The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility In mouse , genes encoding complement regulators CD55 and CD59 have been duplicated . The first described form of CD59 , CD59a , is broadly distributed in mouse tissues , while the later identified CD59b was originally described as testis specific . Subsequent studies have been contradictory , some reporting widespread and abundant expression of CD59b . Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice . Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels . These data confirm that expression of CD59b is essentially restricted to adult testis ; trace expression in other tissues is a consequence of contamination with blood cells , shown previously to express CD59b at low level . In testis , onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome . Ligation of CD59b , but not CD59a , markedly reduced spermatozoal motility , suggesting a specific role in reproductive function . 1 Introduction CD59 , a broadly distributed glycosyl phosphatidylinositol -LRB- GPI -RRB- - anchored protein , is the principal regulator of complement membrane attack complex -LRB- MAC -RRB- assembly on cell membranes -LRB- Meri et al. , 1990 -RRB- . A decade ago , we identified the mouse analogue of CD59 and showed that it was broadly distributed on cells and tissues -LRB- Powell et al. , 1997 -RRB- . Deletion of the gene encoding mouse CD59 caused minimal disturbance in unchallenged animals but markedly enhanced susceptibility to complement-driven pathologies -LRB- Holt et al. , 2001 ; Turnberg et al. , 2003 , 2004 ; Lin et al. , 2004 ; Mead et al. , 2004 ; Williams et al. , 2004 -RRB- . Others demonstrated that the gene encoding CD59 is duplicated in the mouse ; the protein products of these two genes were then termed CD59a for the first described , and CD59b for the product of the new gene -LRB- Qian et al. , 2000 -RRB- . In this first report , it was stated that CD59b message was significantly expressed only in testis . We developed monoclonal antibodies specific for CD59a and CD59b , enabling us to confirm the broad distribution of CD59a and testis restricted expression of CD59b at the protein level ; we concluded that CD59a was the principle regulator of MAC assembly in mouse tissues -LRB- Harris et al. , 2003 -RRB- . This conclusion was challenged in a re-analysis of the pattern of distribution of mRNA encoding the two forms of CD59 ; these studies suggested that both CD59a and CD59b were broadly expressed in the mouse and contended that the latter was the major regulator of MAC in the mouse -LRB- Qin et al. , 2003 -RRB- . These contentions were bolstered by the demonstration that deleting the gene encoding CD59b in mice caused a severe , spontaneous phenotype with anaemia , platelet dysfunction and fertility problems . In light of these data we reassessed the distribution patterns at protein and mRNA levels using a combination of specific monoclonal antibodies and shared primers for PCR in high sensitivity assays -LRB- Baalasubramanian et al. , 2004 -RRB- . These analyses confirmed our earlier work showing that CD59a was broadly expressed while significant expression of CD59b was restricted to testis . Trace amounts of CD59b message and protein were detected in erythrocytes but quantitative assays of protein expression showed that CD59b expression on erythrocytes was less that 5 % that of CD59a . Further , we confirmed that CD59a was dominant in protection of erythrocytes from MAC lysis . The findings of these painstaking studies have recently been challenged yet again -LRB- Qin et al. , 2006 -RRB- . The appropriateness of the techniques used and specificity of probes and primers have all been questioned . These authors concluded that CD59b was broadly expressed and an important regulator of MAC assembly on erythrocytes and in tissues . We , and others , are using CD59a knockout mice in disease models to explore roles of MAC based on our evidence that CD59a is the principal regulator of the MAC in most tissues . If CD59b is indeed widely distributed then the value of studies in CD59a knockouts is in question . It is therefore essential that we test the evidence . Here using multiple methods we revisit these published studies and undertake new analyses to explore the expression patterns of CD59a and CD59b at the mRNA and protein level . We conclude that expression of CD59b at the mRNA and protein level is essentially absent in all tissues other than testis . Low level expression on blood cells was confirmed and trace detection of mRNA in tissues was shown to be likely due to blood contamination . We further analysed expression of CD59b in testis and showed that expression coincided with onset of puberty and was restricted to spermatozoa and their immediate precursors . Ligation of CD59b on spermatozoa with monoclonal antibody markedly inhibited sperm motility , suggesting a specific role in reproductive function . 2 Materials and methods 2.1 Mice Adult -LRB- 8 -- 16 weeks -RRB- and infant -LRB- 1 -- 5 weeks ; pre-puberty -RRB- male mice , and embryos -LRB- day 5 , 10 and 15 -RRB- from C57BL/6 -LRB- H-2b -RRB- background mice were used in our investigation . All experimental procedures were performed in compliance with Home Office and local ethics committee regulations . cd59a − / − mice generated as described previously -LRB- Holt et al. , 2001 -RRB- , and back-crossed 10 generation onto the same background were used as controls . 2.2 Antibodies and reagents Rat anti-mouse CD59a -LRB- CD59a .1 ; IgG1 -RRB- was generated in house -LRB- Harris et al. , 2003 -RRB- . Mouse anti-mouse CD59b -LRB- CD59b .2 ; IgG1 -RRB- was also made and characterized in house -LRB- Baalasubramanian et al. , 2004 -RRB- . Separate aliquots of CD59a .1 and CD59b .2 were labelled with NHS-biotin -LRB- Sigma -- Aldrich , Gillingham , Dorset , UK -RRB- and FITC-NHS -LRB- Perbio Science UK Ltd. , Cramlington , UK -RRB- according to manufacturer 's protocols . As negative controls , rat IgG1 and mouse IgG1 -LRB- purified in-house -RRB- were labelled in the same manner . FITC-labelled streptavidin was purchased from DAKO -LRB- Ely , Cambridgeshire , UK -RRB- . HRPO-labeled donkey anti-rat IgG and HRPO-labeled donkey anti-mouse IgG were purchased from Jackson ImmunoResearch Europe -LRB- Newmarket , Suffolk , UK -RRB- . 2.3 Semi-quantitative RT-PCR Total RNA was purified from all investigated mouse tissues using GenElute kit -LRB- Sigma -- Aldrich -RRB- and controlled for DNA contamination by RT-PCR without using reverse transcriptase and employing a β-actin specific primer pair -LRB- Table 1 -RRB- . Only samples that did not show amplification were used in all analyses . Aliquots of these RNAs -LRB- 1 μg each -RRB- were reverse transcribed using random hexamers and multiscribe reverse transcriptase according to the manufacturer 's instructions -LRB- Applied Biosystems , Warrington , UK -RRB- . Primer pairs specific for either CD59a or CD59b were as described by Qin et al. -LRB- 2006 -RRB- , and a new primer pair common for both CD59a and b and amplifying a sequence in exon 4 comprising 199 bp for CD59a and 162 bp for CD59b , were used in the subsequent amplifying reactions for detection of CD59a and CD59b mRNAs -LRB- Table 1 -RRB- . Either 25 or 35 amplification cycles were used , except where attempting simultaneous detection of both mRNAs when 40 cycles were performed . Amplified products were separated either in 1 % agarose or in 5 % poly-acrylamide gels -LRB- PAAG -RRB- . To obtain semi-quantitative data for expression of the two genes , we used as templates either 2 μl of the original cDNA , or a series of dilutions as indicated in figures . Gels were scanned and band intensity was measured by densitometry using Quantity One 4.3.0 software -LRB- BioRad , Hemel Hempstead , Hertfordshire , UK -RRB- . The quantifications were carried out in triplicate and means and errors calculated . 2.4 Quantitative real-time PCR analysis The cDNAs from testis and liver prepared as described for the semi-quantitative RT-PCR , were subject to a Taqman assay as described previously -LRB- Baalasubramanian et al. , 2004 -RRB- . One of the primer pairs was as described previously -LRB- Baalasubramanian et al. , 2004 -RRB- , a perfect match for the sequence of CD59a mRNA and with a single internal nucleotide mismatch to CD59b mRNA ; the second set was a perfect match for the same sequence in CD59b and with a single internal nucleotide mismatch to CD59a -LRB- Table 1 -RRB- . To measure the relative number of mRNA copies for CD59b in liver , perfused liver and testis we used the primer pair reported by Qin et al. -LRB- 2006 -RRB- to specifically amplify CD59b in a quantitative PCR -LRB- QPCR -RRB- . Because these primers were not designed specifically for this assay , they did not meet the optimal annealing temperature requirements . To correct for this anomaly , the standard amplification programme was modified to include a pre-annealing step at 56 °C for 20 s prior to the extension phase . Primers were designed to β-actin -LRB- Table 1 -RRB- as an internal control for normalization of starting cDNA levels . Quantitative PCR was performed on ABI PRISM 7000 using either TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix according to the manufacturer 's instructions -LRB- Applied Biosystems -RRB- with 50 cycles of amplification . 2.5 Preparation of tissue lysates To obtain tissue lysates , freshly harvested mouse organs were immediately chilled and homogenized with ice-cold lysis buffer -LRB- PBS containing 2 % NP40 , 1 mM phenylmethylsulfonyl fluoride , 10 mM EDTA , 1 μg/ml leupeptin , and 1 μg/ml pepstatin ; 1 g of tissue/1 .5 ml of buffer -RRB- and incubated for 60 min on ice . Insoluble debris was removed by centrifugation -LRB- 5000 × g , 15 min at 4 °C -RRB- and the supernatants stored in aliquots at − 80 °C until use . In some experiments the mouse was perfused with saline at the time of sacrifice to reduce blood contamination in harvested organs . 2.6 SDS-PAGE and Western blot analysis Lysates were mixed 1:1 with sample buffer for SDS-PAGE and separated under non-reducing conditions in 15 % gels . Separated proteins were transferred onto nitrocellulose membranes -LRB- Schleicher & Schuell , London , UK -RRB- , and membranes blocked with 5 % -LRB- w/v -RRB- non-fat milk in PBS -LRB- PBS-M -RRB- . Membranes were then probed with the primary mAb diluted in PBS-M , washed in PBS containing 0.1 % Tween-20 -LRB- PBS-T -RRB- , then probed with HRPO-conjugated donkey anti-rat Ig in PBS-M to detect the rat anti-CD59a or HRPO-conjugated donkey anti-mouse Ig -LRB- Jackson -RRB- to detect the mouse anti-CD59b . After further washing in PBS-T , bands were developed using ECL -LRB- Perbio Science UK Ltd. -RRB- and captured on autoradiographic film -LRB- Kodak Ltd. , Hemel Hempstead , Hertfordshire , UK -RRB- . 2.7 Spermatozoa preparation and analysis Motile cauda epididymal spermatozoa were obtained by a ` swim-up ' technique as previously described -LRB- Mizuno et al. , 2004 , 2005 -RRB- . Briefly , two cauda epididymi from an adult C57BL/6 mouse were roughly minced in 1 ml of DMEM -LRB- Invitrogen , Paisley , UK -RRB- . This suspension was carefully overlayered with 1 ml DMEM and incubated at room temperature for 15 min . The upper layer was removed and spermatozoa pelleted by centrifugation -LRB- 1000 × g -RRB- for 5 min at room temperature . Cells were washed twice by gentle centrifugation as above . For immunofluorescence studies , the re-suspended cells were smeared onto glass slides , air-dried immediately , fixed in acetone at room temperature for 1 min and stored at − 20 °C until use . To prepare lysate , the swim-up spermatozoa from 4 cauda epididymi were pelleted and incubated with mixing in 100 μl of lysis buffer for 30 min on ice . Insoluble debris was removed by centrifugation -LRB- 15,000 × g , 15 min at 4 °C -RRB- and the supernatant stored in aliquots at − 80 °C . To compare CD59b expression in unactivated and acrosome-reacted spermatozoa , the acrosome reaction was induced essentially as described -LRB- Mizuno et al. , 2004 -RRB- . Briefly , swim-up spermatozoa -LRB- 106 cells/ml of DMEM -RRB- were incubated for 1 h at 37 °C with the calcium ionophore A23187 -LRB- Sigma -- Aldrich -RRB- at 1 μM to induce the acrosome reaction . Control cells were incubated without ionophore . Acrosome-reacted and control spermatozoa were smeared on glass slides and immediately air-dried . To observe CD59b distribution , the smears were incubated with FITC-labeled CD59b .2 . Nuclei were counterstained with DAPI -LRB- 4 ′ -6-diamino-2-phenylindole-2 HCl ; 100 ng/ml final concentration ; Sigma -- Aldrich -RRB- . On each slide , at least 100 cells were counted and the assay was carried out in triplicate . 2.8 Functional inhibition assay of CD59a and CD59b in mouse sperm Spermatozoa harvested by swim-up from two epididymi , either from wild type or cd59a − / − mice , were washed and suspended in 2 ml DMEM . Paired aliquots -LRB- 200 μl -RRB- were incubated at 37 °C with mAbs CD59a .1 , CD59b .2 , or isotype-matched control mAb , each at 10 μg/ml . After 4 h of incubation , the sample was immediately placed on a glass haemocytometer slide and the total number of sperm and percentage remaining motile were counted under light microscopy . This experiment was performed in triplicate . 2.9 Statistical analysis All values are expressed as mean ± standard error -LRB- S.E.M. -RRB- . The statistical analysis was performed by one-way ANOVA . When significant differences were observed , statistical analysis was further carried out using unpaired t-test between two groups . Significance between two groups was claimed when P < 0.05 . 3 Results 3.1 CD59b mRNA is highly expressed only in testis We have previously demonstrated that CD59a is the primary regulator of MAC assembly in mouse -LRB- Baalasubramanian et al. , 2004 -RRB- . However , in a recent work -LRB- Qin et al. , 2006 -RRB- , abundant expression of CD59b mRNA in a number of tissues was reported . In an attempt to clarify this issue , which is of key importance for complement studies in mice , we repeated some of the experiments carried out by Qin and co-authors using the primer pairs designed by them . Under routine conditions -LRB- 25 cycles of amplification -RRB- , CD59a mRNA was detected in all tissues tested while CD59b mRNA was found only in testis -LRB- Fig. 1A -RRB- . Increasing the number of amplification cycles to 35 -LRB- Fig. 1B -RRB- revealed weak expression of mRNA for CD59b in liver , blood cells , heart and lung . Amplified sequence was confirmed to be CD59b by sequencing . We next re-visited the primers used in our previous quantitative PCR analysis to assess expression of the two CD59 isoforms in different mouse tissues -LRB- Baalasubramanian et al. , 2004 -RRB- . We had designed a primer pair that annealed to both isoforms and Taqman probes specific for either CD59a or CD59b to enable accurate quantitation . Qin et al. -LRB- 2006 -RRB- criticised these experiments on the basis that , while both primers used in our assay matched perfectly CD59a , each had a single internal nucleotide mismatch compared to the CD59b sequence -LRB- Table 1 -RRB- . They suggested , without evidence , that this mismatch favoured amplification of CD59a and caused our inability to detect CD59b in tissues other than testis and bone marrow . There is a large primer design literature that shows clearly that internal mismatches , unlike those at the 3 ′ end , do not significantly influence amplification -LRB- Sommer and Tautz , 1989 ; Kwok et al. , 1990 ; Christopherson et al. , 1997 ; Löffert et al. , 1998 -RRB- . Nevertheless , we addressed further this issue by repeating our previous QPCR investigation but including a second primer pair from the same sequence matching perfectly CD59b mRNA but with a single mismatch to CD59a mRNA in the same position as in the original primer set -LRB- Table 1 -RRB- . Using the original primer pair that matched perfectly CD59a we obtained very similar results to those we previously published -LRB- Baalasubramanian et al. , 2004 -RRB- . When we used the pair matching CD59b , there was a small increase in threshold cycle -LRB- Ct -RRB- for both CD59a and CD59b , likely a result of altered primer annealing temperature , but there was no difference in the calculated relative amounts for CD59a and CD59b mRNA in testis compared to the original primer set -LRB- Table 2 -RRB- . Of note , QPCR with either primer set did not detect any CD59b mRNA in liver , suggesting that the high cycle number PCR described above was detecting very small amounts of mRNA in these tissues . In summary , the data shows an 8-fold greater expression of CD59a mRNA in liver compared to testis , a 5-fold greater expression of CD59a mRNA compared to CD59b in testis and undetectable expression of CD59b in liver , regardless of primer set used . In light of these results we developed a semi-quantitative PCR to quantify the low levels of CD59b mRNA detected in tissues . In order to be able directly to compare the expression of both isoforms , we designed a new primer pair within exon 4 -LRB- Table 1 -RRB- , which is 100 % homologous to both mRNAs . Amplification of DNA-free mRNA with these primers will result in bands of 199 bp for CD59a and 162 bp for CD59b . In testis we detected both isoforms as expected -LRB- Fig. 2A -RRB- . Densitometric comparison of band intensities at higher dilutions of template , when the amplification reaction is in the linear range , showed approximately 5-fold higher expression of CD59a mRNA as compared to CD59b mRNA in testis , a result compatible with the QPCR data -LRB- Table 2 -RRB- and our published results -LRB- Baalasubramanian et al. , 2004 -RRB- . However , in liver we failed to detect presence of CD59b even after 40 cycles of PCR -LRB- Fig. 2B -RRB- . We reasoned that the large excess of CD59a mRNA in liver out-competes trace amounts of CD59b mRNA for limited reagents in the initial cycles of amplification , thereby reducing the chance for amplification of the low abundance CD59b mRNA . To test this reasoning we performed semi-quantitative PCR in separate tubes , using the primers specific for either CD59a or CD59b mRNA -LRB- Qin et al. , 2006 -RRB- . Firstly , to optimise the annealing and amplification efficiency for both primer pairs , we performed this reaction for testis mRNA -LRB- Fig. 3A -RRB- . Similar band intensities for CD59a and CD59b were obtained for 5 × 105-fold and 105-fold template dilution respectively -LRB- Fig. 3A and B -RRB- . This indicated an approximate 5-fold higher expression of CD59a mRNA compared to CD59b mRNA , supporting our results described above -LRB- Table 2 -RRB- and published -LRB- Baalasubramanian et al. , 2004 -RRB- , and confirming similar amplification efficiency for the specific primer pairs . Amplification of liver mRNA using these primers , and comparison of band intensities in dilutions of templates showed that expression of CD59b mRNA in liver was approximately 105-fold less compared to that of CD59a mRNA -LRB- Fig. 3C and D similar band intensities for CD59a and CD59b at 5 × 105 and 5-fold dilution respectively -RRB- . In these latter experiments we used liver perfused with saline at the time of sacrifice to remove the bulk of entrapped blood ; nevertheless , the trace mRNA detected might be from residual blood cells , which clearly express CD59b protein at low level -LRB- Baalasubramanian et al. , 2004 ; Qin et al. , 2006 -RRB- . To clarify this issue , we compared mRNA from perfused and non-perfused liver by QPCR using CD59b-specific primers and found the amount of CD59b mRNA in liver was reduced 12-fold by perfusion -LRB- Fig. 3E -RRB- . For comparison , expression of CD59b in testis was 2200-fold higher than in unperfused liver and approximately 25,000-fold higher than in perfused liver . Taken together these results strongly suggest that the detectable traces of CD59b mRNA in liver and likely other organs are a consequence of contamination with blood cells . 3.2 Expression of CD59b protein is testis restricted We have previously reported , using immuofluorescence staining , that CD59b protein was highly expressed in testis but absent from all other organs tested -LRB- including liver , lungs , spleen , kidney and heart -RRB- -LRB- Baalasubramanian et al. , 2004 -RRB- . We here extend these studies by Western blotting of testis , liver , lungs , plus aorta , chosen because of the suggested role of CD59 in vascular disease -LRB- Qin et al. , 2004 -RRB- -LRB- Fig. 4 -RRB- . Each organ was perfused with saline prior to preparation of protein lysates . CD59a was strongly detected in all the tissues ; however , CD59b was detected only in testis lysates . These data confirm our published contention that organ expression of CD59b protein is restricted to testis . 3.3 Expression of CD59b mRNA in testis coincides with puberty and plays a role in spermatozoal motility We previously reported that CD59b expression in testis was restricted to developing and mature spermatozoa -LRB- Baalasubramanian et al. , 2004 -RRB- . To confirm this germ cell restricted pattern we examined expression of CD59b mRNA in testis from pre-pubertal mice . CD59b mRNA was absent in testes harvested from mice at days 10 and 20 post-partum and appeared only from day 30 on , coincident with puberty -LRB- Fig. 5A -RRB- . In contrast , CD59a mRNA was present in testes at all timepoints . RNA extracted from embryos at days 5 , 10 and 17 were all positive for CD59a but negative for CD59b mRNA -LRB- Fig. 5A -RRB- . These observations were confirmed by Western blotting -LRB- Fig. 5B -RRB- . Expression of CD59b protein was highest in spermatozoa , intermediate in adult testis and absent from infant testis . CD59a was present in all the lysates , albeit at low level in spermatozoa and with a reduced apparent molecular mass compared with the testis protein , suggesting modification during sperm maturation . We previously reported that CD59b on spermatozoa was focussed on the head region in a highly granular pattern -LRB- Baalasubramanian et al. , 2004 -RRB- . Here we have explored the expression pattern of CD59b in more detail . In freshly isolated spermatozoa , more than 80 % showed this head/granular staining pattern , a minority showing a more diffuse staining on the head region -LRB- Fig. 6 -RRB- . CD59b was weakly expressed on spermatozoal tails and , in about two thirds , strongly in the mid-piece . Initiation of the acrosome reaction -LRB- with A23187 -RRB- caused a precipitous loss of CD59b expression on sperm heads , more than 60 % of cells being negative by 30 min and 90 % by 180 min post-initiation , indicating that CD59b was shed with the outer acrosomal membrane -LRB- Fig. 6 -RRB- . These data suggested that CD59b might play a role in acrosome function , an essential component of sperm capacitation for fertilisation . Therefore , we next investigated whether ligation of CD59b with antibody influenced spermatozoal mobility , a surrogate marker for fertilisation capacity -LRB- Fig. 7 -RRB- . Ligation of CD59b caused a significant suppression of motility of spermatozoa harvested from both wild type and cd59a − / − mice . As a control , CD59a was similarly ligated but did not alter spermatozoal motility compared with the effect of a control antibody . The data suggest that CD59b but not CD59a has a role in regulating spermatozoal motility . These findings are of particular relevance in that a major feature of the CD59b knockout mouse was spermatozoal dysfunction that included diminished motility and infertility -LRB- Qin et al. , 2003 -RRB- . 4 Discussion We have recently examined the distribution patterns of the two isoforms of CD59 in the mouse and concluded , based upon its broad distribution , that CD59a is the primary regulator of MAC assembly in mouse -LRB- Baalasubramanian et al. , 2004 -RRB- . We were unable to detect the expression of CD59b , either at mRNA or protein level , in brain , lungs , heart , liver , spleen and kidney . These data have recently been questioned by Qin et al. -LRB- 2006 -RRB- who first used a BLAST search of the mouse EST database and found ESTs matching the CD59b sequence from several tissues and organs . These in silico data were supported by RT-PCR analyses using primer sets specific for CD59a and CD59b , respectively that showed abundant expression of CD59b mRNA in all tissues tested , indeed , expression in testis was lower than in other organs . The apparent absence of CD59b mRNA in the CD59a knockout mouse in these published data was unexplained . Here we designed a number of quantitative and semi-quantitative assays to comprehensively address this controversial issue . Our data unambiguously demonstrated that expression of CD59b is essentially restricted to testis -LRB- Figs. 3 and 4 -RRB- . A comparative quantitative analysis of expression of CD59b mRNA in perfused and unperfused liver -LRB- Fig. 3 -RRB- showed a 12-fold decrease in expression of CD59b following perfusion , strongly suggesting that the trace detection of this mRNA was a consequence of contamination with blood cells . This would explain why Qin et al. -LRB- 2001 -RRB- were able to detect CD59b in multiple mouse tissues using Northern analysis in which they loaded 10 μg of mRNA in each lane , a huge excess for this sensitive procedure . The contamination likely also explains the presence in multiple tissues of CD59b-specific EST sequences . We further demonstrated that the testis expression of CD59b coincides with puberty , supporting our findings of expression only on spermatozoa and their immediate precursors -LRB- Fig. 5 -RRB- . We found that CD59b was released from spermatozoa heads upon acrosome activation -LRB- Fig. 6 -RRB- , an observation that strongly support a role for this protein in functioning of acrosome . Ligation experiments with antibodies showed that CD59b but not CD59a was involved in spermatozoa motility -LRB- Fig. 7 -RRB- , providing support for an earlier report describing decreased motility and viability of sperm in cd59b KO mice -LRB- Qin et al. , 2005 -RRB- . 5 Concluding remarks The data presented here , obtained using a broad panel of reagents and methods designed to give unbiased and unequivocal results , show that CD59b expression is limited . CD59b mRNA is abundantly expressed only on male germ cells and present in trace amounts in bone marrow and blood cells , but is absent from other organs where trace detection of mRNA is likely due to blood contamination . CD59b protein is abundant only on developing and mature spermatozoa . Erythrocytes express CD59b at low levels that we have previously quantified as less than 200 molecules per cell , irrelevant for protection from complement when CD59a is present at 2500 molecules per cell -LRB- Baalasubramanian et al. , 2004 -RRB- . We show a unique distribution pattern of CD59b protein on spermatozoa , the precipitous loss of this protein with outer acrosomal membranes and effects of CD59b ligation on sperm motility . We conclude that CD59a is indeed the principal regulator of MAC expressed in the mouse and that the CD59a knockout is an appropriate model for studying the roles of MAC and its regulation in disease models . The CD59b knockout mouse might prove of value for studies of fertility ; however , it should be noted that the mouse described by Halperin and co-workers is , for unexplained reasons , also markedly deficient in CD59a -LRB- Qin et al. , 2006 -RRB- , limiting its utility for studying specific roles of CD59b in vivo .

O B B O B O O O O O O O O O O O B O O O O B O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O B O O O O O O O O O B O O O O O B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O


Membrane-seeded autologous chondrocytes : cell viability and characterization at surgery The implantation of chondrocytes , seeded on matrices such as hyaluronic acid or collagen membranes , is a method that is being widely used for the treatment of chondral defects . The aim of the present study was to evaluate the distribution , viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation . Twelve patients who were suffering from articular cartilage lesions were treated by the MACI ® procedure . The residual part of each membrane was tested by colorimetric assay -LRB- MTT -RRB- and histochemical and ultrastructural analyses were carried out . In all of the samples a large number of viable cells , quite homogenously distributed , was detected . The cells expressed the markers of the differentiated hyaline chondrocytes . These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair , in line with the clinical evidences emerging in the literature . Introduction Autologous chondrocyte implantation -LRB- ACI -RRB- is an established method for the treatment of chondral defects -LSB- 6 -RSB- . Some prospective studies have demonstrated subjective and objective improvement in joint function at 12 months after surgery -LSB- 3 , 13 -RSB- . MRI , second-look surgery and biopsies have shown the formation of nearly normal cartilage in a good number of patients -LSB- 13 , 18 , 19 -RSB- . On the basis of these results , ACI may be considered one of the most effective treatments of articular cartilage lesions . However , this method is not devoid of some problems with regard to , for example , the wide surgical approach , the demanding technique , the presence of periosteal flap and the cell-holding in site -LSB- 14 , 16 -RSB- . In order to overcome some of these hurdles , new tissue engineering techniques , widely used nowadays , have been developed using chondrocytes seeded on biological matrices such as hyaluronic acid -LSB- 15 -RSB- or collagen membranes -LSB- 7 -RSB- or atelocollagen gel -LSB- 17 -RSB- . Despite the wide diffusion of these methods , there are still some areas that would need better clarification . The presence of numerous , viable and well-differentiated cells at surgery is an essential requisite for the success of all these methods . It has been shown that autologous chondrocytes in suspension before implantation are able to maintain their differentiated phenotype and are capable of proliferating fairly well -LSB- 9 , 20 -RSB- . On the other hand , little has been reported so far about cell characterization in membrane-seeded ACI techniques . The aim of the present study was to evaluate the distribution , viability and phenotype expression of the cells seeded on a collagen membrane just at the moment of the implantation . Materials and methods In 2003 , 12 consecutive patients , 8 males and 4 females , mean age of 34 years , suffering from cartilage lesions of the knee -LRB- 10 cases -RRB- and the ankle -LRB- 2 cases -RRB- , underwent a collagenic scaffold-based ACI procedure -LRB- MACI ® - Verigen , D -RRB- . Autologous chondrocytes were isolated at the Verigen laboratories from cartilage slices obtained from non-bearing areas of the patients ' joints during a preliminary arthroscopic surgery . Cells were propagated in monolayer cultures in autologous serum for 2 or 3 weeks , according to the cellular growth rate , and were passaged on average 3 times to obtain at least 10 × 106 cells . Cells were then seeded on 20 cm2 type I/III collagen membrane of porcine origin . The cell-seeded membranes were implanted in cartilage defects by means of either arthroscopic or mini-open surgery , using fibrin glue to ensure adhesion . At each implantation , the residual part of the membrane was collected and tested for cell viability , and histochemical and ultrastructural analyses were also performed . Cell viability analysis Cell viability was evaluated by MTT -LRB- dimethylthiazol-diphenyltetrazol bromide ; thiazolil blue -RRB- colorimetric assay . MTT -LRB- Sigma , Italy -RRB- is a water soluble tetrazolium salt that yields a yellowish solution when prepared in medium lacking in phenol red -LRB- RTMI 1640 , Sigma -RRB- . Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by the active mitochondrial dehydrogenases of living cells . The MTT solution -LRB- 5 mg MTT/ml medium -RRB- was added to three samples -LRB- 1 cm2 -RRB- of each membrane , being assayed to equal 1/10 of the original culture medium volume , and incubated for 3 h . The solution was then removed and acidic isopropanol -LRB- 0.04 -- 0.1 N HCl in absolute isopropanol -RRB- was added to solubilize the stain . The results were evaluated by means of the spectrophotometric assay -LRB- 570 nm -RRB- , yielding absorbance as a function of viable cell number . Histochemical and immunohistochemical analysis The samples were fixed by immersion in 4 % para-formaldehyde in 0.1 M phosphate buffer , pH 7.4 , at 4 °C and then embedded in paraffin . Specimens were stained with safranin-O . For the immunohistochemistry , non-specific binding was blocked with 3 % normal goat serum in a phosphate-buffered saline -LRB- PBS -RRB- , pH 7.4 , for 30 min at room temperature ; slides were then incubated overnight with primary antibodies at 4 °C . Sections were incubated with polyclonal antibodies anti S-100 protein -LRB- Dako , Italy -RRB- , a cytoplasmatic marker of chondrocyte phenotype , diluted at 1:3,000 , anti-collagen type I -LRB- Monosan , The Netherlands -RRB- and II -LRB- Calbiochem-Oncogene , CA , USA -RRB- at 1:150 , and monoclonal antibodies anti chondroitin sulphate -LRB- chondroitin-S -RRB- -LRB- Sigma -RRB- at 1:200 . Rabbit and mouse immunoglobulins , at the same dilutions as the primary antibodies , were used as controls . After three washes with Tris -- HCl -LRB- 0.05 M , pH 7.6 -RRB- , revelation of the reactions was accomplished by DAKO LSAB + kit , HRP . Stainings were viewed and photographed with a Leica Microscope -LRB- Leica Cambridge Ltd. , UK -RRB- . Ultrastructural analysis For scanning electron microscopy -LRB- SEM -RRB- , the membranes were fixed in 2 % glutaraldeyde in 0.1 M cacodylate buffer -LRB- pH 7.4 -RRB- , post-fixed in 1 % osmium tetroxide , dehydrated in increasing ethanol concentrations and then CPD-dried . They were mounted on stubs and gold-sputtered . Specimens were observed with a Philips 505 microscope . Results Cell viability analysis The presence of numerous viable cells was observed in all samples , with quite a homogeneous stain distribution , even if some of the areas revealed a greater concentration of converted dye -LRB- Fig. 1 -RRB- . Fig. 1Macroscopic view of human chondrocytes seeded on type I , III collagen membrane after MTT incubation . The converted blue dye shows the presence of numerous viable cells with quite a homogeneous stain distribution Considerable variability was found in mean cell numbers between the samples from different patients , in relation to the different amounts of cells obtained from cartilage slices and seeded on the membrane . Highest values were observed in samples from the younger patients -LRB- Fig. 2 , Table 1 -RRB- . Fig. 2The standard curve obtained from each membrane by MTT colorimetric assay -LRB- see also Table 1 -RRB- Table 1The table shows the mean viable cell number/cm2 obtained from each membrane by MTT colorimetric assayCaseJointAgeSexSeeded cellsViable cells/cm2 -LRB- mean ± SD -RRB- 1 Knee54M19 × 106 -LRB- 7.52 ± 2.79 -RRB- × 1032 Knee22F17 × 106 -LRB- 12.94 ± 2.54 -RRB- × 1033Knee50F15 .4 × 106 -LRB- 17.82 ± 6.25 -RRB- × 1034 Knee23M20 .3 × 106 -LRB- 100.28 ± 22.14 -RRB- × 1035Knee29F20 .4 × 106 -LRB- 110.07 ± 39.18 -RRB- × 1036Knee43M18 .7 × 106 -LRB- 5.34 ± 3.25 -RRB- × 1037Knee48M17 .5 × 106 -LRB- 4.74 ± 2.37 -RRB- × 1038Ankle42F18 .2 × 106 -LRB- 33.13 ± 25.92 -RRB- × 1039Knee19M21 .6 × 106 -LRB- 99.54 ± 25 -RRB- × 10310Knee35M20 × 106 -LRB- 103.81 ± 37.26 -RRB- × 10311Ankle39M19 × 106 -LRB- 79.48 ± 62.57 -RRB- × 10312Knee13M20 .1 × 106 -LRB- 87.88 ± 55.17 -RRB- × 103 Patients ' characteristics and the number of cells initially seeded on each membrane are also reported -LRB- see also Fig. 2 -RRB- Histochemical and immunohistochemical analysis In all of the samples , chondrocytes that were grown on the membrane were arranged in multi-layered sheets , and sometimes invaded deeper into the matrix . The cells appeared to be quite well differentiated , although some had a flattened morphology . They slightly stained metachromatically for safranin O -LRB- Fig. 3 -RRB- , and clearly immunoreacted with anti-S-100 protein -LRB- Fig. 4 -RRB- , type II collagen -LRB- Fig. 5 -RRB- and chondroitin-S antibodies . Only a few cells stained for type I collagen . Fig. 3Human chondrocytes seeded on type I , III collagen membrane before the implantation . Numerous cells are located on the membrane and deeper in the matrix forming a multi-layer engineered tissue . -LRB- Safranin O stain , × 200 -RRB- Fig. 4Human chondrocytes seeded on type I , III collagen membrane . A marked cytoplasmic immunoreaction for S-100 protein is evident -LRB- × 400 -RRB- Fig. 5Human chondrocytes seeded on type I , III collagen membrane . Cells express a positive immunoreaction for type II collagen , while the membrane remains unstained -LRB- × 400 -RRB- Ultrastructural analysis The membranes presented a dual appearance : there was a smooth side with tightly packed fibres and a rougher side , a sparse layer , to which the cells were adhered . Numerous cells firmly adhered to the membrane and showed a round shaped morphology and a rough surface -LRB- Fig. 6 -RRB- . Fig. 6Scanning electron micrograph of human chondrocytes seeded on type I , III collagen membrane . Numerous cells -LRB- arrows -RRB- firmly adhere to the membrane and show a round shaped morphology and a rough surface -LRB- × 7,500 -RRB- Discussion Cell phenotype and proliferation analysis should be an essential step in the evaluation of all tissue-engineered products because the implantation of dedifferentiated or not-proliferating cells would not justify the therapeutic employment of these biotechnologies . The chondrocyte culture may undergo a dedifferentiation process , consisting in a fibroblast-like morphology , a reduction of the type II collagen and aggregating proteoglycans synthesis , and an increase of type I collagen expression -LSB- 1 , 4 -RSB- . Chondrocyte dedifferentiation is common in monolayer cultures , while a long phenotype maintenance is supported by the presence of a tri-dimensional matrix -LSB- 1 , 4 , 11 -RSB- . The chemical and ultrastructural characteristics of the matrix may have great influence on the behaviour of chondrocytes in culture . A Type I collagen membrane appears to be a matrix endowed with such properties which make that ideal and useful for cartilage tissue engineering -LSB- 8 , 10 -RSB- . MACI is a method for the treatment of articular cartilage defects , that employs autologous chondrocytes expanded in monolayer culture , suspended , and then seeded on type I -- III collagen membrane . In a previous study -LSB- 9 -RSB- , we had observed that autologous chondrocytes in suspension before implantation were of `` good cartilaginous quality '' and that they possessed an excellent proliferation capacity . Zheng et al. -LSB- 20 -RSB- confirmed that cells in ACI procedure maintained chondrocyte phenotype and showed a low apoptotic rate . To our knowledge , there is no previous reference to the analysis of the phenotype and the proliferation activity of human chondrocytes before MACI implantation . Data about cell number and viability , but not about cell phenotype and distribution are provided by Verigen 's laboratory for each implant . It should , however , be remembered that it is quite possible that the presence of a membrane like the one found in the MACI technique could interfere with cell behaviour and modify phenotype expression . The present study has demonstrated that the autologous chondrocytes that were analysed adhered to the membrane and had quite a homogenous distribution . These cells were viable in all the cases analysed , as shown by the spectrophotometric assay . The differences observed among patients in the number of cells seeded on the membrane and those found to be viable are probably due to several factors . In the first place , the amount of tissue collected from patients and sent to the laboratory for culture was variable . The cellularity of cartilage tissue and the proliferation capacity of chondrocytes in identical culture conditions are also quite variable between subjects , and this variability can not merely be explained with age , but is more likely to stem from a number of characteristics that define tissue `` quality '' -LSB- 5 , 12 -RSB- . We did not investigate whether during monolayer culture chondrocytes underwent dedifferentiation , however , other authors -LSB- 20 -RSB- demonstrated that cells grown in monolayer with the same culture conditions maintained a differentiated phenotype . Moreover , we felt it was important to establish the phenotypical expression of chondrocytes seeded on the I/III collagen membrane , since those are the cells that are then implanted , independently of what has taken place in the previous steps of the procedure . The positive immunostaining for S-100 protein , chondroitin-S , type II , but not type I collagen , confirms that cells maintained the characteristics of differentiated hyaline chondrocytes at the moment they were implanted . These data reassure that the MACI procedure can indeed provide a suitable engineered tissue for cartilage repair , in line with the mid-term clinical results described in the literature so far -LSB- 2 , 7 -RSB- . Nevertheless , clinical and histological results of this method to attain stable clinical recovery and a hyaline-like cartilaginous scar are auspicated to substantiate these evidences in the long term .

O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

chondrocytes
articular cartilage
cells
tissue engineering


An Analysis of Respondent Driven Sampling with Injection Drug Users -LRB- IDU -RRB- in Albania and the Russian Federation Injection drug users in Tirana , Albania and St. Petersburg , Russia were recruited into a study assessing HIV-related behaviors and HIV serostatus using Respondent Driven Sampling -LRB- RDS -RRB- , a peer-driven recruitment sampling strategy that results in a probability sample . -LRB- Salganik M , Heckathorn DD . Sampling and estimation in hidden populations using respondent-driven sampling . Sociol Method . 2004 ; 34:193 -- 239 -RRB- . This paper presents a comparison of RDS implementation , findings on network and recruitment characteristics , and lessons learned . Initiated with 13 to 15 seeds , approximately 200 IDUs were recruited within 8 weeks . Information resulting from RDS indicates that social network patterns from the two studies differ greatly . Female IDUs in Tirana had smaller network sizes than male IDUs , unlike in St. Petersburg where female IDUs had larger network sizes than male IDUs . Recruitment patterns in each country also differed by demographic categories . Recruitment analyses indicate that IDUs form socially distinct groups by sex in Tirana , whereas there was a greater degree of gender mixing patterns in St. Petersburg . RDS proved to be an effective means of surveying these hard-to-reach populations . Introduction Accessing hard-to-reach populations for HIV prevention and research activities has historically been challenging . Certain high-risk populations are fundamental in the proliferation of HIV , particularly in the early stages of a country 's epidemic . Owing to the stigmatized and often criminalized behaviors of injection drug users , for example , these populations avoid open contact with the rest of civil society . Respondent Driven Sampling -LRB- RDS -RRB- offers a solution to that dilemma , allowing researchers to access hard-to-reach populations through their social networks .2,3 A social network person includes other persons with whom he/she has a social relationship and who tend to engage in the same behaviors as the index person . This paper presents approaches used to implement RDS in Tirana , Albania and St. Petersburg , Russia and lessons learned in these two cities . Materials and Methods The studies in St. Petersburg and Tirana utilized RDS to recruit IDUs into an HIV-related behavioral survey . While the methodology behind RDS has been described elsewhere ,3 in brief it is a referral-based system whereby participants refer a limited number of friends or acquaintances -LRB- who are also members of the target group -RRB- to participate . Study participants receive an incentive both for participating and successfully recruiting others . Data are analyzed using the recruiting linkages and yield a probability sample of the population in the target area . For inclusion in either study , participants had to be at least 15 years old and report having injected drugs recreationally in the past 6 months . Data collection for both surveys included face-to-face interviews and encompassed a 7 -- 8 week period at three sites in each city . In Tirana , data collection took place in offices of two non-governmental organizations -LRB- NGOs -RRB- , conducting outreach to IDU and in one treatment hospital . Data collection in St. Petersburg was conducted in three sites in various parts of the city . One was a fixed site in the center of the city . The other two sites were located just outside the city center and open on weekend afternoons , operating from mobile vans at stable locations . One site was selected due to its proximity to an illegal drug market . The data collection team at each site and shift consisted of two interviewers and one person serving as coupon manager , supervised by a study coordinator . The coupon manager recorded data for each participant on a form that included each individual 's self-reported network size , the coupon number brought to the interview , and the coupon numbers each was given for distribution . The Tirana sites also had a physician , laboratory technician and counselor as data collection included biological samples for HIV and other STIs . Questionnaire topics included sociodemographic indicators , sexual and drug injection risk behaviors , knowledge of HIV and STIs , and previous HIV testing . Survey duration in Tirana was approximately 1.5 hour for both the behavioral and biological portions of the study , while in St. Petersburg data collection lasted approximately 45 minute . In accordance with RDS methodology , the initial participants of the study , known as `` seeds '' ,4 were members of the target IDU population in their respective cities , and were selected to begin recruiting their peers into the study . In Tirana , 15 seeds launched the study , while in Russia recruitment was initiated with 11 seeds ; 2 seeds were added later to generate more recruiting . The seeds selected in both cities were referred from local NGOs conducting outreach with the IDU population , as well as personal IDU contacts of study staff . Each participant in St. Petersburg received three coupons with an eight-week expiration date to recruit their peers while two coupons per participant were used in Tirana -LRB- no expiration dates -RRB- . In St. Petersburg , three coupons continued to be distributed until approximately 200 participants were attained , after which participants were warned that the study would be ending within a few days . In Tirana , coupons were distributed until the desired sample size of 200 was reached ; however , participants were warned from the beginning that once this was reached , no more coupons would be honored . In Tirana , participants received cash incentives of 10 euros for participating and an additional 5 euros for each successful recruit . In St. Petersburg , use of monetary incentives was discouraged by all local stakeholders ; therefore , participants were given a gift package consisting of a box of chocolates and personal hygiene products for completing the survey , and a box of chocolates or a can of coffee for each successful recruit . The gifts were equivalent to U.S. $ 10 for participating and U.S. $ 5 for recruiting . Two hundred ten IDUs -LRB- not including seeds -RRB- were surveyed between July and August 2005 in Tirana , and 200 IDUs -LRB- not including seeds -RRB- were surveyed in St. Petersburg during the same time period . Biometric measures were taken in Tirana to discourage study members from participating in multiple sites . Measurements consisting of wrist circumference multiplied by forearm length were taken and recorded . Participants were told that this was a unique measurement that could be used to determine if they attempted to return or access other data collection sites . Project staff also rotated between sites to decrease the likelihood of participants visiting multiple sites . In St. Petersburg , biometric measurements were not taken since local researchers thought it would induce suspicion among IDUs and consequently discourage participation . Recognition of potential duplicate participants depended upon study staff , who were outreach workers familiar with the target population and operated out of the same site for the entire 8 weeks of data collection . The data were analyzed using RDS Analysis Tool version 5.4 -LRB- RDSAT -RRB- . RDS population estimates and confidence intervals as calculated by RDSAT are reported . The homophily index describes the extent of in-group ties . A homophily index of H = 1.0 reflects perfect homophily , indicating that ties are formed with other members of the same group . In contrast , H = − 1.0 reflects perfect heterophily , indicating that ties are formed completely outside of the group .3 Both studies were approved by the FHI Protection of Human Subjects Committee and the Tirana study was also approved by the Medical Ethical Committee of Albania . In both cities , information about whether anyone refused to accept coupons was elicited from those who returned to collect their compensation for successful recruiting . Approximately 20 % -LRB- Tirana -RRB- and 5 % -LRB- St. Petersburg -RRB- of those approached to participate refused to accept the coupon from participants . Of a total of 450 coupons distributed in Tirana , 210 were returned . In St. Petersburg , 200 out of a total of 629 coupons distributed were returned . Furthermore , in Tirana , only two potential participants were found to be ineligible for participation and once consent had been given , no participants withdrew from the process . This information is not yet available for St. Petersburg . Results Demographic Characteristics Table 1 describes the characteristics of the study samples in Tirana and St. Petersburg . The sample in Tirana consists predominately of male IDUs , with only 14 female IDUs recruited for the survey . This contrasts substantially from the sample in St. Petersburg , where more females were represented compared to Tirana , with 36 % being female and 64 % being male . Table 1Demographic information AlbaniaRussiaSex Males91 % -LRB- 86 , 96 -RRB- 64 % -LRB- 55 , 73 -RRB- Females9 % -LRB- 4 , 14 -RRB- 36 % -LRB- 27 , 45 -RRB- Education completed Did not attend school9 % -LRB- 5 , 16 -RRB- 0 % Primary17 % -LRB- 11 , 26 -RRB- 0 % Secondary/vocational67 % -LRB- 58 , 76 -RRB- 84 % -LRB- 77 , 92 -RRB- University7 % -LRB- 3 , 8 -RRB- 16 % -LRB- 8 , 23 -RRB- Age 15 -- 22 years38 % -LRB- 33 , 48 -RRB- 11 % -LRB- 7 , 15 -RRB- 23 -- 27 years36 % -LRB- 27 , 41 -RRB- 38 % -LRB- 29 , 47 -RRB- 28 years or more26 % -LRB- 19 , 32 -RRB- 51 % -LRB- 42 , 62 -RRB- Current co-habitation situation Married living with spouse12 % -LRB- 6 , 18 -RRB- 9 % -LRB- 4 , 15 -RRB- Married living with other sex partner0 % 4 % -LRB- 2 , 9 -RRB- Married not living with any partner3 % -LRB- 1 , 6 -RRB- 4 % -LRB- 1 , 7 -RRB- Not married , living with sex partner20 % -LRB- 14 , 26 -RRB- 33 % -LRB- 25 , 42 -RRB- Not married , not living with sex partner65 % -LRB- 58 , 72 -RRB- 49 % -LRB- 39 , 60 -RRB- The numbers in parentheses represent the 95 % confidence intervals as calculated by RDS . In Tirana , the majority had completed at least middle school -LRB- eight classes -RRB- , with a third of the sample completing middle school and a third completing high school -LRB- 12 classes -RRB- . Only 7 % in Tirana had completed university . In St. Petersburg , the majority -LRB- 84 % -RRB- had completed either secondary or vocational education , and 16 % had completed university . In Tirana , the sample was comprised of young IDUs with 38 % between 15 and 22 years of age and 74 % below 27 years of age . The St. Petersburg study included an older IDU population , with over half of participants 28 years of age or older and only 11 % between 15 and 22 years of age . In St. Petersburg , males were slightly older than females ; a much higher proportion of males were 28 years of age or older -LRB- 66 vs 26 % of females ; data not shown -RRB- . The majorities of the samples are unmarried -LRB- 85 % in Tirana and 82 % in St. Petersburg -RRB- . In Tirana , the majority also did not live with a sex partner -LRB- 65 % -RRB- , while another 20 % were unmarried but living with a sexual partner . A higher proportion of IDUs in Russia reported being unmarried but living with a sex partner -LRB- 33 % -RRB- . Network Sizes As a part of RDS methodology , respondents were asked about the size of their personal networks . Specifically , participants were asked how many people they know personally who used injecting drugs -LRB- i.e. , `` you know who they are , they know who you are , you have seen them in the last six months '' -RRB- . In Tirana , male IDUs reported a larger average personal network size than female IDUs . In contrast , in St. Petersburg , females had a larger average personal network size than males -LRB- 20 vs 17 , respectively -RRB- . The pattern of network size by age is again very different between IDUs in Tirana and St. Petersburg . In Tirana , network size increases with age -LRB- from 16 among the youngest age group to 21 in the oldest age group -RRB- , whereas in St. Petersburg , network size decreases with age -LRB- from 25 in the youngest age group to 15 in the oldest age group -RRB- . The same trend and differences between the two cities is observed with regard to education , which is likely due to the collinearity between age and education . Similarly , in Tirana , there was a trend of increasing network size with increasing duration of injection drug use . However , in St. Petersburg , the network size was larger for recent initiates and long-term injectors compared to those who injected for 1 -- 5 years -LRB- Table 2 -RRB- . Table 2History of injection drug useDuration of injection drug useAlbaniaRussia < 1 year21 % -LRB- 11 , 24 -RRB- 6 % -LRB- 3 , 9 -RRB- 1 -- 5 years72 % -LRB- 69 , 83 -RRB- 43 % -LRB- 31 , 51 -RRB- > 5 years6 % -LRB- 3 , 11 -RRB- 52 % -LRB- 43 , 63 -RRB- Recruiting Table 3 represents the recruitment patterns of IDUs in Tirana and St. Petersburg with respect to sex , age and length of injection . RDS provides the opportunity to learn about the degree to which people tend to affiliate with others sharing their same characteristics . In Tirana , the gender affiliation patterns demonstrate a strong heterophily in women , whereby women recruited only men -LRB- H = − 1.00 -RRB- , while men recruited women 7 % of the time -LRB- H = 0.16 ; this is probably due to the small number of women in the Tirana sample , with only 14 women participating -RRB- . From a possible 28 coupons given to these women , 16 coupons were returned -LRB- 57 % -RRB- . In contrast , the gender affiliation patterns of IDUs in St. Petersburg reflects neither strong homophily nor heterophily . Both males and females recruited males about the same percentage of times -LRB- 63 and 57 % , respectively -RRB- . The heterophily index in males was − 0.02 and homophily index was 0.12 for females . Table 3 . Recruitment patterns by age , sex , and duration of drug injection in Tirana and St. PetersburgRecruitersRecruiteesTiranaSt . PetersburgYoungest -LRB- 15 -- 22 years -RRB- Middle -LRB- 23 -- 27 years -RRB- Oldest -LRB- ≥ 28 years -RRB- Youngest -LRB- 15 -- 22 years -RRB- Middle -LRB- 23 -- 27 years -RRB- Oldest -LRB- ≥ 28 years -RRB- Age Youngest -LRB- 15 -- 22 years -RRB- 42 % 35 % 23 % 10 % 54 % 36 % Middle -LRB- 23 -- 27 years -RRB- 33 % 38 % 29 % 19 % 36 % 45 % Oldest -LRB- ≥ 28 years -RRB- 29 % 39 % 32 % 12 % 44 % 44 % SexMaleFemaleMaleFemale Male93 % 7 % 63 % 37 % Female100 % 0 % 57 % 43 % Duration of drug injectionNew -LRB- < 1 year -RRB- Mid-term -LRB- 1 -- 5 years -RRB- Long-term -LRB- > 5 years -RRB- New -LRB- < 1 year -RRB- Mid-term -LRB- 1 -- 5 years -RRB- Long-term -LRB- > 5 years -RRB- New -LRB- < 1 year -RRB- 20 % 64 % 16 % 0 % 43 % 57 % Mid-term -LRB- 1 -- 5 years -RRB- 8 % 57 % 35 % 7 % 40 % 54 % Long-term -LRB- > 5 years -RRB- 4 % 65 % 32 % 6 % 40 % 54 % The affiliation patterns of age recruiting relations in IDUs in Tirana reflects neither homophily nor heterophily . The homophily indices were 0.02 for the younger -LRB- 15 -- 22 years -RRB- , 0.05 for the medium age -LRB- 23 -- 27 years -RRB- categories , and 0.09 for the older age group -LRB- ≥ 28 years -RRB- . As can be seen , both youngest and the oldest age categories recruited the medium age group approximately one-third of the time . Further , both the youngest and the medium age groups equally recruited the oldest age group -LRB- approximately 23 -- 29 % of the time -RRB- . Lastly , the medium and the oldest age groups recruited the youngest group with equal vigor at about one-third of the time . Among IDUs in St. Petersburg , the affiliation patterns of age relations reflect neither homophily nor heterophily . The homophily indices were − 0.03 for both the younger -LRB- 15 -- 22 years -RRB- and the medium age -LRB- 23 -- 27 years -RRB- categories , and − 0.15 for the older age group -LRB- ≥ 28 years -RRB- . In Tirana , as with the relations by age , the affiliation patterns of relations based on duration of injection drug use reflects neither homophily nor heterophily . The homophily indices were 0.09 for new injectors -LRB- i.e. , < 1 year of injection experience -RRB- , − 0.10 for the mid-term users -LRB- 1 -- 5 years -RRB- , and 0.09 for the longer-term injectors -LRB- > 5 years -RRB- . New users were recruited the least by the mid - and longer-term injectors -LRB- 8 and 4 % , respectively -RRB- , and both the new and longer term injectors recruited mid-term injectors -LRB- 64 and 65 % , respectively -RRB- . In St. Petersburg , however , the affiliation patterns of relationships by duration of injection drug use differed between new injectors and longer-term injectors . The group of new injectors was completely heterophilous -LRB- H = − 1.0 -RRB- , reflecting a lack of in-group ties . However , both mid-term and longer-term injectors exhibited neither heterophily nor homophily -LRB- H = − 0.07 and H = 0.05 , respectively -RRB- . Discussion These two studies are among the first to report on the level of sexual and drug injection risk behaviors and knowledge about HIV prevention in a probability sample of IDUs in Tirana and St. Petersburg using RDS . The use of a systematic peer recruitment method afforded the possibility of capturing a wider range of IDUs than studies recruiting with outreach workers or from specific venues , such as STI clinics or needle exchange programs . Additionally , these two studies shed light on the social context by identifying social network relations among the IDUs . The recruitment analysis suggests that IDUs in Tirana form socially distinct groups by sex . Female IDUs do not seem to interact with each other ; no female IDU referred another woman . This suggests that intravenous drug use amongst females may be a highly stigmatized activity , or it may reflect their initiation into drug use by men . In contrast , IDUs in St. Petersburg do not form socially distinct groups with regard to sex . The lack of strong ties within sex groups may be a reflection of injection behaviors in that they often inject in group settings which are likely to be of mixed sex and share needles with members of the opposite sex . In Tirana , the lack of strong ties both within and between age groups most likely indicates that younger IDUs are probably not being initiated into injection drug use by injectors who are older in age . However , in St. Petersburg , there were no intra-group ties among younger females or among newer IDUs . All new IDUs appeared to be recruited only by long - and mid-term IDUs , and all younger female IDUs were primarily recruited by older male and female IDUs . This suggests that new IDUs , particularly female IDUs , have been initiated into injection drug use by older IDUs . The high prevalence of HIV and STIs5 -- 7 in the IDU population in Russia places these new injectors at extremely high risk for HIV acquisition . The differences in average network sizes of male and female IDUs between Tirana and St. Petersburg may have implications for prevention programs . The larger network size of male IDUs compared to female IDUs in Tirana is not surprising given that the IDU population in Albania appears to be predominately male and clandestine for women . Therefore , it is apparently rare for female IDUs to know other female IDUs . In contrast , in St. Petersburg , female IDUs on average had a larger network size compared to male IDUs , which may be a reflection of the overlap of this population and the sex worker population . A sizable proportion are engaged in sex work so they likely know other sex workers from working in the same areas . The younger age of IDUs in the Albanian sample indicates a potential for longer term drug use , allowing for greater transmission . Additionally , continued migration between borders with neighboring countries where prevalence amongst high-risk groups is higher -LRB- i.e. , to Greece , Italy or Eastern European countries -RRB- may create a transmission -- migration effect .8 The male-to-female ratio of almost 2:1 in St. Petersburg is consistent with the decreasing trend in the male-to-female ratio of IDUs previously reported , from 7:1 in 1991 to 5:1 in 2001.9 However , it is also possible that previous studies may have underestimated the proportion of female IDUs . RDS may reach female IDUs better than other sampling methodologies . This could be evidence that RDS can capture a more diverse and possibly a more representative sample of IDUs in St. Petersburg . The Tirana sample is proportionately younger than the St. Petersburg sample , perhaps describing an epidemic that is newer or reflecting the population structure in Albania where 40 % is under 25 years of age .10 Records indicate an increase in substance abuse treatment since 2000 and the first HIV case was detected in Albania in 1993 -- lending credence to the possibility of a younger epidemic .11 The co-habitation situation in Tirana differs from that in St. Petersburg , with a far greater proportion in St. Petersburg unmarried but living with a sex partner . It is possible that the more conservative nature of Albanian society and a holdover from the Albanian communist regime policies are reflected in this indicator .12 The finding in St. Petersburg that all new injectors were recruited only by long - and mid-term injectors is of concern . These social relations combined with the high prevalence of HIV in the IDU population in Russia place these new injectors at extremely high risk for HIV acquisition . This scenario is likely exacerbated by their large average network size -LRB- approximately 18 -RRB- . The research conducted in Albania with IDUs is of vital importance . For the first time , policy makers and those involved with drug treatment and prevention of HIV and STIs have information to better plan and target interventions . The pace of recruitment in both countries -LRB- approximately 8 weeks -RRB- seems to be fairly efficient . Differing numbers of coupons -LRB- two in Tirana and three in St. Petersburg -RRB- did not seem to affect overall recruitment with both studies reaching the desired sample size in approximately the same amount of time . In St. Petersburg , slow recruitment particularly at the fixed site at the city center was boosted by the addition of two seeds at week 6 ; participation of only 50 IDUs at week 6 increased to 200 by the eighth week . Over 200 IDUs were reached by the study in 7 -- 8 weeks with 13 -- 15 seeds in each city . The body of literature regarding the use of RDS amongst hard-to-reach populations in developing countries is still in its nascence . Little is known about the effect of incentives on populations in developing countries or if random recruiting of peers -LRB- a fundamental assumption underlying the RDS methodology -RRB- is valid in countries where a large proportion of the population is poor . A greater body of research employing these methods in developing countries is needed . Lessons learned from RDS methodology Data collection during the summer was ideal in St. Petersburg given that some sites were mobile vans and participants had to wait outside . The season was also beneficial in Tirana , with data collection taking place before summer vacation . Given the large size of both cities , having three study sites in different parts of each city facilitated recruitment . Evening and Saturday hours were important to ensure that workers could participate . In St. Petersburg , upon recommendation from local NGOs , letters of support obtained from city police officials helped ensure that IDUs would not be harrassed at the study sites . In Tirana , biometrics were useful in creating a disincentive to participate multiple times . Involving NGO staff to recruit seeds and conduct data collection in the NGO offices enabled participants to feel comfortable about the process . Limitations Possible limitations to the study could have affected the results . In Tirana , the small numbers of women participating in the surveillance may indicate a strong desire to remain hidden , their limited numbers , or a reflection of poor recruiting . Because few women have been arrested or attended treatment facilities , there are no data regarding intravenous drug use amongst women in Tirana , or anywhere in Albania .11 In both cities , it is possible that RDS may have selectively attracted those IDUs desiring the incentives , which may have biased our sample . Formative research was conducted to assess if the amount would be coercive , so this is unlikely . The major limitation of RDS is that it can not control for non-response bias . Barriers to participation due to unknown characteristics of certain sub-groups or to factors beyond the control of the project could occur and are not measurable . Both studies attempted to determine the rate of non-response by asking participants returning to claim recruiting incentives about the number of attempts made before their coupons were accepted and the demographic characteristics of those who refused . They were also asked characteristics of those accepting the coupons . It is hoped that forthcoming analysis can assess if there is a difference between those who refuse , those who accept but do not show up , and those who accept and participate . The coupon return rate in St. Petersburg was considerably lower than that of Tirana most likely due to the greater number of coupons given out by each participant in St. Petersburg -LRB- three coupons -RRB- than in Tirana -LRB- two coupons -RRB- . Conclusion Because IDU participants were recruited through RDS , it is likely that the study sampled from a wide range of IDUs thereby increasing the sample representativeness in these two cities . Our study methodology likely drew a more diverse sample of IDUs compared to traditional recruitment by outreach workers or through needle exchange programs . The studies in Tirana and St. Petersburg greatly enhance our understanding of IDUs and their network characteristics . They also provide evidence for the effectiveness of using RDS in recruiting hard-to-reach populations in environments where stigmatization and criminalization against their behaviors are commonplace .

O O O O O O O B I I O O O O B O O O O B I I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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