Tyrosinase as an autoantigen in patients with vitiligo

Vitiligo is considered an autoimmune disorder due to the generation and presence of autoantibodies directed against melanocyte antigens in the patients' sera. In the present study we point towards a newly defined autoantigen in vitiligo, the enzyme tyrosinase, which participates in the process of melanogenesis. Anti-tyrosinase antibodies were detected in the sera of seven patients with diffuse and 11 patients with localized vitiligo. Employing solid-phase ELISA to mushroom tyrosinase, we found that patients with diffuse vitiligo had significantly higher titres of IgG anti-tyrosinase autoantibodies than patients with localized disease or healthy subjects. These anti-tyrosinase autoantibodies have relatively high functional affinity to tyrosinase and can be recovered from vitiligo patients’ sera by affinity purification. The anti-tyrosinase antibodies do not cross-react with other enzymes recognized as autoantigens in different autoimmune disorders and the autoantibodies do not block the enzymatic activity of tyrosinase, indicating that they are not reacting with the catalytic site of the enzyme. These data point to tyrosinase as an autoantigen in vitiligo and suggest that anti-tyrosinase titres can serve as a marker for disease activity.     

Sutureless amniotic membrane transplantation for ocular surface reconstruction with a chemically defined bioadhesive

The purpose of this study was to evaluate the efficiency and safety of a sutureless amniotic membrane transplantation (AMT) for ocular surface reconstruction with a chemically defined bioadhesive (CDB). The CDB was synthesized from aldehyded polysaccharides and epsilon-poly(L-lysine), two kinds of medical and food additives, as starting materials. Biocompatibility assay indicated that the CDB showed excellent biocompatibility with in vitro and in vivo ocular surface tissues and most of the CDB was histologically degraded within 4 weeks. Sutureless AMT using the CDB was safely and successfully performed onto a rabbit scleral surface. Transplanted amniotic membrane (AM) evaluated by histological, electron microscopic- and immunohistochemical examination indicated that the CDB did not affect normal differentiation of the cells or the integrity of the surrounding tissue. Thus, this newly developed CDB was found to be very useful for sutureless AMT for ocular surface reconstruction, without considering the risk of infection. It has the ability to fix AM to the ocular surface for a long time-period without additional inflammation, scarring, or damage to the surrounding tissues.     

Hoechst 33342 a suitable fluorescent marker for Schwann cells after transplantation in the mouse spinal cord.

Purified rat Schwann cells labeled with Hoechst 33342 were transplanted into a lysophosphatidyl choline induced myelin lesion of the adult shiverer mouse spinal cord. Remyelination by grafted Schwann cells within the lesion was evidenced by codetection of Hoechst labeled Schwann cells and P0 (peripheral myelin protein) immunolabeled myelin on serial cryostat sections and confirmed on adjacent sec ions by electron microscopy. These data show that the Hoechst-shiverer model is an excellent model which can be used in intraspinal transplantation of myelin forming cells to demonstrate the origin of the newly formed myelin. Using this model, we bring the unquestionable evidence that cultured Schwann cells are capable after transplantation to participate with host oligodendrocytes in repair of a myelin lesion of the central nervous system.     

The surface to volume ratio of mitochondria,a suitable parameter for evaluating mitochondrial swelling

Summary Cellular changes occuring in the left ventricular myocardium during ischaemia after different methods of cardiac arrest have been evaluated by morphological and morphometric parameters: volume densities of mitochondria (V_VMi), sarcoplasm (V_VSp), myofibrils (V_VMf), surface densities of mitochondria (S_VMi). The surface to volume ratio of mitochondria (S_Vratio_Mi) has been used as an independent parameter of mitochondrial swelling.Since ischaemic swelling of myocardial cells increases the volume of the reference space and ischaemic swelling of mitochondria decreases the free sarcoplasm, V_VMi and V_VSp cannot be considered as reliable indicators of the degree of oedema. S_VMi/V_VMf remains nearly constant after different forms of cardiac arrest, demonstrating the integrity of mitochondrial outer membranes. The inverse linear ratio between S_Vratio_Mi and the mean mitochondrial volume indicates that the increase in mitochondrial volume is achieved by surface smoothing.Loss of matrix structure and fragmentation of cristae occur at an S_Vratio_Mi of about 5.8, cristolysis at 5.5 to 5.6 and amorphous matrix densities at an S_Vratio_Mi of less than 5.5 m²/m³.The S_Vratio_Mi is a suitable parameter for evaluating mitochondrial swelling both at the onset and during global myocardial ischaemia, independent of the method of cardiac arrest used. It serves as an indicator of the state of structural preservation of mitochondria during ischaemia.Supported by the Deutsche Forschungsgemeinschaft, SFB 330-Organprotektion — Göttingen     

Solid organ transplantation is a reality for patients with HIV infection

Recent policies, guidelines, and laws reflect promising preliminary outcomes among transplant recipients with HIV infection, and ethical analyses suggest that it is not justifiable to deny solid organ transplantation based solely on HIV-infection status. These studies consistently describe stable HIV disease following liver and kidney transplantation. Despite good graft survival, kidney allograft rejection occurs frequently, and serious non-AIDS-defining infections requiring hospitalization are common following antirejection therapy. Profound interactions between immunosuppressants and antiretroviral drugs require careful monitoring, dose adjustment, and highly effective communication between the patient and a multidisciplinary group of health care providers. Despite these scientific and policy advances, many health care providers and patients remain unaware of ongoing progress in this field. The implications are critical, as late referral for liver transplant evaluation increases the pretransplant mortality risk. Because important patient selection and clinical management questions remain, it is critical that ongoing studies are completed quickly.     

Clinical outcome of renal transplantation in patients with positive pre-transplant hepatitis B surface antigen

The clinical outcomes of 2,054 renal recipients were examined retrospectively based on pre-transplant hepatitis B surface antigen (HBsAg) status to investigate the efficacy of lamivudine treatment in HBsAg positive recipients. Pre-transplant HBsAg positivity was documented in 66 recipients. The 10-year patient and graft survival rates in Ag positive group were significantly lower than those of Ag negative group (64.4/36.6% vs. 88.2/70.5%, respectively, P < 0.0001). Since 1997, lamivudine was used when hepatitis B virus polymerase chain reaction (HBV PCR) was positive or when the level of post-transplant viral load rose. Lamivudine given to 27 recipients markedly improved both 10-year patient and graft survivals compared to Ag positive renal recipients who did not take lamivudine (85.3/59.2% vs. 49.9/22.7%, respectively, P < 0.0001). Overall, 13 viral breakthroughs among 24 lamivudine-responsive patients were observed. The cumulative incidence of viral breakthrough at 60 months was 53.3%. Adefovir rescue in three viral breakthroughs patients induces virological response and restoration of liver function. In 10 patients who did not changed to adefovir, 6 patients are alive with elevated liver enzymes. In conclusion, in the era of lamivudine and adefovir, renal transplantation in HBsAg positive end-stage renal disease patients should not be abandoned.     

Extending options for highly sensitized patients to receive a suitable kidney graft

Highly sensitized patients (anti-HLA) on the kidney waiting list wait longer for a suitable crossmatch negative organ. At the moment there are two strategies to enhance transplantation of these patients. One approach is the determination of acceptable HLA mismatches and application of this knowledge for the selection of crossmatch negative donors, and the second is the desensitization of patients with intravenous immunoglobulin-based protocols to enable transplantation of an organ from a donor towards which antibodies were originally present. Both approaches have advantages and disadvantages and are only successful in a proportion of the patients. The optimal solution is an integrated strategy whereby desensitization is used for those patients for whom the acceptable mismatch approach is not successful.     

Development of a measure of informed choice suitable for use in low literacy populations

OBJECTIVE: To assess the reliability and validity of a simplified questionnaire-based measure of informed choice in populations with low literacy. The measure comprises (a) knowledge about the test and (b) attitudes towards undergoing the test. Responses to (a) and (b) together with information on test uptake, are used to classify choices as informed or uninformed. METHODS: A cross-sectional study of 79 pregnant women (46 women with higher, and 33 with lower education levels) completed a simplified questionnaire, a standardised questionnaire and a semi-structured interview about antenatal sickle cell and thalassaemia (SCT) screening. The measures used were: (a) informed choice, based on knowledge about the test, attitudes towards undergoing the test, and uptake of the test and (b) ease of completion measures. RESULTS: The simplified measures of knowledge and attitudes were able to distinguish between women classified according to interview responses as having good or poor knowledge (knowledge scores 6.8 versus 3.2, p<0.001), and positive or negative attitudes towards undergoing the test (attitude scores 20.6 versus 16.2, p=0.023). There was no difference in rates of informed choice derived from the simplified or standardised measures (54% versus 51%, 95% CI difference -11 to 19). Women with lower levels of education found the simplified questionnaire easier to complete than the standardised version (11.0 versus 9.6, p=0.009). Those with higher levels of education found no difference in ease of completion between the two versions of the questionnaire (11.8 versus 11.6, p=0.54). CONCLUSION: A simplified questionnaire-based measure of informed choice in antenatal SCT screening is as reliable and valid as a more complex standardised version and for those with less education, easier to complete. PRACTICE IMPLICATIONS: The simplified questionnaire-based measure of informed choice is suitable for use in populations with low and high levels of education.     

Association of Interleukin-10 gene promoter polymorphisms in Saudi patients with vitiligo

The promoter region of human Interleukin -10 gene is highly polymorphic and has been associated with numerous autoimmune diseases. Recent studies have linked vitiligo with defective autoimmune system. This study is aimed to explore a possible association between IL-10 gene polymorphism and vitiligo in Saudi population. This case control study consisted of 184 Saudi subjects including 83 vitiligo patients (40 males, 43 females mean age 27.85 +/- 12.43 years) and 101 matched controls. Genomic DNA was extracted from the blood samples of healthy controls and Vitiligo patients visiting out patient clinic of Department of Dermatology, Riyadh Armed Forces Hospital, using QIA ampR DNA mini kit (Qiagen CA, USA). Interleukin-10 gene was amplified by polymerase chain reaction (PCR) using Arms primers to detect any polymorphism involved at positions -592, -819 and -1082. The frequencies of GG genotype at -1082, and CC genotype at positions -592 and 819 were significantly higher in vitiligo patients compared to healthy subjects suggesting that GG and CC genotypes might be susceptible to vitiligo in Saudis. On the other hand genotypes -1082 GA, -819 CT, and -592 CA of IL-10 were more prevalent in healthy controls suggesting protective effects of GA, CT and CA genotypes against vitiligo. This study indicates that the IL-10 gene may play a significant role in the etiology of vitiligo among Saudis.     

Bone marrow transplantation for patients with chronic myeloid leukemia

Between February 1981 and December 1984 we treated 52 patients with chronic myeloid leukemia in the chronic phase and 18 patients with more advanced disease by high-dose chemoradiotherapy followed by allogeneic bone marrow transplantation using marrow cells from HLA-identical sibling donors. In addition, the 40 patients who had not previously undergone splenectomy received radiotherapy to the spleen. To prevent graft versus host disease, cyclosporine was given either alone or in conjunction with donor marrow depleted of T cells. Of the 52 patients treated in the chronic phase, 38 are alive after a median follow-up of 25 months (range, 7 to 50); the actuarial survival at two years was 72 percent, and the actuarial risk of relapse was 7 percent. Of the 18 patients with more advanced disease, 4 have survived; the actuarial two-year survival was 18 percent, and the actuarial risk of relapse was 42 percent. We conclude that the probability of cure is highest if transplantation is performed while the patient remains in the chronic phase of chronic myeloid leukemia. T-cell depletion may have reduced the incidence and severity of graft versus host disease. The value of irradiation to the spleen before transplantation has not been established.     

Generation of EBV-specific CTLs suitable for adoptive immunotherapy of EBV-associated lymphoproliferative disease following allogeneic transplantation

Lymphoproliferative disease induced by EBV (EBV-LPD) is a complication of allogeneic transplantation caused by T-cell immunodeficiency. EBV-LPD responds poorly to conventional treatment modalities. Recently, adoptive transfer of EBV-specific, HLA-class I-restricted CTL lines was shown to be effective both as prophylaxis and as treatment of EBV-LPD. The CTL lines are produced by in vitro selection of EBV-specific memory T cells contained in MNC of EBV-seropositive individuals by repeated stimulation with irradiated EBV-transformed cells. The non-EBV-reactive and potentially alloreactive cells remain unactivated. Twenty-one CTL lines were initiated. All were successfully generated and shown to be HLA-restricted and EBV-specific as well as CD8/CD4 and CD45RO positive T cells. The majority of the CTL lines were generated from BMT donors, but two CTL lines were obtained from solid organ transplant recipients receiving immunosuppression. Evidence of probable in vivo efficacy is presented. Development of efficient adoptive T-cell strategies for EBV-positive malignancies could serve as a model for treatment of other viral or malignant disorders.     

Development and experience with an integrated system for transplantation telepathology

Rapid and accurate interpretation of allograft biopsies influences the outcome after organ transplantation. Expert histopathologic interpretation can also determine whether a donor organ should be used for transplantation or disposed. These and similar considerations in the field of Transplantation Pathology prompted us to develop a static image, store-and-forward telepathology system capable of rendering accurate, robust, and confidential communication by using readily available equipment and bandwidth capabilities for interactive real-time second opinion consultation. Between July 1999 and October 2000, 102 cases were transmitted, including 78 for second opinion and 1 for primary diagnosis with 6 (5 real-time) frozen sections. Full agreement with the original diagnosis was obtained in 67 of 78 (86%) cases; in 11 (14%) cases, teleconsultation resulted in 8 minor and 3 clinically significant differences of opinion. This led to a change in therapy in 1 case and further evaluation in 2 other cases. We conclude that static image, store-and-forward telepathology can enhance the practice of transplantation pathology, but a multidisciplinary team for ongiong support and development is required. This technology has the potential to promote case sharing, conduct continuing education, build consensus, and standardize readings of biopsies in multicenter trials in which histopathologic findings represent important outcome measures.     

Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing

Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval. Although it is a rare phenomenon, antibody-mediated pure red cell aplasia (PRCA) is a serious complication than can result from antibodies that develop and neutralize EPO as well as endogenous erythropoietin. Currently, there are no universally accepted analytical methods to detect the full repertoire of binding and neutralizing anti-EPO antibodies. A number of different methods that differ in terms of antibodies detected and assay sensitivities are used by different manufacturers. There is also a lack of antibody reference reagents, and therefore no consistent basis for detecting and measuring anti-EPO antibodies. Reference reagents, with established ranges, are essential to monitor the safety and efficacy of all erythropoiesis-stimulating agents (ESAs) structurally related to human erythropoietin. This is the first report of the development and characterization of a panel of fully human antibodies against EPO suitable as reference reagents. The characteristics of antibodies within the panel were selected based on the prevalence of non-neutralizing IgG and IgM antibodies in non-PRCA patients and neutralizing IgG antibodies, including IgG1 and IgG4, in antibody-mediated PRCA subjects. The reference panel includes antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing erythropoietin epitopes. The subclass of human antibodies in this reference panel includes an IgG1, IgG2, and IgG4, as well as an IgM isotype. This antibody panel could help select appropriate immunogenicity assays, guide validation, and monitor assay performance. Further, this human anti-ESA antibody panel may help set the limits of each assay platform in terms of the full repertoire of the anti-ESA antibodies, and may facilitate standardization of ESA immunogenicity reporting across assay platforms.     

Function-blocking autoantibodies to the melanin-concentrating hormone receptor in vitiligo patients

Vitiligo is a common depigmenting skin disorder resulting from the loss of melanocytes in the cutaneous epidermis. Although the cause of the disease remains obscure, autoimmune mechanisms are thought to be involved. Recently, melanin-concentrating hormone receptor (MCHR)-binding autoantibodies have been identified in vitiligo patients. In the present study, we aimed to determine if MCHR autoantibodies could also affect receptor function either by direct activation or by blocking its response to melanin-concentrating hormone. The results indicated that 10/18 (56%) vitiligo patient IgG samples inhibited the function of MCHR expressed in a Chinese hamster ovary cell line. In contrast, neither control (n=20) nor SLE patient (n=10) IgG samples blocked receptor function. Compared with healthy controls, MCHR function-blocking autoantibodies were found at a significantly increased frequency in the vitiligo patient group (P=0.0004). No MCHR-activating autoantibodies were detected in any of the vitiligo patient, SLE patient or control IgG samples that were analysed. In addition, vitiligo patient IgGs were tested for MCHR autoantibodies that could mediate antibody-dependent cell-mediated cytotoxicity via the receptor. However, this could only be demonstrated in two vitiligo patient sera. Overall, this work has provided additional evidence that MCHR is a B-cell autoantigen in vitiligo and has demonstrated the existence of MCHR function-blocking autoantibodies further to the receptor-binding autoantibodies previously reported.     

Peripheral blood as a preferable source of stem cells for salvage transplantation in patients with graft failure after cord blood transplantation: a retrospective analysis of the registry data of the Japanese society for hematopoietic cell transplantation

To compare the different stem cell sources used in salvage transplantation for graft failure (GF) after cord blood transplantation (CBT), we retrospectively analyzed data of 220 patients who developed GF after undergoing CBT between January 2001 and December 2007 and underwent a second hematopoietic stem cell transplantation (HSCT) within 3 months. The donor sources for salvage HSCT were cord blood (n = 180), peripheral blood stem cells (PBSCs; n = 24), and bone marrow (BM; n = 16). The cumulative incidence of neutrophil engraftment on day 30 after the second HSCT was 39% with CB, 71% with PBSCs, and 75% with BM. Multivariate analysis revealed that PBSC and BM grafts were associated with a significantly higher engraftment rate than CB (hazard ratio [HR], 7.77; P < .001 and HR, 2.81; P = .016, respectively). Although the incidence of grade II-IV acute?graft-versus-host disease was significantly higher in the PBSC group than in the CB group (HR, 2.83; P?= .011), the incidence of 1-year nonrelapse mortality was lower in the PBSC group than in the CB group (HR, 0.43; P = .019), and 1-year overall survival was superior in the PBSC group compared with the CB group (HR, 0.45; P = .036). Our results suggest that PBSC is the preferable source of stem cells in salvage HSCT for GF after CBT.     

Establishing a transport protocol for the delivery of melanocytes and keratinocytes for the treatment of vitiligo

We have previously developed a cell delivery and transfer technology for delivering autologous keratinocytes and melanocytes to patients with vitiligo. However, for this technology to benefit many patients geographically distant from the cell culture facility transportation issues need to be overcome. In this study we begin to investigate this by looking at what role surface chemistry and medium supplements, including fetal calf serum, CO? gassing, and temperature, play in influencing cell viability. Cells were maintained on carriers for up to 48 h outside of a CO? incubator at 37 °C and their subsequent ability to adhere and become organized into a new epithelium with appropriately located melanocytes was assessed. Consistently good viability and performance on an in vitro wound bed model was achieved by maintaining cells for 48 h adherent to a 20% acrylic acid coated carrier at lower (around 23 °C rather than 37 °C) temperatures in the medium preperfused with CO? before transport. Under these circumstances fetal calf serum was not required. In summary, the surface chemistry of the transport substrate and an appropriately CO? buffered medium at near room temperature can extend the effective performance life of these cultured cells to at least 48 h from when they leave standard incubator conditions.     

Pretreatment of human lymphocytes with interferon enhances the synthesis of interferon in cocultures with allogeneic cells

Human lymphocytes pretreated with interferon (IFN) alpha, beta, or gamma produced 17 times more IFN alpha (600-10,000 units/ml) than nontreated lymphocytes when cocultivated with allogeneic cells. Significant increases in IFN production (500-3,000 units/ml) were observed when lymphocytes were treated with IFN for just 2 h, and peak levels (10,000 units/ml) were produced after a 4-h treatment. The amount of IFN required to show the maximum priming effect was between 100 and 1,000 units; higher levels of IFN were inhibitory. The levels of IFN increased as the lymphocyte-to-target-cell ratio increased from 2:1 to 10:1 and decreased at higher ratios. The decrease in IFN production at higher ratios of lymphocytes to target cells could not be attributed to the presence of a soluble suppressor substance. The additional IFN found in supernates was attributed to enhanced production of IFN by the same cells, rather than recruitment of more cells to produce IFN. This conclusion is based on the fact that no increase in the number of cells staining positive for IFN production was observed in primed lymphocytes. The increased amount of IFN due to priming enhanced both nonsensitized cytotoxic activity and the transfer of antiviral activity, which could be prevented by antibody to IFN. The data suggest that priming may be an important biological mechanism for obtaining significant levels of IFN more rapidly in the vicinity of transformed cells or virus-infected tissues.