factors associated with medical student career choice. Plastic and Reconstructive Surgery. 2008;121(3):1049–1053. [PubMed]
8. Chen H, Hardacre JM, Martin C, Lillemoe KD. Do medical school surgical rotations influence subspecialty choice? Journal of Surgical Research. 2001;97(2):172–178. [PubMed]
9. Davis CR, O’Donoghue JM, McPhail J, Green AR. How to improve plastic surgery knowledge, skills and career interest in undergraduates in one day. Journal of Plastic, Reconstructive and Aesthetic Surgery. 2010;63(10):1677–1681. [PubMed]

Articles from Plastic Surgery International are provided here courtesy of Hindawi Publishing Corporation
studentjournalofmedicine

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

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Sara Salinas, Editor
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

Introduction

Clostridial neurotoxins bind to nerve terminals and deliver their zinc-endopeptidase (Light Chain, LC) [1] inside the cytosol, where they specifically cleave one of the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins leading to inhibition of neuroexocytosis [2][6]. Botulinum neurotoxin serotype A (BoNT/A) causes prolonged, reversible muscle weakness by entering motor nerve terminals and cleaving 9 amino acids from the C-terminus of the SNARE protein SNAP25 (SNAP25206) to yield SNAP25197 [7], disrupting exocytosis and blocking neurotransmitter release [5], [8], [9]. Because of its potency and specificity for pre-synaptic nerve terminals, BoNT/A is used to treat numerous clinical conditions [10][13].

Detection of BoNTs in drug products, contaminated foods, and clinical and environmental samples is challenging because of their potency (i.e., low quantities leading to symptoms). The currently approved method for measuring BoNT biological activity is the mouse LD50 (mLD50) bioassay [14][19], which represents inhibition of the respiratory muscles. The mLD50 is highly sensitive (7–20 pg/mL) and has been adopted by all BoNT-based products manufacturers to test drug product potency. The mouse bioassay presents several challenges including assay time required, inability to differentiate between serotypes, sample capacity, and need for highly trained personnel and special animal facilities. Alternatives (i.e., refinements) include the localized muscle paralysis (abdominal ptosis) [20] and Digit Abduction Score assays [21] that are less severe but still require BoNTs injection in animals. Ex vivo alternatives include the rat or mouse phrenic nerve diaphragm [22] and the rat intercostal muscle strips assays [23], [24] that allow several tests from tissues of a single animal. For over 25 years there has been a strong desire to develop in vitro assays that could replace animals or animal tissues [14], [25] and still enable sensitive evaluation of all key steps in BoNT/A action. A sensitive cell-based potency assay (CBPA) is the preferred alternative [16][19], [25].

A replacement to the mouse bioassay poses challenging limit of detection (LOD) requirements, in the low pM, because of the minute quantity of BoNT in drug products, and the required sensitivity, accuracy, precision, and reproducibility for Quality Control (QC) standards [14], [18], [25]. Light Chain assays (ELISA [26][28], Endopep-MS [29], FRET [30], [31], HPLC-UPLC [32], and DARET [33], [34]) only measure activity of the catalytic domain and cannot detect non-functionality in other BoNT domains (i.e., translocation or binding domains). Previous cell-based assays to screen BoNT inhibitors relied on cells with low toxin sensitivity such as SH-SY5Y [35] or Neuro-2a cells [36], [37]. A reported cell-based FRET assay [30] requires treatments with 50 nM BoNT/A for 48–96 h. Embryonic chicken neurons [38] lack the sensitivity of mammalian neurons. Primary neurons from spinal cord or dorsal root ganglia [39][43] are sensitive to BoNT but technically challenging, time-consuming, and highly variable [14], [25]. Sensitive assays that use embryonic stem cell-derived neurons [44][47] rely on Western blot read-out with intrinsic variability and their extensive differentiation protocols pose challenges to QC validation. We report here a functional CBPA with differentiated human neuroblastoma SiMa cells [48] that fulfills all the requirements for a replacement assay [14], [25]. It reflects all steps in BoNT/A mechanism of action, its sensitivity (EC50∼1-0.4 U/well) is equivalent or better than the mLD50, and improving the mLD50, it is specific for BoNT/A by measuring SNAP25197. It is based on a neuronal cell line and a sandwich ELISA read-out, it is accurate, reproducible, and amenable to QC validation. Moreover, it measures BoNT/A biological activity in BOTOX® (onabotulinumtoxinA) vials.

Results

Monoclonal antibody specific for SNAP25197

Enzymatic activity of the BoNT/A-LC generates SNAP25197 by cleaving 9 amino acids at the C-terminus of SNAP25206 [7]. One of the breakthroughs in the development of the present BoNT/A CBPA was the generation of a monoclonal antibody, 2E2A6 (IgG3.k), recognizing SNAP25197. Monoclonal 2E2A6 is highly specific for SNAP25197 with no detectable cross-reactivity to SNAP25206 in Western blot (Figure 1A), and ELISA (OD405 = 0.892 for SNAP25134–197 vs. OD405 = 0.036 for SNAP25134–206 peptides). 2E2A6 has been characterized using Surface Plasmon Resonance (SPR) analysis and compared with a commercial antibody, MC-6053 (Research & Diagnostic Antibodies), claiming to be specific for SNAP25197, but that was found to bind SNAP25206 with a KD of 240 nM in the SPR analysis demonstrating some cross-reactivity. SPR analysis demonstrated that the 2E2A6 antibody has excellent affinity and specificity for SNAP25197 (KD = 0.075 nM for SNAP25197 and no binding to SNAP25206 at doses up to 10 µM) with a very low dissociation constant (1.06×10−4 s−1, Figure 1B) that makes it ideal for the development of an assay specific for BoNT/A. This monoclonal antibody ensures a constant and reliable supply of a critical reagent for the CBPA.

Figure 1

Characterization of anti-SNAP25197 monoclonal antibody 2E2A6.

Identification of BoNT/A sensitive SiMa cells

Neuronal-derived cell lines were obtained from the American Tissue Culture Collection (ATCC, 24 cell lines), European Collection of Cell Cultures (ECACC, 11 cell lines), and German Collection of Microorganisms and Cell Cultures (DSMZ, 7 cell lines) and screened for their sensitivity to BoNT/A. Differentiated Neuro-2a cells were previously identified as BoNT/A sensitive and served as comparison for screening additional cell lines. The primary screen was performed using BoNT/A complex at 0 and 1 nM with 6 h treatment followed by overnight incubation in toxin free medium to allow for cleavage of SNAP25. Samples were analyzed in Western blots (WB) using anti-SNAP25 antibodies (mAb SMI-81 or pAb S9684) that detect both SNAP25206 and SNAP25197 bands, allowing the calculation of % cleaved SNAP25. The best cell lines for BoNT/A uptake were Neuro-2a, LA-1-55n, PC12, N18, and SiMa (Figure 2A and Figure 3A). Undifferentiated SiMa cells [48] were more sensitive to BoNT/A than undifferentiated Neuro-2a cells (Figure 2B) with 22.4% SNAP25 cleavage at 0.11 nM and 38% at 0.33 nM BoNT/A after overnight treatment, while no cleavage was detected on undifferentiated Neuro-2a cells under these treatment conditions.

Figure 2

SiMa cells were selected from forty-two cell lines screened for BoNT/A complex uptake.
Figure 3

SiMa outperformed other candidate neuronal cell lines in the WB and ELISA assays.

BoNT/A uptake by SiMa cells was compared to the other candidate cell lines using a SNAP25197 WB-assay (Figure 3A). Neuro-2a, PC12, LA-1-55n, and SiMa cells were differentiated in collagen IV plates in EMEM serum-free medium with 25 µg/mL GT1b for three days before treatment with BoNT/A complex (0.005 to 300 pM) for 24 h followed by two-day incubation to allow for SNAP25197 accumulation. These optimal differentiation and treatment conditions were established previously for Neuro-2a and PC12 cells and were applied to LA-1-55n and SiMa cells. The dose-response curve in figure 3A demonstrates that SiMa cells, with an EC50 of 6.5 pM, were very sensitive to BoNT/A (Figure 3B). No EC50 values were obtained for the other cell lines since no upper asymptote was achieved. Moreover, a clear signal above background was detected at 0.04 pM (Figure 3A) demonstrating that differentiated SiMa cells possess a high affinity uptake system comparable to primary neurons [39], [42]. SiMa cells displayed better efficacy for BoNT/A uptake at every concentration tested. At the highest BoNT/A dose tested, the signal-to-background (S/B) ratio was 412 for SiMa cells (Figure 3B) with a clear upper asymptote facilitating curve fitting. The S/B indicates the sensitivity of the assay, especially at low doses. At 1.2 pM BoNT/A the S/B ratio was 85 (∼2–6 for the other cell lines). After three days of differentiation in the optimized medium, SiMa cells stopped dividing and acquired morphological characteristics of neurons extending neurites. Additionally, there was an increase in the mRNA levels for SV2A and SV2C, and the differentiated cells expressed SV2B that was not present in undifferentiated cells. A breakthrough in the development of a CBPA for BoNT/A was achieved with the identification of these sensitive SiMa human neuroblastoma cells [48]. Differentiated SiMa cells possess the sensitivity of embryonic spinal cord neurons (eSC) [39][41] and embryonic stem cell-derived neurons [44][47] and, being an established cell line, they are feasible for use in a QC environment [14].

Development of an ELISA read-out for the BoNT/A-CBPA

The SNAP25197 WB-assay with differentiated SiMa cells described above is the most sensitive assay developed with an established cell line described to date, but Western blot is not viable for high-throughput screening or QC validation. In contrast, sandwich ELISA assays, based on two antibodies which bind to different sites on the antigen, are robust, sensitive, and amenable to validation. The antibody binding affinity for the antigen is usually the main determinant of immunoassay sensitivity; therefore, monoclonal anti-SNAP25197 antibodies were used for capture. Twenty-four combinations of capture and detection antibodies were tested for the sandwich ELISA in the MSD® (Meso Scale Discovery) electrochemiluminescence (ECL) detection platform. The combination producing the best results consisted of 2E2A6 monoclonal for capture and anti-SNAP25 polyclonal S9684 (N-terminus) for detection (Figure 3D).

The newly developed ECL-ELISA was compared to the Western blot read-out. Lysates from the four candidate cell lines (half of the lysates from figure 3A) were tested. MSD High Bind plates were coated with 5 µL/well of 2E2A6 at 20 µg/mL. The S9684 polyclonal antibody was labeled with sulfo-tag and 25 µL/well at 5 µg/mL were used for detection. The ECL-ELISA produced excellent signal to background for all the cell lines at the highest dose tested indicating that robust assays could be developed (Figure 3B & 3C). Neuro-2 and LA-1-55n cells produced EC50 values of 55 and 58 pM respectively, while the PC12 cells curve did not reach an upper asymptote. Differentiated SiMa cells were very sensitive to BoNT/A with an EC50 of 3.3 pM and a plateau at ∼100 pM. The S/B ratios for SiMa cells in the ECL-ELISA were ∼500 at the 100 pM dose and ∼160 at the 1.2 pM dose. All the elements to develop a CBPA for BoNT/A that is sensitive, specific, and amenable to validation were in place.

Sensitivity and specificity of SiMa cells

The hallmarks of high affinity BoNT/A uptake are rapid binding and internalization combined with sensitivity to low toxin concentrations. Those are exemplified in embryonic spinal cord neurons (eSC) exposed to 0.4 pM BoNT/A for two days rendering 50% SNAP25 cleavage [39] and eSC treated with 500 pM BoNT/A for 4 min followed by 2.5 h incubation producing 10–20% SNAP25 cleavage [40]. SiMa cells detect BoNT/A activity at sub-pM toxin concentrations with 24 h treatments (Figure 3). To demonstrate fast uptake, differentiated SiMa cells were treated with 1 nM BoNT/A from 1 to 60 minutes. SiMa cells produced significant cleavage of SNAP25 over background after treatments as short as one minute (Figure 4A).

Figure 4

SiMa cells are sensitive and specific for BoNT/A uptake.

The specificity of a method defines its ability to measure the analyte of interest and differentiate it from similar compounds. This CBPA is specific to BoNT/A by design since the 2E2A6 monoclonal antibody only recognizes SNAP25197. To replace the bioassay, the CBPA must distinguish a fully active neurotoxin from altered or damaged neurotoxins. In the specificity studies, differentiated SiMa cells were treated with recombinant LHN/A, lacking the binding domain but containing the Light Chain and Translocation domains, and a recombinant iBoNT/A containing an inactivating mutation in the LC [49] (Figure 4B). SNAP25197 was only detected at the higher doses of LHN/A tested, suggesting a non-specific internalization of LHN/A (signals at 100 nM LHN/A were similar to BoNT/A at 0.31 pM) and there was no SNAP25197 detected after iBoNT/A treatments. LHN/A uptake was at least 60,000 fold lower than 150 kDa BoNT/A (EC50 = 1.6 pM). To determine the effects of higher concentrations of LHN/A in the SiMa CBA, differentiated SiMa cells were treated with BoNT/A complex (at pM concentrations) or LHN/A with a highest dose of 50 µM. The data in figure 4C confirms specificity of the CBPA to fully active toxin and defines the effects of LHN/A in the assay at concentrations ∼106 higher than those of active BoNT/A. The EC50 for the LHN/A molecule was 2.1 µM versus 0.85 pM for the fully active BoNT/A. Moreover, the assay can measure the potency of pure neurotoxin (150 kDa) as well as BoNT/A complex. These results demonstrate that the CBPA mirrors BoNT/A mechanism of action in vivo: binding, internalization-translocation, and catalytic activity [14].

Optimization of the CBPA for BoNT/A

Three major experimental steps require optimization in a CBPA: cell growth and differentiation conditions, drug treatment, and read-out parameters. Factors influencing performance at each step were evaluated individually with BoNT/A uptake as the end-point, measured as the presence of SNAP25197, and are summarized in Table 1. The final conditions chosen for the optimized assay were plating 50,000 cells/well in EMEM serum-free medium supplemented with N2 and B27 (Figure 5A) in poly-D-lysine plates for ≥48 h (Figure 5B) followed by 0.004–25 pM BoNT/A treatment for 24 h and two-day incubation in toxin free medium to allow for SNAP25197 accumulation (Figure 5C). For the ECL-ELISA, High Bind ELISA plates were spotted with 5 µL of 2E2A6 at 20 µg/mL (Figure 5D), dried and then blocked with 2% ECL (Enhanced Chemiluminescence) with 10% goat serum for 1 h followed by lysate incubation overnight at 4°C (Figure 5E). Sulfo-tag labeled detection antibody was incubated at room temperature for 1 h, reading buffer was added, and the plates were imaged using a SECTOR® Imager 6000.

Figure 5

Optimization the SiMa CBPA.
Table 1

Assay Optimization and Standardization.

Edge effects, a source of variability, are sometimes due to evaporation from wells that are incubated for long periods of time during differentiation or treatment, mainly in the edges of the plates, rows A and H, and columns 1 and 12. Edge effects were evaluated in the tissue culture plates with CellTiter-Glo® to measure cell viability after cell differentiation, BoNT/A treatment, and incubation. The variability (% CV) of rows A and H and columns 1 and 12 were compared to the inner wells in the plate. Edge effects were detected (p<0.001) and the assay was designed avoiding the use of the plate edges (Figure 6A).

Figure 6

Development of a sensitive screening CBPA.

System suitability studies (linearity), to ensure that small differences in potency between samples can be detected in the assay, were performed by two operators. To simulate samples with different potencies, samples from 2-fold more concentrated to 0.1 (10-fold) diluted compared to the BoNT/A reference preparation were tested in the assay. For these assays 150 kDa BoNT/A was used. Accuracy was evaluated by calculating the relative potency (RP) and 95% confidence intervals (CI) between the 1× and the concentrated or diluted samples, and by obtaining a percent recovery from the expected and the experimental relative potencies. The data obtained with dilutions from 0.5 (2-fold) to 0.1 (10-fold) (Table S1) demonstrates that the assay can differentiate from 0.5 (2-fold) to 0.16 (6-fold) dilutions, but samples diluted further cannot be distinguished among them. Since this assay is intended to replace the bioassay for product release, the most relevant range is 0.5 to 1.75 relative potency. Results in Table 2 demonstrate that the assay differentiates the 1× sample from both the diluted and the more concentrated samples. Of special interest are the 0.75 and 1.25 relative potencies in which none of the four independent assays performed contained 1× in the 95% CI and were therefore different from the 1× reference preparation. The CBPA can identify samples with 20% difference in potency. Accuracy was excellent with percent recoveries from 96 to 112% in a research laboratory setting. Similar suitability studies were performed with BOTOX® during assay validation in the GMP laboratory leading to assay approval by regulatory agencies.

Table 2

System suitability-Sensitive Assay: Excellent accuracy and linearity.

To measure the quality and power of the CBPA, the z-prime (Z′ = 1−[3(SDmax+SD0)/(Meanmax−Mean0)) values for 49 assays conducted by three operators were calculated. The Z′ parameter compares the assay dynamic range to data variation and measures how statistically different the experimental values are from the negative control [50]. A Z′ value between 0.5 and 1.0 is defined as an excellent assay suitable for screening with 1.0 being ideal, while a value <0.5 is a marginal assay not suitable for screening. The average Z′ value for the CBPA was 0.82 with values ranging from 0.6 to 0.96 for individual assays, demonstrating an excellent assay.

The CBPA described here is extremely sensitive, accurate, and amenable for screening but requires six days to complete from cell plating to results (3½ days from treatment to data, figure 6A). When screening in process or environmental samples in which botulism patients need to be diagnosed quickly, speed is more important than sensitivity [25]. Therefore, a screening CBPA was designed (Figure 6A) in which cells were plated in optimized differentiation medium overnight and treated the next day with BoNT/A (0.01 to 10 nM) for 6 h followed by an overnight incubation in toxin-free medium to allow for SNAP25197 accumulation. To complete the ECL-ELISA in one day, lysates are incubated 2 h at 4°C (cell plating to data two days, treatment to data 1½ days). For both assays hands-on time is approximately 4 h. The screening CBPA is sensitive, EC50∼120 pM, and possesses excellent S/B>200 at 120 pM and ∼40 at 10 pM (Figure 6B). The screening assay has been routinely conducted by three operators in our laboratory (125 independent assays performed) with average Z′ of 0.79 (from 0.99 to 0.51) indicating fitness for screening. System suitability and accuracy were evaluated by dilutional linearity studies focusing on relative potencies of 0.5 to 1.75 (Figure 6C). For the 0.5 and 1.75 relative potency samples, all the individual assays differentiated the sample from the 1× reference preparation. Some individual assays for the other dilutions had confidence intervals overlapping 1× and therefore could not be differentiated from the 1× reference preparation running a single assay. However, when at least four independent assays were run and their data combined utilizing PLA software, all the concentrated and diluted samples tested were determined to be different from the 1× reference preparation. Excellent accuracy was obtained with percent recoveries ranging from 85.6 to 117% in a research laboratory setting.

The CBPA can also utilize standard chemiluminescence as an alternate read-out (Figure 7A) allowing the use of a luminometer to read the plates. The table in figure 7B summarizes the optimized parameters and the final conditions for the BoNT/A sensitive CBPA. A representative run with BoNT/A complex (Figure 7A) demonstrates sensitivity and low variation among replicates. The S/B at 25 pM was ∼400 on average and the S/B at 0.03 pM was ∼10 fold.

Figure 7

Sandwich ELISA assay with chemiluminescence is as sensitive as the ECL assay.

BOTOX® biological activity can be evaluated in the CBPA

BOTOX® (onabotulinumtoxinA) contains 0.9 mg NaCl and 0.5 mg human serum albumin (HSA) in each 100 U vial (Units in each vial are determined utilizing Allergan’s validated mLD50 assay, are specific to BOTOX®, and are not interchangeable with other commercial Botulinum neurotoxin preparations). Reconstitution of BOTOX® (the nominal value of 100 U was used) with isotonic culture medium (BoNT/A concentration 500 U/mL or ∼25 pM) results in a hypertonic medium with detrimental effects on cells in culture. Testing each excipient separately in the assay demonstrated that only NaCl at the 500 U/mL dose negatively affected SiMa cells and reduced toxin uptake and/or cell viability. To circumvent hypertonicity, a custom EMEM medium was designed and used to reconstitute 100 U BOTOX® vials (500 U/mL or ∼25 pM). The matrix was kept constant for all concentrations along the dose-response curve by adding NaCl and HSA to the dilution medium to match the amount of excipients present at 500 U/mL. The assay became more sensitive with an EC50 of 9 U/mL (0.9 U/well) and an S/B ratio of 50 at 2 U/mL (0.2 U/well) (Figure 8A). The data clearly demonstrates that the CBPA can measure BoNT/A biological activity in a formulated product with identical sensitivity to the mouse bioassay. In order to determine the utility of the assay with pharmaceutical preparations, two lots of BOTOX® were tested in the research laboratory (Figure 8B) generating a relative potency of 0.83 with a confidence interval of 0.7 to 1.1 indicating that the potency of the two lots is indistinguishable since the confidence interval included the number one. Moreover, to determine the reproducibility of the assay by several operators, a single lot of BOTOX® was tested in the optimized assay in several plates by two operators in the research laboratory (Figure 8C). The average EC50 was 3.96±0.16 U/mL for operator 1 (n = 8) and 4.44±0.16 U/mL (n = 9) for operator 2. To compare the performance of the CBPA when different forms of BoNT/A are tested, the ECL-ELISA assay with differentiated SiMa cells was used to test the biological activity of BoNT/A complex, 150 kDa neurotoxin, and BOTOX® in different plates (Figure S1). The EC50 values obtained in the assays were 1.1±0.3 pM for BoNT/A complex, 1.5±0.2 pM for 150 kDa BoNT/A, and 1.35±0.05 pM for BOTOX®, demonstrating that when BOTOX® was diluted in the custom medium, designed to overcome the effects of the excipients present in the formulation, BoNT/A biological activity in the formulated product can be accurately measured. In conclusion, a cell-based potency assay with excellent sensitivity, specificity, accuracy, and precision has been developed to fully replace the mouse bioassay to measure BoNT/A biological activity.

Figure 8

The CBPA can measure BoNT/A biological activity in BOTOX® vials.

Discussion

The sensitive BoNT/A CBPA reported here utilizing differentiated human neuroblastoma SiMa cells and an ECL sandwich ELISA satisfies all the requirements for a fully in vitro replacement of the mouse bioassay for BoNT/A [14], [16][19], [25] and fulfills our long-standing commitment to the “3R” principles of refinement, reduction and eventual replacement of the bioassay [18] in the final product release. The replacement CBPA for BoNT/A potency testing has a sensitivity similar to the mouse bioassay (EC50∼1-0.4 U/well) and is specific for BoNT/A, by the use of monoclonal antibodies specific for SNAP25197. The assay utilizes a stable cell line of neuronal origin [48] that can be differentiated in 48 h and a sensitive sandwich ELISA read-out that can be validated in a QC laboratory. This CBPA represents the multi-step pharmacological mode of action of BoNT/A at pre-synaptic terminals [3], [4], [8]; it is accurate, robust, reproducible, amenable to validation, and can measure BoNT/A biological activity in pharmaceutical preparations (containing less than a nanogram of BoNT/A formulated with excipients).

For over 25 years there has been a strong desire to replace the mouse bioassay with a fully in vitro assay that enables sensitive evaluation of all key steps in BoNT/A action [14], [18], [25]. It was the dogma that continuous cell lines lacked the sensitivity necessary to develop an assay that could replace the mouse bioassay [47], but at the same time the use of primary neurons or embryonic cell derived neurons pose their own challenges as they have to be freshly derived from animal tissue [39][42] or they require complicated protocols and long time to be fully differentiated [44][46]. Moreover, when replacing a bioassay approved by regulatory agencies with a new in vitro assay, the sensitivity of the method is not the only consideration as the assay has to be validated and cross-validated against the mouse bioassay [18], [25]. Our team set in place a rigorous evaluation process of continuous cell lines for their sensitivity to BoNT/A that culminated in the identification of several sensitive cell lines that could be amenable for developing potency assays, with SiMa cells being the most sensitive even when undifferentiated (Figure 2). However, to achieve the sensitivity needed to replace the mouse bioassay (pM concentrations), optimization of the cells’ growth and differentiation conditions was essential. After the optimization process with Neuro-2a, PC12, LA1-55n, and SiMa cell lines, we achieved great sensitivity with EC50 values in the mid and low pM (Figure 3) that were excellent to develop BoNT/A activity assays. A breakthrough was achieved with the identification of the SiMa cells that were very sensitive (EC50 = 6.5 pM in WB and 3 pM in ELISA) to BoNT/A and produced excellent S/B at all doses tested, especially at sub-pM concentrations, comparable to the primary and embryonic cell derived neurons [40], [41], [44][47] while providing a continuous and reliable source of cells (allowing preparation of cell banks) for the CBPA.

The development of a read-out for the cell-based assay that could be validated in a QC environment was essential since Western blots, with intrinsic variability, are difficult to validate. Sandwich ELISA assays are robust, sensitive, and amenable to validation. The antibody binding affinity for the antigen is usually the main determinant of immunoassay sensitivity. The second breakthrough was achieved with the generation of a highly specific high affinity anti-SNAP25197 monoclonal antibody. The 2E2A6 antibody, displaying high affinity and a very low dissociation constant (Figure 1) was ideal to capture cleaved SNAP25197 from cell lysates treated with BoNT/A. The high specificity for SNAP25197 resulted in extremely low background signal from untreated lysates; excellent signal to background ratios, even at femtomolar amounts of BoNT/A; and Z′ values that support the use of the assay for screening.

A very sensitive CBPA is needed for measuring BoNT/A biological activity in pharmaceutical products and in other situations in which the toxin is present in very low concentrations. But during outbreaks when patients are waiting for a diagnosis or for research to identify BoNT/A inhibitors, the speed of the assay becomes more important. Our team was able to develop a shorter, but yet sensitive assay (Figure 6, EC50 = 120 pM) that can produce reliable measurement of BoNT/A activity in only 2½ days. Additionally, we rigorously optimized and characterized both CBPAs (sensitive and screening, figure 6A). For the sensitive CBPA we optimized and standardized twenty parameters comprising cell growth and differentiation, BoNT/A treatment, and ECL-ELISA (Table 1). Moreover, dilutional linearity/recovery experiments performed with both assays by three operators demonstrated excellent accuracy and precision (Tables 2 and S1). The optimized CBPA was slightly modified to test BOTOX® by designing a custom medium to overcome the adverse effects of the excipients found in the formulation (Figure 8A). In conclusion, this is the first CBPA utilizing an established cell line reported to measure BoNT/A biological activity in BOTOX® possessing sensitivity equal or superior (EC50∼1-0.4 U/well) to the mouse bioassay and being specific for BoNT/A by design. Further development, validation, and cross-validation of a version of this CBPA has resulted in FDA, Health Canada, and European Union approval for use in the potency testing of BOTOX® (onabotulinumtoxinA), BOTOX® Cosmetic, and Vistabel®.

Materials and Methods

Cell Lines and Growth Conditions

Unless otherwise stated, tissue culture reagents were from Invitrogen, Carlsbad, CA

PC-12- Rat pheochromocytoma (CRL-1721; ATCC) were cultured in collagen IV plates in ATCC’s recommended medium. Differentiation medium: RPMI medium with 2 mM GlutaMAX™, 1× B27 supplement, 1× N2 supplement, 10 mM HEPES, 1 mM Sodium Pyruvate, 50 ng/mL NGF, 100 U/mL Penicillin, and 100 µg/mL Streptomycin. Neuro-2a- Murine neuroblastoma (CCL-131; ATCC) were cultured in Costar flasks in ATCC’s recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAX™, 0.1 mM NEAA, 10 mM HEPES, 1× N2 supplement, and 1× B27 supplement. SH-SY5Y- Human neuroblastoma (94030304; ECACC) were cultured in Costar tissue culture flasks in ECACC’s recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAX™, 0.1 mM NEAA, 10 mM HEPES, 1× N2 supplement, and 1× B27 supplement. N18- Mouse neuroblastoma×Rat glioma hybrid (88112301; ECACC) were cultured in Costar tissue culture flasks, 162 cm2 (CLS3150; Corning). Growth medium: DMEM with 2 mM glucose, 2 mM glutamine, and 10% heat-inactivated FBS. LA1-55n- human neuroblastoma (06041203; ECACC) were cultured in Costar flasks in ECACC’s recommended medium. SiMa- human neuroblastoma (ACC 164, DSMZ) were cultured in collagen IV flasks in DSMZ’s recommended medium. Differentiation medium: Minimum Essential Medium with 2 mM GlutaMAX™ I with Earle’s salts, 0.1 mM Non-Essential Amino-Acids, 10 mM HEPES, 1× N2 supplement, and 1× B27 supplement. For optimal differentiation, PC-12, Neuro-2a, LA1-55n, and SiMa cells were plated in 96-well plates at 5×104 cells/well in 100 µL differentiation medium for three days.

Monoclonal antibody to SNAP25197

Murine monoclonal antibodies specific for SNAP25197 were generated with the (C)DSNKTRIDEANQ peptide utilizing standard immunization protocols. Antibodies to SNAP25197 were screened in WB and ELISA. Antibodies were affinity purified from ascites before use in the SPR and ELISA assays.

Surface Plasmon Resonance Binding Analysis

Experiments were performed on a BIAcore 3000 instrument (GE Healthcare). Ligands, SNAP25134–197 and SNAP25134–206 peptides, were immobilized on a CM5 chip using amine coupling. Analytes, Anti-SNAP25197 2E2A6 (1.1 mg/mL stock solution, AGN) or MC-6053 (15 µg/mL stock solution, Protein A purified, Research and Diagnostics Antibodies) antibodies, were injected at 0–125 nM. Sensorgram curves were fitted to a 1[ratio]1 Langmuir binding model (BIAevaluation 3.0 software, GE Healthcare). The association, ka (1/Ms), the dissociation, kd (1/s) and the equilibrium, KD (KD = kd/ka) (M) constants were determined.

BoNT/A treatment

Cells were treated with BoNT/A complex, 150 kDa BoNT/A (Metabiologics, Madison, WI), recombinant LHN/A or iBoNT/A, or with BOTOX® (Allergan, Irvien, CA) for different amounts of time as specified in each assay. The medium containing BoNT/A was then replaced with fresh medium without toxin and cells were incubated for additional periods of time as specified in each assay. Each concentration of BoNT/A was tested in triplicate.

Cell Lysis and Western Blot Analysis

Cells were washed once with PBS and lysed in freshly prepared Triton X-100 Lysis Buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, and one tablet of EDTA-free protease inhibitors) on ice for 20 min. Lysed cells were centrifuged in the plate at 4000 rpm for 20 min at 4°C. Western blots (WB) for detection of SNAP25206 and SNAP25197 or only SNAP25197 were described previously [51]. For WB analysis the lysates were transferred to a 96-well PCR plate, 2× SDS-PAGE loading buffer (Invitrogen) was added, and the plate was heated to 95°C for 5 minutes. The gels were run in 1× MOPS-SDS running buffer (Invitrogen) at 200 V for 55 min. Proteins were transferred to nitrocellulose membranes (Bio-Rad) pre-wet in Western blot transfer buffer (Invitrogen) containing 20% Methanol (Burdick and Jackson). The Western blot transferred at 800 mA for 2 h using the TE-62 transfer cell apparatus (GE Healthcare). Blots were blocked in 2% ECL blocking agent (GE Healthcare) in 1× TBS/0.1% Tween 20 (Bio-Rad) (TBS-T) for 1 h at room temperature. The following primary antibodies were used: anti-SNAP25197 polyclonal antibody [51] diluted 1[ratio]1000, SMI-81 antibody (Sternberger Monoclonals Inc) diluted 1[ratio]5000 to evaluate the intact and cleaved SNAP25 products (i.e. SNAP25206 and SNAP25197 were detected). Antibodies were diluted in 2% blocking agent/TBS-T buffer and incubated overnight at 4°C with gentle shaking. Secondary antibodies were anti-rabbit or anti-mouse IgG H+L HRP conjugated (Invitrogen) diluted 1[ratio]5,000 or 1[ratio]10,000 in 2% blocking agent/TBS-T buffer for 1 h at room temperature. The membranes were washed and exposed for 5 min to ECL Plus Western Blotting System Detection Reagents (GE Healthcare). Chemifluorescence was captured by scanning the blots in the Typhoon 9410 Imager (GE Healthcare) at λex 452/λem 520. The intensity of the gel bands were calculated using ImageQuant TL software (GE Healthcare). The data was analyzed using SigmaPlot v 10.0 (Systat Software Inc.) or PLA2.0 (Stegmann Systems). Intensity values were plotted against concentration of BoNT/A in log scale and fitted to a 4-parameter logistics function (Y = Y0+a/[1+(X/X0)b]) without constraints. Based on the fitted curves the EC50 values, corresponding to “X0”, were determined.

ECL and chemiluminescence sandwich ELISA

For the ECL sandwich ELISA, MSD High Bind plates (Meso Scale Discovery) pre-spotted with anti-SNAP25197 MAb 2E2A6 were blocked with 150 µL blocking buffer for 1 h at RT. After blocking, the buffer was discarded and 25 µL of cell lysate were added to each well of the plate followed by incubation as detailed in text. Plates were washed with PBS-T, and SULFO-TAG NHS-Ester labeled detection pAb anti-SNAP25 antibody (Antibody to N-terminus of SNAP25, Cat# S9684, Sigma) in diluent buffer was added. Plates were sealed and shaken at room temperature for 1 h, washed with PBS-T, and 150 µL of 1× Read Buffer was added per well. Plates were immediately read on the SI6000 Image plate reader. For the chemiluminescence sandwich ELISA, white plates (Greiner) were coated with 100 µL/well of anti-SNAP25197 2E2A6 MAb at 4°C overnight. Plates were blocked with 2% ECL blocking with 10% goat serum for 1 h at RT. Fifty microliters of cell lysate were added to each well and the plates were incubated at 4°C. S9684 anti-SNAP25 pAb conjugated with HRP was used for detection. The plates were developed with SuperSignal ELISA Pico 1[ratio]1 mixture (Pierce) and read at 395 nm on a Luminometer (Molecular Devices). Data was fitted to a 4PL model as detailed above.

Supporting Information

Figure S1

The ECL-ELISA CBPA with SiMa cells can be used to test the biological activity of BoNT/A complex, 150 kDa neurotoxin, and BOTOX®. Comparison of CBPAs performed with A. BoNT/A complex, B. 150 kDa BoNT/A, and C. BOTOX® (the nominal value of 100 U was used) utilizing the optimized assay conditions. The EC50 values obtained in the assays are very similar demonstrating that the assay is robust, versatile, and can detect BoNT/A biological activity at very low concentrations in the presence of formulation excipients.

(TIF)

Table S1

(TIF)

Acknowledgments

Authors wish to thank P.E. Garay for the development of the BOTOX® reconstitution medium and characterization of the 2E2A6 monoclonal antibody. R. Lewis and L. Moller for technical assistance with assay development. T. Terrell, D. Hodges, L. Wong, L.E. Steward, and J. Francis for scientific discussions. L.E. Steward, M. Gilmore, M. Spillane, S. Liu, and S. Ghanshani for the recombinant LHN/A and iBoNT/A. D. Hodges for 2E2A6 monoclonal antibody scale-up and purification. K. Abel and M. Gilmore for review of the manuscript.

Supplementary data

Available at PLoS One Online (http://www.plosone.org).

Funding Statement

The research reported in this manuscript was solely funded by Allergan Inc (www.allergan.com). The funders had no role in study design, data collection and analysis, or preparation of the manuscript. Allergan upper management reviewed the manuscript and approved its submission for publication. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Incidence of End-Stage Renal Disease in the Turkish-Cypriot Population of Northern Cyprus: A Population Based Study

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Leighton R. James, Editor
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Abstract

Background

This is the first report of the incidence and causes of end-stage renal disease (ESRD) of the Turkish-Cypriot population in Northern Cyprus.

Methods

Data were collected over eight consecutive years (2004–2011) from all those starting renal replacement therapy (RRT) in this population. Crude and age-standardised incidence at 90 days was calculated and comparisons made with other national registries. We collected DNA from the entire prevalent population. As an initial experiment we looked for two genetic causes of ESRD that have been reported in Greek Cypriots.

Results

Crude and age-standardised incidence at 90 days was 234 and 327 per million population (pmp) per year, respectively. The mean age was 63, and 62% were male. The age-adjusted prevalence of RRT in Turkish-Cypriots was 1543 pmp on 01/01/2011. The incidence of RRT is higher than other countries reporting to the European Renal Association – European Dialysis and Transplant Association, with the exception of Turkey. Diabetes is a major cause of ESRD in those under 65, accounting for 36% of incident cases followed by 30% with uncertain aetiology. 18% of the incident population had a family history of ESRD. We identified two families with thin basement membrane nephropathy caused by a mutation in COL4A3, but no new cases of CFHR5 nephropathy.

Conclusions

This study provides the first estimate of RRT incidence in the Turkish-Cypriot population, describes the contribution of different underlying diagnoses to ESRD, and provides a basis for healthcare policy planning.

Introduction

In recent years end-stage renal disease (ESRD) has become an increasing public health challenge for high and middle income countries, with an associated escalation of the cost of providing renal replacement therapy (RRT) [1][4]. The collection of accurate epidemiological data is of great importance for healthcare policy planning [1][3]. This is especially true for places where the infrastructure delivering RRT is improving, such as the island of Cyprus.

There are considerable differences in the incidence and prevalence of RRT within Europe. Registry data demonstrate a North-South gradient, with higher incidence of RRT and lower mortality around the Mediterranean [2], [5]. Several factors have been suggested to contribute to this variation [6], [7]. In order to understand the aetiology of ESRD and chronic kidney disease, which affects many more people, it is important to determine the primary renal diagnosis [8]. ESRD attributed to type 2 diabetes and hypertension continues to rise throughout the world, and this is increasingly true for countries such as Cyprus [2], [9][11].

Cyprus is an island in the eastern Mediterranean that has been occupied by a series of historical powers, in particular the Greeks and Ottoman Turks. The Cypriot population is genetically distinct from mainland populations of either Greece or Turkey, although environmental factors, such as diet and lifestyle are broadly similar [12], [13].

Turkish-Cypriots form a distinct ethno-linguistic community centered on Turkish administered Northern Cyprus (TRNC). The aim of this study was to describe the incidence and prevalence of RRT in this ethnically-defined Mediterranean population by type of primary renal disease. To put these data into context we compared these data with reported RRT incidence from Greece, Turkey, and the white population of England. An important, and probably unique, aspect of our registry is that every RRT patient has provided a DNA sample for research. Because the Greek and Turkish communities share many genetic characteristics [14], [15], we initially undertook genetic testing for two conditions that have been identified in Greek Cypriots, CFHR5 nephropathy and thin basement membrane nephropathy [16], [17].

Materials and Methods

Ethics Statement

This study was approved by the ethics committee of Lefkosa Burhan Nalbantoğlu State Hospital. All participants provided informed consent in writing, in accordance with the Declaration of Helsinki.

Study Population

The study was conducted at Nicosia State Hospital in Northern Cyprus. Nicosia State Hospital is a multi-specialty tertiary care hospital, and provides renal services to the whole of Turkish administered Northern Cyprus (TRNC). All citizens of TRNC are entitled to free RRT, regardless of ethnicity. All patients with symptomatic chronic kidney disease (CKD) from within this population present either through the village practitioners, private hospitals or directly to Nicosia State Hospital. Details of every individual admitted with acute or chronic renal failure in the last decade were collected with a unique identifier number, thus preventing any duplication of records or redundancy of referrals. ESRD was defined as chronic renal disease with an eGFR of <10 ml/min/1.73 m2; but patients with diabetic nephropathy often started RRT with eGFR<15 ml/min/1.73 m2.

The most recent census of the TRNC population in 2006 showed that of 178,031 de jure citizens, 120,007 were born to parents who were both themselves born in Cyprus, providing an effective measure of the size of the ethnic Turkish-Cypriot population that has been applied throughout this paper [18].

Variables

Basic demographic data (age at ESRD, sex, ethnic group, and probable diagnosis) were recorded for all patients receiving RRT at day 1, and again after 90 days on the renal replacement programme. Diabetic nephropathy was defined as ESRD in the presence of diabetes without evidence of an alternative diagnosis. Family history of ESRD was defined as a first or second degree relative with ESRD.

Data Sources for International Comparisons

European data was taken from the European Renal Association – European Dialysis and Transplant Association (ERA-EDTA) Registry report for 2008 [2]. Additional data for Greece, Turkey, and England were taken from the Hellenic Renal Registry (Dr GA Ioannidis personal communication), the National Haemodialysis, Transplantation and Nephrology Registry report of Turkey 2008 [19], and the 2008 Renal Registry Report [1]. The denominator population for international comparisons was calculated from the relevant national statistics [20], [21].

Statistical Analysis

The incidence of RRT in this population was averaged over eight consecutive calendar years (2004–2011). Age-standardised incidence rates were calculated using the Eurostat EU27 population figures [22]. All comparisons with other populations were made using data for chronic RRT; that is those still on RRT at 90 days. The emphasis of this report will be on age-specific comparisons as these are not affected by age-referral biases. We calculated 95% confidence intervals assuming a Poisson distribution for incidence rates. Statistical analysis was carried out using Stata version 11.

Genetic Testing of All Adult Patients on RRT in North Cyprus

After informed consent for this study, blood was taken from all patients on renal replacement therapy on 01/01/2011. Genetic analysis was performed at University College London, UK. DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen, Stanford, CA, USA). We designed primers to screen for the G1334E and 3854delG mutation in COL4A4 and the G871C mutation in COL4A3 [23]. Screening PCR was also performed to amplify both wild-type and mutant CFHR5 alleles in a single reaction as described previously [16].

Results

A total of 225 Turkish-Cypriot patients were maintained on renal replacement therapy beyond 90 days during the study period (01/01/04–31/12/11). More males than females started RRT (139 vs 86), but the age distribution was similar between the sexes (Table 1). 18.2% of the incident population had a first or second degree relative with ESRD, and this rose to 27% in those with ESRD due to uncertain aetiology. The average crude and age-standardised incidence rates at day 1 for this period were 311.4 and 456.9 per million population, respectively. The Turkish-Cypriot case mix at 90 days included 36.0% with ESRD due to diabetic nephropathy, 29.8% with unknown diagnosis, and 3.6% with polycystic kidney disease (Table 2). Tetra primer PCR identified two individuals on RRT with the G871C mutation in COL4A3 previously reported in Greek-Cypriot pedigrees [17]. We did not detect the other previously cited mutations in COL4A3 and COL4A4 or the CFHR5 duplication in the RRT population [16], [17].

Table 1

Baseline characteristics of the incident Turkish-Cypriot renal replacement therapy (RRT) population at 90 days.
Table 2

Provisional renal diagnosis in the Turkish-Cypriot renal replacement therapy (RRT) population at 90 days, by incidence per million population and percentage, in comparison with 2008 registry data for Greece, Turkey, and the UK.

The majority (91.1%) of Turkish-Cypriot patients started RRT on haemodialysis; only a few started peritoneal dialysis (4.4%) or had pre-emptive renal transplantation (3.5%) (Table 1). The crude and age-standardised prevalence of RRT in Turkish-Cypriots was 1216 and 1543 pmp, respectively. The median age of the Turkish-Cypriot prevalent population was 60 for both men and women, and the mean duration on RRT was 5.0 years. The majority of patients on RRT in Northern Cyprus are treated with hemodialysis, which is carried out in two centers, Nicosia and Famagusta. The proportion of prevalent patients on peritoneal dialysis (14%) is similar to other countries [24]. Renal transplantation is performed in either Turkey or the Republic of Cyprus (Greek Cypriot), and benefits a high proportion of patients (prevalence 376 pmp).

We carried out international comparisons. Turkey, Greece, and Tunisia have an age-adjusted incidence rate at day 90 of 349.1, 159.5, and 238.7 pmp respectively [2]. In comparison the crude and age-adjusted incidence rates of chronic RRT for Turkish-Cypriots were 234.4 and 327.2 per million population, respectively. Figure 1 shows that the incidence of RRT in each age group in Turkish-Cypriots is comparable to that seen in Turkey. Despite the small number of cases, the incidence of RRT in the 45–64 age group in Turkish-Cypriots (412.0 pmp) is significantly higher than that seen in the white population of England (123.2 pmp).

Figure 1

Incidence of renal replacement therapy (RRT) at 90 days by age and gender.

There is a high rate of RRT for ESRD attributed to diabetic nephropathy in all eastern Mediterranean countries (84.4 pmp in Turkish-Cypriots, 53.5 pmp in Greece, and 61.6 pmp in Turkey) (Table 2). Table 2 also shows that the code ‘uncertain aetiology’ is more common in the Turkish-Cypriot registry (69.8 pmp) than in other populations. However there were lower levels of hypertensive nephropathy in Turkish-Cypriots than in Greece or Turkey (1.0 pmp vs 19.1 pmp and 53.4 pmp respectively). The incidence of polycystic kidney disease was broadly similar across all registries (range 6.7–10.1 pmp).

The incidence of RRT in the minority Turkish population of Northern Cyprus is broadly in line with the rate seen in Turkish-Cypriots. An additional 76 Turkish patients were maintained on renal replacement therapy beyond 90 days during the study period, giving an overall incidence of 211.3 pmp in the de jure population of Northern Cyprus. However data on this population is less accurate due to significant migration to and from the mainland of Turkey [18].

Discussion

High Incidence of RRT

This study presents the first population-based RRT incidence figure from Cyprus, and reveals a high incidence of RRT that is RRT is higher than other countries reporting to the ERA-EDTA, with the exception of Turkey [2]. Diabetes is a major cause of ESRD overall and specifically in those under 65, with rates comparable to those seen in the USA [3]. We found that the high incidence of RRT in Turkish-Cypriots is not due to the specific mutations in COL4A3, COL4A4, and CFHR5 assessed in this study [16], [17]. Finally, a third of Turkish-Cypriot patients start RRT with unknown primary diagnosis. This highlights both the need for earlier detection of these cases and the possibility that there may be other uncharacterised conditions causing ESRD in this population.

Diabetic Nephropathy

The Turkish-Cypriot case-mix, with a high incidence of RRT for ESRD due to diabetic nephropathy, is similar to that seen in Turkey and Greece (Table 2). Diabetes is also a common cause of ESRD in developing countries around the Eastern Mediterranean, such as Egypt, Kuwait, Lebanon and Saudi Arabia [11]. The incidence of RRT for ESRD attributed to diabetic nephropathy seen in Turkish-Cypriot less than 65 years old is striking (Figure 2). Currently data concerning the incidence of diabetes in Turkish-Cypriots is lacking. Diabetes is common in Greek-Cypriots and mainland Turkish patients [25][27], but not to the extent seen in some other populations with high levels of diabetic nephropathy, such as the Pima Indians of Arizona [28], [29]. Moreover, childhood levels of obesity in Cyprus are much closer to those seen in other European countries than in the US [30], [31]. It is therefore possible that, in common with other registries, some of these cases reflect a co-occurrence of Type 2 diabetes with ESRD due to an alternative aetiology [32].

Figure 2

Incidence of renal replacement therapy (RRT) for end stage renal disease (ESRD) due to diabetic nephropathy.

Genetic Renal Disease in the Turkish-Cypriot Population

Congenital factors may also be important in the aetiology of ESRD in this population [33]. Previous estimates of the prevalence of family history of ESRD amongst incident dialysis patients, suggest that 7–15% Caucasians have a first or second-degree relative with ESRD [34], [35]. This proportion is highest in young adults, non-Caucasians, and for those where ESRD is caused by diabetes or hypertension [34]. We observed a similar rate of familial ESRD in Turkish-Cypriot patients on RRT, which was even higher (27%) in the group with unknown diagnosis, suggesting the existence of conditions that are not yet characterised in this population. For comparison, the rate of RRT for ESRD due to polycystic kidney disease was similar across all registries.

In order to assess the contribution of inherited renal disease we collected DNA from the entire Turkish-Cypriot population on RRT. Previous work has demonstrated a number of important founder mutations and significant geographic clustering for several monogenetic diseases affecting the Greek-Cypriot population of Cyprus, including mutations of COL4A3, COL4A4, PKD2, and MEFV [17], [36], [37]. Although the CFHR5 duplication is common in Greek-Cypriots originating from the southern half of the island [38], we did not detect it in our sample of prevalent patients. However, due to the small size of our sample the allelic frequency may still lie within that observed for Greek Cypriots [39]. The familial clustering and high incidence of RRT in Turkish-Cypriots is therefore due to other monogenetic or polygenic diseases in this population.

Coding ‘Uncertain Aetiology’

There is a significant group in the Turkish-Cypriot RRT population with unknown diagnosis (69.8 pmp). This group partly reflects late presentation and limited diagnostic investigations. Comparative data from the Eastern Mediterranean is difficult to obtain, and information on primary renal disease is less robust [8], [32] but there is evidence for a reciprocal relationship with coding for hypertension in many registries [8], [32], [40]. The incidence of hypertensive nephropathy is conspicuously lower in Turkish-Cypriots than in neighbouring countries. In the absence of clear diagnostic criteria for hypertensive nephropathy, it may be appropriate to combine ‘hypertension’ with ‘uncertain aetiology’ in Table 2, which would imply that there is no established primary renal diagnosis in 40% of patients on RRT in the Greek, Turkish, and Turkish-Cypriot registries.

The majority of patients with an unknown diagnosis present clinically with minimal proteinuria (<1 g protein/day) and asymptomatic disease, consistent with a pathological process primarily affecting the renal tubules [8] and similar to the features of medullary cystic kidney disease type 1. A number of families presenting this way in the Greek Cypriot population have shown linkage to the region 1q21, but the gene responsible has not yet been identified [41]. It is possible that the medullary cystic phenotype reflects the final common pathway of a number of genetic and environmental factors that are common in this population.

Alternative Explanations for the Incidence of RRT in Turkish-Cypriots

Regional variations in RRT incidence may reflect both genetic and environmental factors [7], [28], [42]. Macroeconomic factors that influence regional variations in RRT incidence include per capita GDP and health-care expenditure [7]. Northern Cyprus has a developing economy, with per-capita GDP that is 76% of that in the Republic of Cyprus, and it is dependent on aid from the Turkish government [43]. RRT has only recently become widely available in TRNC and there is no long-term provision of private dialysis. Economic factors also influence the management of CKD and co-morbidities, as well as the competing risk of mortality, in the general population [7]. The high incidence of RRT in the Turkish-Cypriot population may therefore reflect suboptimal management of diabetes, hypertension, and associated complications. This has significance for healthcare planning on the island, and underlines the importance of prospective assessment of kidney function in this population.

Wider Relevance of these Findings

Our findings have several implications. First, these data highlight the need to examine the care of diabetics who are at risk of renal disease in the Turkish-Cypriot population, particularly in young people, to prevent rising rates of RRT. To this end, we are examining case records of patients who were diagnosed with diabetic nephropathy and reached ESRD at a young age. Second, there is a large diaspora of Turkish-Cypriots who may carry with them an increased risk of ESRD. Third, this study provides a template for other adult registries across the Middle-East, and highlights the proportion of patients with unknown diagnosis [8]. It is hoped that with greater access to diagnostic investigations this number will be reduced further.

The strength of this study is that we have a complete dataset from the study period, including DNA samples from the entire prevalent RRT population. Many countries with new RRT programs, such as Bangladesh or Malaysia, show increasing take-up of services with time [24]. However the incidence rate, mean age, and sex-ratio of patients on RRT at 90 days shown in Table 1 are broadly in line with other European populations [2]. Moreover, by examining incidence at 90-days in those aged <65 years we have sought to avoid misclassification and bias due to referral patterns, availability of RRT, and prevalence of co-morbidities.

The main limitations of this study are factors affecting the calculation of incidence rates. This study used the same definition of ethnicity as the 2006 census, and the size of the population remained stable over the study period. Referral bias is unlikely as RRT is freely available to all citizens, and because all ERSD is managed through one centre we were able to achieve a remarkably complete dataset.

In conclusion, this study provides a complete dataset of RRT in the Turkish-Cypriot population and shows that incidence and prevalence of RRT are high. Diabetes is a major cause of ESRD overall and specifically in those under 65. With the prevalence of diabetes and hypertension projected to rise further, facilities to target the earlier stages of diabetes and CKD need to be developed further in this population. Earlier identification of CKD together with long-term follow-up will enable more accurate determination of renal diagnosis before the onset of ESRD. Familial renal disease is common in this population, and this study represents the first complete collection of DNA from an ethnically-defined population on RRT. This population therefore provides an opportunity to look for genetic factors associated with an increased risk of ESRD in Cyprus. The high prevalence of RRT in Turkish-Cypriots has implications for healthcare policy planning.

Acknowledgments

We thank Meral Yükseliş for assistance with data collection, Dr G.A. Ioannidis for data from the Hellenic Renal Registry, and Professor Constantinos Deltas and Dr Konstantinos Voskarides for provision of DNA samples.

Funding Statement

This work was supported by a Clinical Research Training Fellowship to TMFC from the Medical Research Council (http://www.mrc.ac.uk/index.htm) and a grant from the St Peter’s Trust for Kidney, Bladder and Prostate Research (http://www.stpeterstrust.org.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Angiotensin Converting Enzyme Inhibitor and HMG-CoA Reductase Inhibitor as Adjunct Treatment for Persons with HIV Infection: A Feasibility Randomized Trial

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Geneviève Chêne, Editor
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Abstract

Background

Treatments that reduce inflammation and cardiovascular disease (CVD) risk among individuals with HIV infection receiving effective antiretroviral therapy (ART) are needed.

Design and Methods

We conducted a 2×2 factorial feasibility study of lisinopril (L) (10 mg daily) vs L-placebo in combination with pravastatin (P) (20 mg daily) vs P-placebo among participants receiving ART with undetectable HIV RNA levels, a Framingham 10 year risk score (FRS) ≥3%, and no indication for ACE-I or statin therapy. Tolerability and adherence were evaluated. Longitudinal mixed models assessed changes in blood pressure (BP), blood lipids, and inflammatory biomarkers from baseline through months 1 and 4.

Results

Thirty-seven participants were randomized and 34 [lisinopril/pravastatin (n = 9), lisinopril/P-placebo (n = 8), L-placebo/pravastatin (n = 9), L-placebo/P-placebo (n = 8)] attended at least one follow-up visit. Participants were 97% male, 41% white, 67% were current smokers, and 65% were taking a protease inhibitor. Median age was 48 years, CD4 count 483 cells/mm3, FRS 7.79%, total cholesterol 184 mg/dL, and LDL-C 95 mg/dL. There was no treatment difference for pravastatin vs P-placebo in total cholesterol, LDL-C, or any of the inflammatory biomarkers. Participants randomized to lisinopril vs. L-placebo had significant declines in diastolic BP (−3.3 mmHg, p = 0.05), hsCRP (−0.61 µg/mL, p = 0.02) and TNF-α (−0.17 pg/mL, p = 0.04). Participants taking lisinopril vs L-placebo were more likely to report missed doses (88 vs 35%; p = 0.001) and have adherence <90% by pill count (42 vs. 0%; p = 0.02). Few participants from either group reported side effects (n = 3 vs. n = 1).

Conclusions

The modest BP changes and decreased adherence with lisinopril and absence of lipid differences with pravastatin suggest future studies of these drug classes should consider a run-in period to assess adherence and use a different statin. Our results also indicate that ACE-I therapy may have anti-inflammatory benefits for ART-treated persons with HIV infection and this should be further evaluated.

Trial Registration

ClinicalTrials.gov NCT00982189

Introduction

Individuals with HIV infection are at increased risk for premature cardiovascular disease (CVD) due to the higher prevalence of traditional risk factors (e.g., smoking), toxicity from antiretroviral therapy (ART; e.g., metabolic complications), as well as direct effects of HIV itself [1]. Specifically, HIV-related inflammation persists despite effective viral suppression with ART treatment and this may further amplify CVD risk [2], [3], [4], [5]. CVD prevention strategies that encompass both anti-inflammatory benefits as well as traditional risk factor modification may be uniquely beneficial in this context.

Similar to the general population, high blood pressure (BP) and cholesterol account for a significant proportion of CVD risk among patients with HIV infection and remain a key component of prevention strategies [6]. In the general population, epidemiologic data demonstrate a consistent graded relationship between BP and cholesterol with CVD, which persists through normal BP values (down to at least 115/75 mmHg) and moderate total cholesterol levels (155 to >200 mg/dL) [7], [8]. For a target population at higher absolute CVD risk, such as individuals with HIV infection, these data suggest risk factor reductions may be beneficial irrespective of whether individual BP or cholesterol levels exceed current thresholds for treatment [9], [10].

Angiotensin converting enzyme inhibitors (ACE-I) and HMG-CoA reductase inhibitors (‘statins’) have been shown to reduce CVD risk through their BP and cholesterol lowering properties, respectively [11], [12]. However, both classes of medications appear to have additional anti-inflammatory pleotropic effects that may be uniquely beneficial for HIV positive patients [13], [14], [15]. Prior to expanding the use of ACE-I and/or statins for HIV-infected persons to patients for whom these treatments are not currently indicated, safety and tolerability data are needed to inform large-scale trials that more clearly define the net risk-benefit balance.

The goal of this study was to determine if a strategy using lisinopril (an ACE-I) at 10 mg daily and pravastatin (a ‘statin’) at 20 mg daily as adjunctive treatment was feasible, well tolerated, and led to risk factor reductions when given alone or in combination to virologically suppressed patients receiving ART. We also explored the potential treatment effect on biomarkers of systemic inflammation: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α).

Methods

The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.

Participants

Participants with HIV infection receiving ART with HIV RNA levels <200 copies/mL and a FRS ≥3% for 10-year coronary heart disease risk were enrolled after written informed consent at one of two HIV clinics (Hennepin County Medical Center [HCMC] and Clinic 42, Allina Hospitals and Clinics, Minneapolis, Minnesota) from January 2010 through February 2011. Exclusion criteria included known CVD, hypertension or BP ≥140/90 mmHg, low-density lipoprotein cholesterol (LDL-C) >160 mg/dL (or >130 mg/dL with a FRS >10%), triglycerides >500 mg/dL, diabetes, cirrhosis, serum creatinine ≥2.0 mg/dL, or a contra-indication to taking ACE-I or statin therapy. Our criteria specifying a FRS ≥3% both eliminates those at very low risk for CVD and efficiently selects for a target population at moderate CVD risk but for whom BP or cholesterol lowering therapy were not typically indicated. When applying data that HIV infection is associated with approximately a 2-fold increased CVD risk, our target population should have at least a 5% risk for coronary event in next 10 years [16]. FRS used for analyses was calculated from the published algorithm that considered age, gender, systolic BP, total cholesterol, high-density lipoprotein [HDL-C], and current smoking and diabetes status [17]. For screening purposes, FRS was estimated through application of a point-of-care online calculator (hp2010.nhlbihin.net/atpiii/calculator.asp). The study was approved by the institutional review board at each clinical site (HCMC Human Subjects Research Committee and Allina Hospitals and Clinics Institutional Review Board) and the protocol was registered at ClinicalTrials.gov (NCT00982189).

Study Design

The study design was a randomized, double-blinded 2×2 factorial design of lisinopril (L) 10 mg daily versus matched L-placebo daily in combination with pravastatin (P) 20 mg daily versus P-placebo (Figure 1). The goal of the study was to inform planning for larger studies by first assessing ability to recruit for the proposed interventions, and assessing tolerability and adherence. Planned sample size was 40 participants, which provided 80% power to detect a 12.6 mmHg difference between the lisinopril and L-placebo groups for systolic BP (assuming a standard deviation of 14 mmHG) and a 27 mg/dL difference in LDL cholesterol between the pravastatin and P-placebo groups (assuming a standard deviation of 30 mg/dL). The sample size and recruitment period was limited by budget and time restrictions associated with the American Heart Association funding mechanism supporting the study.

Figure 1

Study Design Flow-Diagram.

In order to blind both participants and study investigators to the treatment assigned, active study drugs were over-encapsulated to match the respective placebo capsules. Treatment allocation was balanced in blocks of 4 or 8 providing four groups of approximately equal size. Treatment schedules were only known to the unblinded statistician and the pharmacist responsible for preparing study medication bottles; neither had contact with study participants. After the baseline assessment, participants were instructed to take 1 capsule from each study medication bottle by mouth daily and returned for repeat study visit procedures at 1 and 4 months.

Outcomes

Adherence was assessed via participant self-reported estimates of number of missed doses per week during the study, and then objectively at month 4 by pill count. Tolerability and safety were ascertained through participant history at both 1 and 4 month visits. A fasting lipid profile (total cholesterol, LDL-C, HDL-C, and triglycerides) was obtained at the site clinical laboratory at baseline, 1 and 4 months. Research nurses measured BP in triplicate at each study visit, with mean values used for analyses. Plasma biomarkers of systemic inflammation were also assessed at baseline and the 1 and 4-month follow-up visits. Inflammatory markers were measured by the Laboratory for Clinical Biochemistry Research at the University of Vermont; hsCRP was measured with a NB™II nephelometer, N Antiserum to Human CRP (Siemens Diagnostics, Norwood, MA); IL-6 with Chemiluminescent Sandwich ELISA (R&D Systems, Minneapolis, MN); and TNF-α with Millipore Panel B multiplex (Billerica, MA). The lower level of detection for hsCRP, IL-6, and TNF-α were 0.16 µg/mL, 0.16 pg/mL, and 0.32 pg/mL. All samples were analyzed blinded to treatment group. The assay coefficient of variance (CV) using these methods is 5% for hsCRP, 7% for IL-6, and 8% for TNF-α. In addition to these measures we also obtained a basic metabolic panel, aspartate aminotransferase, alinine aminotransferase, serum creatine kinase, and a complete blood count, and HIV clinical labs (HIV RNA level and CD4+ T-cell count) at each visit.

Statistical Methods

All analyses are based on the participants who had at least one follow-up visit. Descriptive statistics were calculated to compare self-reported side-effects and medication adherence and pill counts for each treatment (lisonopril versus L-placebo and pravastatin versus P-placebo); p-values for those comparisons are from Fisher’s exact tests. A missed follow-up visit was assigned the worse adherence category (>3 misses per week). Longitudinal mixed effects regression models which used all available follow-up data were used to assess treatment effects on change in BP, lipids, and inflammatory biomarkers averaged over both month 1 and 4 visits. Models included the baseline level of the outcome of interest, main effects for lisinopril and pravastatin, and the interaction between those treatments. None of the interaction terms were significant and therefore they were removed from the final models; only the results of main effects models are presented. Levels of hsCRP, IL-6 and TNF-α were loge–transformed before analysis; results are reported on the original scale after back-transformation. A global assessment measure described by O’Brien was also considered for the inflammatory biomarkers since we expected the treatments to have a similar effect on all of markers [18]. With this “rank-sum” method the values of hsCRP, IL-6 and TNF-α were ranked and then summed for each treatment group. Models as described above were used to compare the treatments for sum of the ranks. The comparisons of the inflammatory markers are considered exploratory. Two-sided p-values and 95% confidence intervals (CIs) are cited, with no adjustments for multiple comparisons. Analyses were performed using SAS version 9.2 (SAS Institute) and R version 2.9.

Results

Study Population

Forty-seven participants completed a screening visit and 37 of these were randomized (Figure 1). The reason for screening failure (n = 10) was a FRS <3%. Three persons who were randomized withdrew consent prior to starting study medication and had no follow-up data (2 allocated to lisinopril/P-placebo group and 1 allocated to L-placebo/P-placebo group). Of the remaining 34 participants in the final analysis cohort, one missed the month 1 visit (from L-placebo/P-placebo group) and one missed the month 4 visit (from Lisinopril/P-placebo group). Baseline characteristics for the study population are presented in Table 1. Median age was 48, only one female was randomized, the majority (68%) reported smoking cigarettes, median CD4 count was 482 cells/mm3, and most (65%) participants were taking protease-inhibitor-based ART. No participants had a prior history of injection drug use. Median FRS was 7.9%, and BP and cholesterol levels were below treatment thresholds for persons without prior CVD. There were no significant differences in baseline characteristics between lisinopril and L-placebo groups, pravastatin and P-placebo groups, or when compared across all 4 of the individual treatment combinations.

Table 1

Baseline Characteristics.

Tolerability and Adherence

Few participants reported side effects to study medication during the 4-month study period (Table 2). One participant each in the lisinopril and L-placebo groups reported cough. The participant receiving active lisinopril was unblinded and stopped the study medication. Additional specific side effects reported include nausea (n = 1) and runny nose (n = 1). Despite no difference in self-reported side effects, participants taking lisinopril vs. L-placebo reported lower rates of perfect adherence (i.e., no missed doses) at month 1 (59% vs. 100%; p = 0.007) and month 4 (13% vs. 65%; p = 0.007). Of the participants who returned study medication (n = 25), adherence of >90% of possible doses (assessed by pill count) was achieved by 58% of those randomized to lisinopril and 100% of those taking L-placebo (p = 0.01). Of participants who did not return study medications at month 4, one discarded his remaining supply and the others failed to return the bottles after repeated requests by the study coordinator. Side effects and adherence (assessed by self report or via pill count) did not differ between pravastatin and P-placebo groups (Table 2).

Table 2

Toxicity and Adherence During Follow-up.

Treatment Effect of Lisinopril on Blood Pressure

Compared to L-placebo, declines in systolic and diastolic BP were greater at both 1 and 4 months and averaged across both visits. For the latter, the treatment difference averaged across follow-up was −2.6 mmHg (95% CI −8.1, 2.8; p = 0.33) for systolic BP and −3.3 mmHg (95% CI −6.5, −0.1; p = 0.05) for diastolic BP (Table 3 and Figure 2).

Figure 2

Median (IQR) Levels of Blood Pressure and Biomarkers of Inflammation for Lisinopril versus L-placebo Treatment Groups.
Table 3

The Treatment Effect of Lisinopril (n = 17) Versus L-Placebo (n = 17) on Blood Pressure.

Treatment Effect of Pravastatin on Cholesterol

There were no significant differences in total cholesterol, LDL-C, triglycerides, HDL-C, or total-to-HDL-C ratio between pravastatin and P-placebo groups across follow-up visits (Table 4).

Table 4

The Treatment Effect of Pravastatin (n = 18) Versus P-Placebo (n = 16) on Blood Lipid Levels (n = 34).

Inflammatory Biomarkers

Baseline levels of hsCRP, IL-6 and TNF-α are reported (table 1), and there were no differences between treatment groups at study entry. At baseline, 13 (35%) participants had hsCRP levels categorized as low-risk for CVD (<1.0 mg/L), with the remaining either average- (n = 18, hsCRP 1.0–3.0 mg/L) or high- (n = 6, hsCRP >3.0 mg/L) CVD risk [19]. Figure 2 presents the median values during follow-up. The average change (decrease) from baseline during follow-up (estimated from longitudinal models) was greater for lisinopril compared to L-placebo for hsCRP (−0.54 µg/mL, 95% CI −0.90, −0.32; p = 0.02), IL-6 (−0.88 pg/mL, 95% CI −1.21, 0.64; p = 0.41), TNF-α (−0.84 pg/mL, 95% CI −0.99, −0.71; p = 0.04). These differences correspond to relative reductions for the lisinopril group of 42% for hsCRP and 23% for TNF-α. The treatment effect for hsCRP was not apparent until month 4 (−0.34 µg/mL; p = 0.02), whereas the decline in TNF-α was present by month 1 (−0.81 pg/mL; p = 0.02). Finally, the inflammatory score improved over follow-up for the group randomized to lisinopril vs. L-placebo (−2.81, 95% CI −5.68 to 0.05; p = 0.054).

There was no evidence of a pravastatin treatment effect on any of the inflammatory biomarkers at month 1, month 4, or averaged over both follow-up visits. There were also no differences in the lisinopril treatment effect on biomarker changes between those receiving or not receiving pravastatin (data not shown).

Discussion

This feasibility study of lisinopril and/or pravastatin as a CVD prevention strategy for HIV positive persons taking effective ART with viral suppression provides important information for future trials directed at BP and lipid changes among HIV positive patients who do not have an indication for these drugs. We found that adherence to lisinopril (at 10 mg daily) was less than for matched L-placebo, and as a consequence blood pressure lowering was modest. Improvements in blood lipids were not evident with pravastatin (at 20 mg daily) though this effect may have been limited by the relatively low potency at this dose as well as the small sample size. Importantly, among these virologically suppressed patients, we also found that lisinopril led to short-term improvements in biomarkers of systemic inflammation (hsCRP and TNF-α).

Epidemiologic data suggests persons with HIV infection have an approximate 2-fold increased risk for CVD, when compared to the general population [16], [20], [21]. Whether absolute CVD event rates will continue to differ and widen further over time is unclear, given some evidence that more aggressive management of traditional risk factors in contemporary HIV care has attenuated CVD risk [22]. Newer antiretroviral medications may also continue to reduce metabolic complications. However, even with optimal management of BP and cholesterol to levels below clinical treatment thresholds based on risk, factors unique to HIV disease still appear to result in excess CVD events [4], [23], [24]. Furthermore, by focusing on extreme elevations in individual risk factors there are missed opportunities to reduce CVD risk through modest simultaneous reductions in multiple risk factors. This, combined with the observation that CVD risk can be reduced among persons without clinically overt CVD by lowering BP within normotensive values or lowering LDL-C beyond 130 mg/dL [25], [26], [27], [28], motivates the strategy of combining low-doses of various CVD prevention medications into a singly daily pill as prevention (i.e., the polypill) [9], [10], [29]. Our data provide some support that such a study is feasible in individuals with HIV infection, but, consistent with data from general population [30], [31] issues of tolerability, adherence, and potency will need to be carefully considered. Future studies like this should consider a run-in period to assess adherence or use of better-tolerated medications with similar mechanisms (e.g., angiotensin receptor blockers).

The proportion of ART-treated HIV infected patients in clinical practice that currently have no indication for treatment with an ACE-I or a statin likely varies widely by setting. The reported prevalence for dyslipidemia (e.g., cholesterol >200–240 mg/dL, HDL <35 mg/dL, receipt of lipid-lowering therapy or clinical diagnosis) has ranged from 30–35% [16], [22], [23], [24]. For hypertension (BP ≥140/90 mmHg, receiving BP lowering therapy or a clinical diagnosis), it is between 10–30%, and for a prior history of CVD between 5–10% [16], [22], [23], [24]. When one also excludes persons at very low risk CVD risk (e.g., unlikely to benefit from aggressive prevention efforts), the target population for a pre-emptive CVD prevention strategy will likely include much less than half of patients in most HIV clinical settings. Furthermore, CVD prevention treatments will likely be most effectively implemented if they target patients receiving ART whose risk for AIDS complications is low. Despite these potential exclusions, the difference between a low-burden of CVD risk factors and optimally managed risk factors still has substantial implications for longer-term CVD risk over a lifetime [32]. Defining the appropriate target population that optimizes the net benefit-risk balance will be an important goal for future HIV-related CVD prevention studies.

Inflammation is a key factor in the pathogenesis of cardiovascular disease and a hallmark of HIV infection that persists despite effective treatment with ART for years [2], [5]. The reasons for chronic immune activation and inflammation are multi-factorial, but potential drivers include residual low-level HIV replication, translocation of microbial products across damaged mucosal barriers, the presence of co-pathogens (e.g., herpes viruses or hepatitis B or C), as well as metabolic complications (e.g., increased visceral adiposity) [33], [34], [35], [36]. In this context, anti-inflammatory treatments are particularly attractive candidates for HIV-related CVD prevention, whether or not they target HIV-specific mechanisms or down-regulate inflammatory pathways more broadly. ACE-I and statins have been associated with anti-inflammatory effects [13], [14], [15]. We found that among persons with HIV infection, lisinopril use was associated with a decline in biomarkers of systemic inflammation. Favorable changes were evident in spite of suboptimal adherence. High-sensitivity CRP, specifically, is elevated with HIV infection and associated with risk for CVD among both HIV-infected and uninfected persons [2], [3], [20].

Our findings were limited by the small sample size. Confidence intervals are wide and we may have missed important treatment effects. Low power also limited our ability to detect treatment interactions. The lack of a treatment effect of pravastatin may be due to the low potency of this statin, as we did not detect changes in cholesterol or lipoproteins. Given the approach we were studying (i.e., adding pravastatin as primary prevention to asymptomatic patients) we chose a starting dose (e.g., 20 mg) to minimize risk/tolerability and our short-term follow-up duration precluded dose escalation. Other HIV studies using higher doses (i.e., pravastatin 40 mg daily) or other statins (e.g., atorvastatin and rosuvastatin) have demonstrated reductions in measures of immune activation or inflammation [13], [37].

In summary, our results support the feasibility of conducting further studies of similar adjunct treatments that may have multiple beneficial effects such as reducing BP and systemic inflammation among HIV positive patients. Adherence concerns with lisinopril in this context suggest other, more tolerable medications with similar effects on the renin-angiotensin-aldosterone-system (e.g., angiotensin receptor blockers), may be a more effective strategy. Ultimately, in addition to larger feasibility studies, HIV clinical outcome trials will have to performed to assess the risk/benefit of such adjunctive treatment strategies.

Supporting Information

Checklist S1

CONSORT Checklist.

(DOC)

Protocol S1

Trial Protocol.

(PDF)

Acknowledgments

The authors would like to thank all study participants, and our research staff: Bette Bordenave, Edie Gunderson, Rachel Givot, Deb Woods, Mark Olson, Miki Olson, Chelsea Robinson, Jack Hall and Candace Nelson.

Funding Statement

Funding for study provided by the American Heart Association Clinical Research Program (grant# CRP2230357). More information on AHA funding can be found on their website: www.my.americanheart.org/professional/research/research_ucm_316889_subhomepage.jsp. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Bone Micro-CT Assessments in an Orchidectomised Rat Model Supplemented with Eurycoma longifolia

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Copyright © 2012 Rosmaliza Ramli et al.
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Abstract

Recent studies suggested that Eurycoma longifolia, a herbal plant, may have the potential to treat osteoporosis in elderly male. This study aimed to determine the effects of Eurycoma longifolia supplementation on the trabecular bone microarchitecture of orchidectomised rats (androgen-deficient osteoporosis model). Forty-eight-aged (10–12 months old) Sprague Dawley rats were divided into six groups of sham-operated (SHAM), orchidectomised control (ORX), orchidectomised + 7 mg/rat testosterone enanthate (TEN) and orchidectomised + Eurycoma longifolia 30 mg/kg (EL30), orchidectomised + Eurycoma longifolia 60 mg/kg (EL60), orchidectomised + Eurycoma longifolia 90 mg/kg (EL90). Rats were euthanized following six weeks of treatment. The left femora were used to measure the trabecular bone microarchitecture using micro-CT. Orchidectomy significantly decreased connectivity density, trabecular bone volume, and trabecular number compared to the SHAM group. Testosterone replacement reversed all the orchidectomy-induced changes in the micro-CT parameters. EL at 30 and 60 mg/kg rat worsened the trabecular bone connectivity density and trabecular separation parameters of orchidectomised rats. EL at 90 mg/kg rat preserved the bone volume. High dose of EL (90 mg/kg) may have potential in preserving the bone microarchitecture of orchidectomised rats, but lower doses may further worsen the osteoporotic changes.

1. Introduction

Eurycoma longifolia, known locally as “Tongkat Ali” in Malaysia is a native herb in the Southeast Asia region, especially in Malaysia, Indonesia, Cambodia, Laos, and Vietnam [1]. This small plant from the Simaroubaceae family can grow up to more than 15 meters and starts to bear fruits after 2-3 years of cultivation. Since decades ago, almost all parts of this evergreen plant including the fruits, root, and leaves have been sought after for its medicinal uses. However, the most valuable component is the root which has been more commonly used as an aphrodisiac and also to treat a wide range of diseases, including aches, fever, malaria, sexual insufficiency and glandular swelling [24]. The root extract of Eurycoma longifolia has been used as herbal ingredients to enhance blood flow and restore vitality and energy following childbirth [5]. Due to these therapeutic values, Eurycoma longifolia has been commercialized in various forms of health supplement [6], and added to beverages such as coffee and tea. Since decades ago, various researches have been conducted to study and explore the active components which are responsible for these claimed benefits. These components are the quassinoids for example eurycomanone [3, 4, 7], canthine-6-one alkaloids [4, 812], squalene derivatives [13], and byphenylneolignans [14]. Considering the fact that this herbal extract increased the level of testosterone in sexually insufficient individuals, there was an effort to explore the role of Eurycoma longifolia in treatment of androgen-deficiency-related diseases. An earlier study observed the potential of Eurycoma longifolia for the treatment of androgen-deficient osteoporosis [15].

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and increased fracture risk [16]. Male osteoporosis which was not recognized earlier has now become an important public health issue. Although hip fractures involve less than one-third of males, the mortality rate has been reported to be 31% and 17% in males and females, respectively [17]. Osteoporotic fracture in males is associated with significant morbidity and mortality, which have huge emotional and financial impacts on their families and the society [18].

The treatment of choice for men with androgen-deficient osteoporosis is testosterone-replacement therapy. This therapy is recommended based on the increasing understanding on the role of androgens in bone physiology and remodeling, which is made evident by numerous animal and human studies [19]. However, testosterone replacement therapy is contraindicated in patients with prostate or breast cancer [20]. Some physicians even perceived that testosterone replacement therapy is associated with increased risk of prostate cancer. A study reported that almost 35% of hypogonadal men did not receive treatment due to fear of developing prostate cancer [21]. Another contraindication which is worth mentioning is sleep apnoea as hypogonadal patients may have worsening of sleep apnoea with testosterone replacement [20]. Some may also relate testosterone replacement therapy with cardiovascular disease, liver damage, and erythrocytosis [22]. Thus, it is necessary and essential to find an alternative treatment which not only gives protection to the bone but at the same time possesses fewer side effects compared to the testosterone therapy.

Over the past few decades, Eurycoma longifolia has been recognized internationally especially as an aphrodisiac and is being used widely to enhance virility and sexual performance in male [23, 24]. Many studies reported that this ability of Eurycoma longifolia is associated with the increased level of testosterone in subjects supplemented with Eurycoma longifolia. Recent human trials showed that Eurycoma longifolia supplementation increased the level of testosterone, together with the increase in the superoxide dismutase (SOD), an antioxidant which plays an important role in slowing down the process of aging [2528]. Despite the documented history of Eurycoma longifolia as a testosterone-raising herb, the underlying mechanisms for its androgenic effects remain unclear. Shawn and Kraemer (2007) in their study suggested that Eurycoma longifolia increased testosterone levels by way of promoting the dissociation of testosterone from sex-hormone-binding globulin (SHBG) [29]. This SHBG is known to increase in aging men and causes a decrease in the bioavailability of the active fractions of testosterone. One of the bioactive components of Eurycoma longifolia, eurypeptide may enhance the biosynthesis of different androgens, by activating the CYP17 (17 α-hydroxylase) enzyme, thereby boosting further the metabolism of pregninolone and 17-OH-progesterone to dehydroepiandrosterone (DHEA) [30]. It also enhances the metabolism of progesterone and 17-OH-progesterone to 4-androstenedione and testosterone [30].

The World Health Organization (WHO) defines osteoporosis as bone mineral density (BMD) value of 2.5 standard deviations or more below the young adult women mean value [31]. BMD measurement by dual energy X-ray absorptiometry (DEXA) is currently the most common technique for assessing the risk of osteoporosis. However, this BMD measurement may not reflect the actual bone strength and the risk of fragility fracture. In a prospective study involving 699 subjects, it was observed that there was an overlapping of BMD measurements between subjects with and without fractures [32]. In the same study, they found that the relationship between bone mass and fracture risk was not linear. The trabecular bone, which is predominantly affected in osteoporotic changes, is a continuous three-dimensional network of bars and plate, with varying densities and orientations. Several studies have suggested that connectivity density of the trabecular bone, rather than bone density, may be the parameter mostly affected by osteoporotic changes [3335]. Connectivity density, with regard to bones microarchitecture, is defined as the maximal number of branches that can be broken before the microarchitecture is separated into parts [36]. Apart from trabecular bone density, histomorphometry analysis of the bone microarchitecture also contributes to the prediction of fracture risk [33, 37]. However, this conventional and static two-dimensional histomorphometry gives only limited information on bone microarchitecture [38]. A three-dimensional, nondestructive method of bone microarchitecture measurement pioneered by Feldkamp et al. in 1989, has become feasible only in recent years [39]. This microcomputed tomography (micro-CT) imaging system gives a detailed picture of three-dimensional bone architecture together with bone connectivity density. This high-resolution imaging technique has become more popular and largely applied in the field of bone research, both in basic as well as preclinical. Thus in this study, we aimed to observe the effects of Eurycoma longifolia supplementation on the trabecular bone microarchitecture in orchidectomised rats using micro-CT.

2. Materials and Methods

2.1. Experimental Animals and Treatment

Forty-eight Sprague Dawley rats aged 10–12 months old (300–400 mg) were divided into six groups of sham-operated (SHAM), orchidectomised control (ORX), orchidectomised + testosterone enanthate 7 mg/rat (TEN), orchidectomised + Eurycoma longifolia 30 mg/kg (EL30), orchidectomised + Eurycoma longifolia 60 mg/kg (EL60) and orchidectomised + Eurycoma longifolia 90 mg/kg (EL90). Eurycoma longifolia standardized aqueous extract (EL) was given 6 days a week via oral gavages, while testosterone was injected intramuscularly weekly throughout the six weeks duration of the study. The rats were housed singly in plastic cages at room temperature with a 12-hour light-dark cycle. They were fed with commercial rat chow (Gold Coin, Selangor, Malaysia) and tap water ad libitum. Ethical approval was obtained from the Universiti Kebangsaan Malaysia Animal Ethics Committee (UKMAEC) (PP/FAR/2011/NAZRUN/22-MARCH/362-JUNE-2011-MAY-2012).

2.2. Materials and Bone Preparation

Eurycoma longifolia standardized aqueous extract (EL) was obtained from Phytes Biotek Sdn, Bhd. (Shah Alam, Malaysia). The extract was in brownish powder form and the bioactive components were eurypeptide (22.0%), glycosaponin (41.1%) and eurycomanone (1.6%). EL aqueous powder was dissolved in deionized water and given via oral gavages at doses of 30, 60, and 90 mg/kg rat weight at 9 am six days a week for six weeks [40]. Testosterone enanthate (Jesalis Pharma, Germany) was diluted in peanut oil and 7 mg/rat was administered via intramuscular injection once a week throughout the study period [41]. At the end of the treatment, the rats were euthanized by overdose of diethyl ether. Femora were dissected and cleaned from all soft tissues. They were then stored in 10% formalin solution until analyzed.

2.3. Micro-CT Analysis

The effect of EL supplementation on trabecular bone was assessed using micro-CT (μCT80 scanner, Scanco Medical, Switzerland). Before scanning, measurement protocols to define parameters such as source energy and image resolution were created. The left femur was placed in a sample holder in a vertical direction with the epiphyseal head facing downward. The source energy selected for this study were 70 KVp and 114 μA with image resolution set as “high” to obtain the best contrast between bone and soft tissues. The trabecular bone parameters were obtained from the distal end of the left femur. Scanning was done at the metaphyseal area located 1.5 mm below the lowest point of the epiphyseal growth plate and extending 2.0 mm in the proximal direction. This is the secondary spongiosa area, which is rich in high-turnover trabecular bone. Trabecular bone was chosen because its remodelling process is more dynamic than the cortical bone [42].

2.4. Statistical Analysis

The results were expressed as mean ± standard error of the mean (SEM). The data analysis was performed using the Statistical Package for Social Sciences software (SPSS 19.0; SPSS, Chicago, IL, USA). The data were tested for normality using the Kolmogorov-Smirnov test. For normally distributed data, the statistical tests used were the analysis of variance (ANOVA), followed by Tukey’s Honestly Significant Difference (HSD) test. For data that were not normally distributed, Kruskal-Wallis and Mann-Whitney tests were used.

3. Results

There were no significant differences in body weight between the different groups at the beginning of the study. The body weight increased steadily throughout the period of the study. At the end of the study, there was a significant decrease in the body weight of the ORX group compared to the SHAM group (P < 0.01). The body weights of all the other groups showed no significant difference compared to each other (Figure 1).

Figure 1

Mean body weight throughout the study. SHAM, sham-operated; ORX, orchidectomised-control; TEN, orchidectomised + 7 mg/rat testosterone enanthate; EL30, orchidectomised + Eurycoma longifolia 30 mg/kg; EL60, orchidectomised + Eurycoma longifolia

In the present study, orchidectomy significantly decreased (−37.56%, P < 0.05) the trabecular bone connectivity density compared to the SHAM group (Figure 2). In contrast, the group supplemented with testosterone enanthate (TEN) showed an increase (+57.30%, P < 0.05) in trabecular bone connectivity density compared to the ORX group. Generally, the groups supplemented with EL showed a dose-dependent increase in the trabecular bone connectivity density. However, the bone connectivity density of these groups were significantly lower (P < 0.05) than the SHAM, ORX, and TEN groups. Only the group with the highest dose of EL (EL90) showed similar trabecular bone connectivity density compared to that of the ORX group.

Figure 2

Trabecular bone connectivity density for all the groups after 6 weeks of treatment. SHAM, sham-operated; ORX, orchidectomised-control; TEN, orchidectomised + 7 mg/rat testosterone enanthate; EL30, orchidectomised + Eurycoma longifolia 30 mg/kg;

Figure 3 depicts the three-dimensional image of the distal femur metaphysis. The bone tissue chosen as the region of interest (ROI) shown in white has been reconstructed in three dimension to see the structural context of trabecular bone. The loss of trabecular bone connectivity density is apparent in the groups treated with EL (EL30, EL60 and EL90) compared to the SHAM and TEN groups, with associated changes in other indices of trabecular bone microarchitecture.

Figure 3

Three dimensional micro-CT images of the trabecular microstructure of distal femur metaphysis. (a) sham-operated; (b) orchidectomised-control; (c) orchidectomised + 7 mg/rat testosterone enanthate; (d) orchidectomised + Eurycoma longifolia 30 mg/kg;

Bone volume was significantly decreased in the ORX, EL30 and EL60 groups (−24.97, −40.72, and −33.11%, resp., P < 0.05 for all) compared to the SHAM group (Figure 4). EL supplementation at 90 mg/kg was effective in preserving bone volume as it showed no significant difference compared to the SHAM group.

Figure 4

Bone volume for all the groups after 6 weeks of treatment. SHAM, sham-operated; ORX, orchidectomised control; TEN, orchidectomised + 7 mg/rat testosterone enanthate; EL30, orchidectomised + Eurycoma longifolia 30 mg/kg; EL60, orchidectomised

There was a significant decrease in the trabecular number in the ORX and all EL treated groups (−29.77, −47.23, −47.79, and −39.91%, resp., P < 0.05 for all) compared to the SHAM group (Figure 5). Similarly, all EL treated groups showed significantly higher trabecular separation (+103.36, +92.96 and +87.36%, resp., P < 0.05 for all) compared to the SHAM group (Figure 6).

Figure 5

Trabecular number for all the groups after 6 weeks of treatment. SHAM, sham-operated; ORX, orchidectomisedcontrol; TEN, orchidectomised + 7 mg/rat testosterone enanthate; EL30, orchidectomised + Eurycoma longifolia 30 mg/kg; EL60, orchidectomised
Figure 6

Trabecular separation for all the groups after 6 weeks of treatment. SHAM, sham-operated; ORX, orchidectomised control; TEN, orchidectomised + 7 mg/rat testosterone enanthate; EL30, orchidectomised + Eurycoma longifolia 30 mg/kg; EL60,

As for the trabecular thickness parameter, there were no significant differences between the groups (Figure 7).

Figure 7

Trabecular thickness for all the groups after 6 weeks of treatment. SHAM, sham-operated; ORX, orchidectomised control; TEN, orchidectomised + 7 mg/rat testosterone enanthate; EL30, orchidectomised + Eurycoma longifolia 30 mg/kg; EL60,

4. Discussion

In males, androgen deficiency caused severe loss of bone [43], muscle, and fat mass [44, 45]. These may have contributed to the low body weight of rats in the orchidectomised control group. The body weights of rats supplemented with EL or receiving testosterone were preserved from the weight loss effects of orchidectomy. This may be due to the anabolic effects of testosterone and EL on the bone and body compositions. Androgen replacement therapy on both orchidectomised rats and hypogonadal men were found to reverse the effects of androgen deficiency on muscle mass [4547]. The androgenic effect of EL used in this study was supported by a five-week human study, which showed that supplementation of water soluble extract of Eurycoma longifolia increased muscle mass and strength [48].

In the present study, the trabecular bone microarchitecture was assessed using the state of the art micro-CT. It is capable of three-dimensional bone analysis which produces high quality images, thus well accepted by the scientific communities. A comparison study revealed that all radiographic parameters (bone thickness and length) were significantly correlated to the corresponding micro-CT measurements [49]. Another study comparing three-dimensional micro-CT and two-dimensional histomorphometry, found good correlations between these two methods [50]. The same study also showed an excellent correlation in the connectivity density estimation using ConnEuler principle and micro-CT.

As expected, the present study showed that androgen withdrawal induced by orchidectomy had caused significant deteriorations of the bone microarchitecture of the rats at six weeks post-orchidectomy. These findings were supported by Yao et al. (2005) who reported that orchidectomy caused reduction in trabecular bone connectivity density but caused no change in trabecular thickness [51]. Meanwhile, studies by Yarrow et al. (2008) and Libouban et al. (2008) showed that orchidectomy caused deterioration in all trabecular bone microarchitectures which include trabecular bone number, thickness and separation [41, 52]. Testosterone replacement was able to reverse all the orchidectomy-induced changes. This was in agreement with Yarrow et al. (2008) which showed that testosterone administration to orchidectomised rats significantly improved the trabecular number, width and separation [41].

In a previous study, EL supplementation at 15 mg/kg was able to prevent bone calcium loss in orchidectomised rats, although not as effective as testosterone treatment [15]. In the present study, higher EL doses of 30, 60, and 90 mg/kg were used to determine whether EL supplementations would affect bone microarchitecture in a dose-dependent manner.

Surprisingly, data from this study showed that EL at doses of 30 and 60 mg/kg failed to protect bone from orchidectomy-induced changes. Supplementation of 90 mg/kg EL showed some protection on the bone volume. Meanwhile, EL doses of 30 and 60 mg/kg worsened all the bone microarchitecture indices such as the bone connectivity density, volume, number, and separation. There is paucity of literature on the effects of Eurycoma longifolia on osteoporosis. However, it can be postulated that EL may directly damage bone cells or they may be involved in the regulation of osteoblasts and osteoclasts activities, resulting in increased bone resorption. The standardized water extract of EL used in this study contains eurycomanone, which was found to be toxic to human cells [10]. However, in vitro toxicity studies often used methanolic root extracts of Eurycoma longifolia [10, 53], which is more potent, and thus may explain its toxic effects on human cells. Apart from eurycomanone, there are other Eurycoma longifolia constituents, which were shown to possess cytotoxic activities such as β carboline alkaloid, which was active against human lung and breast-cancer cells and canthine-6-1 alkaloid, which was active against multiple human cancerous cells [1, 10, 54]. The toxic effects of these EL constituents may have caused the deterioration of the bone microarchitecture found in this study.

Bone is composed of supporting cells (osteoblast and osteocytes), remodeling cells (osteoclasts) osteoid and inorganic mineral salts (hydroxyapatite). Osteoblasts synthesize new collagenous matrix and regulate its mineralization by concentrating calcium and phosphate, and destroying mineralization inhibitors such as pyrophosphate and proteoglycan [55, 56]. Approximately 30 to 50% of osteoblasts become osteocytes or bone-lining cells, while the majority of them undergo apoptosis [57]. The bone-lining cells differentiate into osteoblasts upon exposure to parathyroid hormone or mechanical forces [58]. The formation, activation, and resorption of osteoclasts, the bone-resorbing cells, are regulated by the ratio of receptor activator of NF-κβ ligand (RANKL) to osteoprotegerin (OPG), Interleukin-1 (IL-1) and Interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), parathyroid hormone, 1,25-dihydroxyvitamin D, and calcitonin [59, 60]. Both RANKL and M-SCF are produced by osteoblasts and marrow stromal cells, and the presence of these two cells is very important in osteoclastogenesis [61, 62]. Bone remodeling is the combination of bone resorption and formation by osteoclasts and osteoblasts, respectively, to maintain bone mass and bone strength, which can be assessed by measuring the bone microarchitecture indices. Thus, any interruption to these cytokines or bone cells regulating the activities of bone resorption and bone formation, will affect these measurements. The present study showed that EL30 and EL60 groups have suffered deterioration in the bone microarchitectures. Studies have shown that estrogen withdrawal following ovariectomy caused deterioration of trabecular bone microarchitecture which was related to perforation of trabecular plates and loss of connectivity density [6365]. In the present study, this loss of connectivity density was reversible only with testosterone replacement but not with EL30 and EL60 supplementations. A study by Yao et al. (2005) on the long bone (tibia) reported that ovariectomy caused reduction in total trabecular bone volume, connectivity, and trabecular number while trabecular thickness did not change significantly [51]. In the current study, testosterone depletion following orchidectomy showed similar results. Yao et al. (2005) also reported that connectivity density of the tibia showed contradictory findings compared to the vertebra, partly due to the slower bone loss rate in the vertebra than in the tibia: there was only 20% loss of trabecular bone in the vertebra four months postovariectomy versus more than 80% in the tibia [51]. In the vertebra, estrogen depletion-induced fenestration of plates, which increased the connectivity density, and treatments with anabolic or antiresorptive agents filled in the fenestration, resulting in a lower connectivity density of the bone [51].

There were no significant changes in the trabecular thickness for all the groups. Physiologically, with the loss of bone connectivity density, reduction in trabecular number and widening of the trabecular separation, there would be a compensatory trabecular thickening. This finding is in agreement with Gasser et al. (2005) who found that zoledronic acid (antiresorptive agent) increased trabecular thickness without improving the bone connectivity density [66]. Other studies have also reported compensatory increase in the remaining trabecular following the loss of bone mass during adulthood [6770]. This compensatory mechanism may account for the nonsignificant changes in the trabecular thickness for the orchidectomised rats seen in the present study.

Supplementation of EL at 90 mg/kg showed preservation in the trabecular volume although it was less effective than testosterone. As mentioned earlier, EL may have indirectly improved bone volume by increasing the testosterone level. Although there is paucity in literature on the effects of EL on bone microarchitecture, there are many studies on the relationship between testosterone and bone metabolism and structure. A study on hypogonadal men given testosterone treatment showed significant increases in trabecular bone volume fraction and thickness after 24 months of treatment [71]. Meanwhile, another study on supraphysiological administration of testosterone enanthate also showed improvements on the trabecular number and width, and reduction in trabecular separation [41].

Although the exact mechanisms are still unclear, it was suggested that Eurycoma longifolia increased free testosterone level by enhancing the dissociation of testosterone from SHBG [29]. Eurycoma longifolia has also been shown to enhance the metabolism of different types of androgens, thus increasing the levels of more biologically active androgens, testosterone, and DHEA [30]. The latter mechanism may be supported by recent human trials on Eurycoma longifolia supplementation, which showed that water extract of Eurycoma longifolia increased the total testosterone and DHEA levels [2528, 72]. In animal studies involving rats and mice, both the water and ethanolic extracts of Eurycoma longifolia were found to increase the sexual motivation, performance, and frequency of sexual activity [2, 23]. In one of the studies, it was shown that at 800 mg/kg, Eurycoma longifolia promoted the growth of the reproductive organs (seminal vesicles and ventral prostate) in male rats [23]. These effects resembled those induced by testosterone administration [73]. Testosterone may exert its effects directly via activation of the androgen receptor (AR) found on bone skeletal surfaces or indirectly via aromatization of androgens (testosterone) to estrogen [19]. Thus, the increase in androgens may play an important role in maintaining the bone volume of the rats supplemented with 90 mg/kg dose of EL.

5. Conclusion

In conclusion, EL supplementations at the dose of 30 and 60 mg/kg deteriorated the bone microarchitecture of orchidectomised rats. At higher dose of 90 mg/kg, EL supplementation was only able to preserve bone volume. Further studies are required to determine if low dose of EL is toxic to bone cells, and if higher dose than 90 mg/kg would provide better protection to bone microarchitecture.

Acknowledgment

The authors would like to thank Mrs Sinar Suriya Muhammad, Mrs Farhana Mohd Fozi, Ms Juliana Abdul Hamid, Ms Nurul Hafizah Abas, and Mr Fadhlullah Zuhair from the Department of Pharmacology for their technical assistance. The authors would also like to thank Mrs Zarina Jaafar from School of Dental Sciences, USM for her help during the micro-CT analysis. This study was made possible through the grant UKM-FF-03-FRGS0047-2009 provided by the Faculty of Medicine UKM.

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