Long-term results of desensitization protocol with and without rituximab in sensitized kidney transplant recipients.

BACKGROUND: Desensitization protocols have been developed in order to overcome the immunological barrier of donor-specific anti-HLA antibodies (DSA). METHODS: During 2006-2012, we implemented a program for desensitizing sensitized (positive DSA, negative NIH-CDC crossmatch) living-donor recipients. The long-term outcome of 36 sensitized recipients, treated with IVIG and plasmapheresis (PP), with or without rituximab (added when > 7500 MFI), was compared to 252 non-sensitized living-donor recipients. RESULTS: Median peak DSA level before desensitization was 7223 (range 3567-16 000) MFI. During a mean follow-up of 121.9 months, graft loss occurred in 6/36 (17%) of the sensitized and 15/251 (6%) of the non-sensitized recipients (P = 0.021). Five-year and 10-year death-censored graft survival rates were 85% and 81% compared to 95% and 92%, respectively, for the non-sensitized recipients. There was no difference in recipients' survival. Slightly more episodes of acute rejection occurred in the sensitized group but had not influence on graft survival. At the last follow-up, 28 recipients had functioning graft; seventeen (47%) did not have detectable DSA. Eleven recipients had excellent graft function despite having detectable DSA. CONCLUSION: The long-term outcomes of sensitized recipients who underwent desensitization are encouraging. Adding rituximab to PP + IVIG in candidates with very high titers may result in improved outcome.

Serum Lactate Dehydrogenase is Elevated in Ischemic Acute Tubular Necrosis but Not in Acute Rejection in Kidney Transplant Patients.

BACKGROUND: Serum lactate dehydrogenase (LDH) levels may help to distinguish ischemic acute tubular necrosis (ATN) from acute rejection after kidney transplantation. METHODS: All kidney biopsies performed in the years 2010 to 2012 were reviewed. Serum LDH, creatinine level, clinical variables, and presence of donor-specific antibodies were recorded before the biopsy. RESULTS: Overall 150 biopsies were included. Ischemic ATN was diagnosed in 45 biopsies and acute cellular-mediated rejection and/or antibody-mediated rejection in 59 biopsies, 38 of which were accompanied by ATN. Serum LDH was elevated in 23 (51%) of 45 cases with ischemic ATN versus 15 (14%) of 105 cases with other diagnoses ( P < .0001). Median serum LDH was 478 U/L (range 277-2018) for ischemic ATN and 372 U/L (range 191-748) for all other diagnoses ( P < .001). When delayed graft function or primary nonfunctioning grafts were caused by ischemic ATN, serum LDH was elevated in 58% of cases, but when caused by acute rejection, LDH was normal in 88% of cases ( P = .02). CONCLUSIONS: There is a strong association between elevated serum LDH 1 to 3 days before performing kidney biopsy and the diagnosis of ischemic ATN after kidney transplantation, especially at the immediate posttransplantation period. Normal serum LDH at this period should raise a suspicion of acute rejection.

Luminex LiquiChip System for the Evaluation of Cytokine Levels in Patients with Contact Dermatitis to Potassium Dichromate.

BACKGROUND: Allergic contact dermatitis (ACD) is associated with increased production of cytokines. The patch test is the "gold-standard" diagnostic method, but it poses a risk of false results. OBJECTIVE: To evaluate a novel laboratory technique, the Luminex LiquiChip, which simultaneously measures blood levels of multiple cytokines, as a diagnostic tool in patients with chrome-induced ACD. METHODS: The study group included 20 patients with ACD and relevant patch test results for potassium dichromate and 19 patients with ACD for nickel or fragrance as control. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence and absence of potassium dichromate. The Luminex LiquiChip was used to measure levels of the following cytokines: granulocyte-macrophage colony-stimulating factor, interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon-γ, and tumor necrosis factor (TNF)-α. RESULTS: Potassium dichromate-stimulated PBMCs secreted significantly higher amounts of all cytokines except TNF-α than nonstimulated PBMCs. PBMCs from patients with ACD to chromium secreted significantly higher amounts of all cytokines tested, except IL-4, compared to PBMCs from patients with ACD to nickel or fragrance. CONCLUSIONS: Potassium dichromate stimulates the production of both Th1- and Th2-type cytokines in patients with chrome allergy. The Luminex LiquiChip is a promising in vitro method and may serve as a diagnostic tool for ACD.

Transglutaminase 6 Antibodies in the Serum of Patients With Amyotrophic Lateral Sclerosis.

IMPORTANCE: Celiac disease is an autoimmune disorder triggered by gluten in genetically predisposed individuals. Gluten sensitivity can cause neurologic manifestations, such as ataxia or neuropathy, with or without gastrointestinal symptoms. Many patients with gluten ataxia produce antibodies toward the newly identified neuronal transglutaminase 6 (TG6). Two case reports described patients initially diagnosed with amyotrophic lateral sclerosis (ALS) and ultimately with celiac disease who improved with a strict gluten-free diet. OBJECTIVE: To evaluate the prevalence of celiac disease-related antibodies and HLA antigen alleles, as well as TG6 antibodies, in patients with ALS and healthy individuals serving as controls to determine whether a neurologic presentation of a gluten-related disorder mimicking ALS might occur in some patients. DESIGN, SETTING, AND PARTICIPANTS: In a case-control study conducted in an ALS tertiary center, we measured serum levels of total IgA antibodies, IgA antibodies to transglutaminase 2 (TG2) and endomysium, as well as IgA and IgG antibodies to deamidated gliadine peptide and TG6 and performed HLA antigen genotyping in 150 consecutive patients with ALS and 115 healthy volunteers of similar age and sex. Participants did not have any known autoimmune or gastroenterologic disorder and were not receiving any immunomodulatory medications. The study was conducted from July 1, 2010, to December 31, 2012. MAIN OUTCOMES AND MEASURES: Antibody levels and frequency of individuals with abnormal antibody values as well as frequency of HLA antigen alleles were compared between patient and control groups. RESULTS: All patients and control group participants were seronegative to IgA antibodies to TG2, endomysium, and deamidated gliadine peptide. Twenty-three patients (15.3%) were seropositive to TG6 IgA antibodies as opposed to only 5 controls (4.3%) (P = .004). The patients seropositive for TG6 showed a classic picture of ALS, similar to that of seronegative patients. Fifty patients and 20 controls were tested for celiac disease-specific HLA antigen alleles; 13 of 22 TG6 IgA seropositive individuals (59.1%) were seropositive for celiac disease-related alleles compared with 8 (28.6%) of the 28 seronegative individuals (P = .04). Mean (SD) levels of IgA antibodies to TG2 were 1.78 (0.73) in patients and 1.58 (0.68) in controls (normal, <10). In a subset of study participants, mean levels of deamidated gliadin peptide autoantibodies were 7.46 (6.92) in patients and 6.08 (3.90) in controls (normal, <16). Mean levels of IgA antibodies to TG6 were 29.3 (30.1) in patients and 21.0 (27.4) in controls (P = .02; normal, <26). CONCLUSIONS AND RELEVANCE: The data from this study indicate that, in certain cases, an ALS syndrome might be associated with autoimmunity and gluten sensitivity. Although the data are preliminary and need replication, gluten sensitivity is potentially treatable; therefore, this diagnostic challenge should not be overlooked.

Lichen planopilaris is associated with HLA DRB1*11 and DQB1*03 alleles.

There are no studies of the possible association of the human leukocyte antigen (HLA) system with lichen planopilaris (LPP). To determine whether the HLA system is associated with LPP, 40 consecutive Jewish Israeli patients with LPP (study group) and 252 volunteers (controls) were typed for DRB1*and DQB1* loci by molecular methods. Compared with controls, the study group had a significantly higher frequency of the DRB1*11 allele (62% vs. 21%, corrected p-value (pc) = 0.001) owing to increased frequencies of DRB1*11: 01 and DRB1*11: 04. The DQB1*03 allele was also expressed at a significantly higher frequency in the study group (70% vs. 33%, pc = 0.0005); specifically, the frequency of DQB1*03: 01 was increased. The majority (82.5%) of the patients were of non-Ashkenazi origin. We conclude that LPP appears to be over-represented in non-Ashkenazi Jewish patients and is associated with an increased frequency of HLA DRB1*11 and DQB1*03 alleles. These findings suggest that immunogenetic factors play a role in LPP.

Age-dependent HLA profiles of the Israeli population: impact on hematopoietic cell donor recruitment and availability.

Approximately three million people have immigrated to the state of Israel since it was founded. Consequently, the immunogenetic profile of the younger generation may consist of a genetic mixture of formerly distinct population groups. We aimed to investigate whether HLA profiles in the Israeli population are age dependent and how this influences representation of various age groups in local donor registries. We determined HLA-A*, HLA-B*, and HLA-DRB1* low-resolution phenotypes of three age groups (n = 4,169 in each): (1) cord blood units collected between 2009 and 2013 (BABIES) and adult registry donors (2) aged 18-28 years (YOUNG) and (3) aged 49-60 years (OLD). We compared the results with virtual groups that simulate the offspring of the actual study groups. None of the three actual age groups were in Hardy-Weinberg equilibrium. The YOUNG presented four HLA-B alleles that were absent in the OLD and BABIES. A significantly higher percentage among the OLD and BABIES had a "matched" individual within their group in comparison to the YOUNG. In the YOUNG, the 10 most common haplotypes account for 16.7 % of the population, in comparison to 18.2 % in the OLD or 19.8 % in the BABIES group. The BABIES group was genetically remote from all other groups. Further disparities were found between the actual and the corresponding virtual groups. We conclude that discrete age groups in Israel present distinct immunogenetic profiles, where the younger generation is more heterogeneous. The population dynamics of the age-dependent HLA profile is multifactorial: gradual intersubgroup admixture, nonrandom mating, and entry of new alleles.

Genetic considerations in human sex-mate selection: partners share human leukocyte antigen but not short-tandem-repeat identity markers.

PROBLEM: Previous studies support a role for MHC on mating preference, yet it remains unsettled as to whether mating occurs preferentially between individuals sharing human leukocyte antigen (HLA) determinants or not. Investigating sex-mate preferences in the contemporary Israeli population is of further curiosity being a population with distinct genetic characteristics, where multifaceted cultural considerations influence mate selection. METHOD OF STUDY: Pairs of male-female sex partners were evaluated in three groups. Two groups represented unmarried (n = 1002) or married (n = 308) couples and a control group of fictitious male-female couples. HLA and short-tandem-repeat (STR) genetic identification markers were assessed for the frequency of shared antigens and alleles. RESULTS: Human leukocyte antigen results showed that Class I and/ or Class II single antigen as well as double antigen sharing was more common in sex partners than in control group couples (P < 0.001). Married versus unmarried pairs were not distinguishable. In contrast, STR-DNA markers failed to differentiate between sex-mates and controls (P = 0.78). CONCLUSION: Sex partnerships shared HLA determinants more frequently than randomly constituted male-female pairs. The observed phenomenon does not reflect a syngenetic background between sex-mates as STR markers were not selectively shared. Thus, sex-mate selection in man may contravene the evolutionary pressure for genetic diversity in regard to HLA.

Utility of immune monitoring in heart transplant recipients on everolimus-based immune suppression.

BACKGROUND: Everolimus provides effective immune suppression (IS) after heart transplant (HTx). Its pharmacologic properties differentiate everolimus from other IS drugs. A non-invasive immune monitoring (IM) assay test appears to predict the immune state in HTx recipients on standard calcineurin-inhibitor-based IS. The utility of IM in HTx recipients on everolimus-based IS was evaluated. METHODS: Between June 2005 and June 2011, 34 adult HTx recipients followed up at our center received everolimus and had 381 IM assays that were performed at six months to 16-yr post-transplant. Results of the IM assay were correlated with infection and rejection episodes that occurred during the IM testing. RESULTS: In the everolimus-based IS group, there were 18 infectious episodes and four rejection episodes. The average IM score was significantly lower during infection than at steady state (188 ± 122 vs. 338 ± 137 ng/mL ATP, p < 0.001) and not significantly different during rejection when compared with steady state (430 ± 132 vs. 338 ± 137 ng/mL ATP, p = 0.5). CONCLUSIONS: The non-invasive IM assay predicts infectious risk in HTx recipients on everolimus-based IS. Its inconclusive association with rejection was probably due to the small number of rejections. Serial longitudinal IM may allow proper adjustment of everolimus doses.

Periodic fever, aphthous stomatitis, pharyngitis and cervical adenopathy syndrome is associated with activation of GM-CSF and burst-like expression of IL-8 in peripheral blood.

INTRODUCTION: Periodic fever, aphthous stomatitis, pharyngitis and cervical adenopathy (PFAPA) is an autoinflammatory syndrome characterized by periodic fever with aphthous stomatitis, cervical lymphadenopathy, myalgia, and abdominal pain. Peripheral blood concentrations of selected cytokines of PFAPA patients during and between febrile episodes were analyzed in a search for PFAPA-specific molecular signature. METHODS: 23 children with PFAPA (age 6.07 ± 2.94 years, range 5-9 years) and three control children with severe oropharyngeal infections (age 6.2 ± 7.95 years, range 1-17 years) participated in the study. Peripheral blood concentrations of IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-γ, GM-CSF, TNF-α were measured using Luminex technology. RESULTS: PFAPA febrile episodes were characterized by detection of GM-CSF - 134.07 ± 315.5 pg/mL; significant (P < 0.001), compared to baseline and controls, elevation of concentrations of IL-8 (3193.7 ± 2508 pg/mL vs. 100.36 ± 119. pg/mL vs. 2.04 ± 4.08 pg/mL, respectively), IL-6 (1355.38 ± 2026.53 pg/mL vs. 28.8 ± 44.2 pg/mL and 27.13 ± 26.42 pg/mL, respectively). IL-1ß was detected only in febrile and afebrile PFAPA patients (922.8 ± 1639 pg/mL vs. 10.98 ± 19.4 pg/ml, P < 0.002, respectively), but not in controls. Peripheral blood concentration of TNFα did not differ significantly between study groups. IL-2, IL-4, IL-5, and IL-10 were negligible in all study subjects. DISCUSSION: PFAPA febrile episodes are characterized by activation of GM-CSF and IL-8 with Th1 skewing. We propose a molecular mechanism governing this phenomenon.

Trends and challenges in searching for HLA-matched unrelated donors in Israel.

Despite the large local donor registries, for some Israeli patients an HLA-matched unrelated donor cannot be identified within a favorable time period. This study aims to retrospectively analyze donor search processes in our transplantation center in order to provide a basis to enhance the efficacy of these procedures. We analyzed 121 donor searches performed in the last 5 years in our center and investigated various parameters characterizing these searches. For over 80% of patients, a donor matched at least at 9/10 alleles was identified, mostly from the local donor registries. The probability of finding a 10/10 HLA-matched donor in a local registry for patients of the Arab minority was significantly lower than patients of Jewish origin (25% versus 64%, respectively; p<0.05). Furthermore, we found that identifying a fully matched donor for an adult patient is significantly more probable than for a pediatric patient (66% versus 43%, respectively; p<0.05). These data indicate of dissimilarities between HLA profiles of the older versus the younger patients, which influence their probability to find a suitable donor. In conclusion, in spite of the large size of local donor registries, they do not optimally reflect the immunogenetic profiles of patients of ethnic minorities and of pediatric patients.

The immune-modulator AS101 reduces anti-HLA antibodies in sera of sensitized patients: a structural approach.

BACKGROUND: Significant efforts are dedicated to identification of agents that eliminate anti-HLA antibodies (Ab) from sera of transplant candidates. Antibody titer following in vitro incubation of sera with desensitizing agents has shown to reflect the probability that a patient would benefit from clinical de-sensitization protocols. AS101 is a non-toxic, synthetic, organic tellurium compound. The aim of this research was to assess the ability of AS101 to reduce anti-HLA Abs and to identify patients likely to benefit from this effect. METHODS: Sera of sensitized patients awaiting transplant were incubated in the presence of AS101. Measured mean fluorescence intensity (MFI) represents reactivity of anti HLA Abs in the serum, as detected by the Luminex platform. The repertoire of HLA antigen epitopes was recognized using HLA Matchmaker software. RESULTS: AS101 Incubation caused a significant Ab titer decrease in approximately two thirds of the samples. The median Class I and II MFI decrease among the responding samples was 16.7% and 14%, respectively (p<0.05). HLA Matchmaker analysis of the patients' class I epitope sequences revealed apparent amino-acid differences between the patterns of the responding and non-responding patients. CONCLUSION: In vitro incubation of sera in the presence of AS101 causes a decrease in the anti-HLA Ab's reactivity in several patient samples. Sera most likely to demonstrate this effect are characterized by a moderate MFI level and a distinct antibody reactivity pattern specific for defined HLA antigen epitopes. These results support further investigation of AS101 as a potential agent for desensitization of humoral reactivity prior to transplantation.

Confronting the challenge: individualized immune monitoring after organ transplantation using the cellular immune function assay.

The survival of a transplanted organ is dependent on avoidance of rejection, achieved through continuous immuno-suppression. Management of the transplant recipient confronts the clinician with a key challenge of post-transplant immune monitoring. Early detection of an activated allo-immune response is a harbinger of incipient rejection. Thus, timely intervention may prevent acute and chronic injury to the transplanted organ. Similarly, over immune-suppression can lead to infections or malignancies, hence the importance of early detection of the precarious suppression. The need for non-invasive systemic immune monitoring of the transplant recipient is therefore imperative. This review describes the cellular immune function assay--a non-invasive diagnostic method for evaluation of the net state of the recipient's cellular immune function. We describe the background that brought about the need for a reliable diagnostic tool for serial immune monitoring, and we overview the main mile-stones in the assimilation of the assay and its implementation in the clinic. The arising conclusion presents a novel non-invasive diagnostic bio-marker for post-transplant immune monitoring which enables the clinician to intervene prior to manifestation of clinical complications. The usefulness of the assay in detecting a state of over-suppression has been consensually described in multiple publications while its contribution in detection and management of under-suppression conditions remains to be determined by means of prospective interventional studies. The cellular immune function assay can be useful and beneficial for patient care only if used for longitudinal monitoring through serial testing.

Influence of histocompatibility genes on disease susceptibility and treatment response in patients with relapsing-remitting multiple sclerosis treated with interferon ß-1a.

Multiple sclerosis (MS) is the most common, non-traumatic cause of neurological disability in young adults. The aim of this study was to investigate the influence of HLA class II alleles DRB1* and DQB1* on susceptibility to relapsing-remitting (RR) MS and response to interferon (IFN) ß-1a treatment. A prospective observational study was conducted. Seventeen patients with clinically definite RRMS, attending a tertiary referral center for multiple sclerosis in Israel and receiving treatment with subcutaneous IFN ß-1a, 22 mcg three times weekly were recruited between December 1998 and February 2000 and observed for 12 months. HLA genotyping was performed and clinical characteristics (relapse rate and disability progression) assessed at baseline and after 12 months. HLA data for a healthy control group were also used for comparison. HLA and the success of treatment with IFN ß-1a in this group of RRMS patients were assessed. The frequency of DRB1*03 was six times higher in patients treated with IFN ß-1a than in the healthy control group (n=100): 29% (5/17) versus 5% (5/100), respectively. Additionally, DQB1*03 and DQB1*02 were present in 82% (14/17) and 41% (7/17) of RRMS patients, but in only 33% (33/100) and 18% (18/100) of control patients, respectively. A better response to IFN ß-1a treatment was seen in patients carrying these alleles than in patients without these alleles. Our results indicated that DRB1*03, DQB1*03 and DQB1*02 alleles may contribute to MS susceptibility and IFN ß-1a responsiveness, and warrant further verification in a larger population.

TCR-independent killing of B cell malignancies by anti-third-party CTLs: the critical role of MHC-CD8 engagement.

We previously demonstrated that anti-third-party CTLs (stimulated under IL-2 deprivation against cells with an MHC class I [MHC-I] background different from that of the host and the donor) are depleted of graft-versus-host reactivity and can eradicate B cell chronic lymphocytic leukemia cells in vitro or in an HU/SCID mouse model. We demonstrated in the current study that human allogeneic or autologous anti-third-party CTLs can also efficiently eradicate primary non-Hodgkin B cell lymphoma by inducing slow apoptosis of the pathological cells. Using MHC-I mutant cell line as target cells, which are unrecognizable by the CTL TCR, we demonstrated directly that this killing is TCR independent. Strikingly, this unique TCR-independent killing is induced through lymphoma MHC-I engagement. We further showed that this killing mechanism begins with durable conjugate formation between the CTLs and the tumor cells, through rapid binding of tumor ICAM-1 to the CTL LFA-1 molecule. This conjugation is followed by a slower second step of MHC-I-dependent apoptosis, requiring the binding of the MHC-I α2/3 C region on tumor cells to the CTL CD8 molecule for killing to ensue. By comparing CTL-mediated killing of Daudi lymphoma cells (lacking surface MHC-I expression) to Daudi cells with reconstituted surface MHC-I, we demonstrated directly for the first time to our knowledge, in vitro and in vivo, a novel role for MHC-I in the induction of lymphoma cell apoptosis by CTLs. Additionally, by using different knockout and transgenic strains, we further showed that mouse anti-third-party CTLs also kill lymphoma cells using similar unique TCR-independence mechanism as human CTLs, while sparing normal naive B cells.

Individualized immune monitoring of cardiac transplant recipients by noninvasive longitudinal cellular immunity tests.

BACKGROUND: Common immunosuppression strategies after heart transplantation (HTx) are based on accepted target drug levels, disregarding that drug levels do not correlate with the individual patient's pharmacokinetics or with the actual immunosuppressive drug effect on the patient. The Immuknow assay is used for immune monitoring and management of organ transplant recipients. This study evaluated the Immuknow assay for longitudinal immune monitoring of HTx patients throughout various clinical settings. METHODS: The functional immune response as measured by the Immuknow assay was determined in 327 samples collected from 50 HTx patients at the Rabin Medical Center and was analyzed together with common clinical parameters. RESULTS: The median Immuknow levels measured throughout the infection episodes and the episodes of biopsy-proven acute rejection were 129 and 619 ng ATP/mL, respectively. These values were significantly dissimilar to the median Immuknow level measured during clinical quiescence, which was 351 ng ATP/mL (P<0.05). Calcineurin inhibitors drug-level measurements did not provide a reliable depiction of the patients' immune function, because the median deviation from the recommended drug trough levels range was significantly higher than the median deviation of Immuknow levels from their expected immune response zones. Longitudinal monitoring of Immuknow levels through serial testing proved to be a reliable method for individual patient immune management. CONCLUSIONS: The Immuknow assay reliably reflects the cellular immune function of HTx patients, thereby supporting the immune monitoring and management of these patients. Serial longitudinal Immuknow monitoring allows immune management of therapy according to the individual patient's immune status.

ImmuKnow: a new parameter in immune monitoring of pediatric liver transplantation recipients.

Lifelong immunosuppression is mandatory for optimal graft and patient survival following liver transplantation. Nevertheless, graft rejection or numerous adverse events associated with overimmunosuppression or underimmunosuppression cannot be completely avoided. The ImmuKnow assay measures cell-mediated immunity and is able to discern between conditions of overimmunosuppression and underimmunosuppression. The aim of this study was to evaluate the ImmuKnow assay in the evaluation of the immune function in pediatric liver transplant recipients and to assess its correlation with the patients' clinical and biochemical status. Eighty-nine whole blood samples were collected from 23 liver transplant recipients that were 1 to 18 years old. The net state of immune function was determined by the quantitative measurement of the intracellular adenosine 5-triphosphate level in CD4+ lymphocytes after phytohemagglutinin stimulation. Comprehensive clinical data were correlated with the ImmuKnow assay results. In 23 of the 28 samples collected during clinical quiescence, ImmuKnow results were correlated with the clinical status, expressing the patient's moderate immune function. However, a correlation between measured therapeutic drug levels and clinical quiescence was found in only 18 of the 28 samples. In 6 patients who suffered from clinical complications, ImmuKnow measurements showed a wide range of deviations, expressing the unstable immunological status of these patients. In conclusion, the ImmuKnow assay correlates with the clinical status of liver-transplanted children. It serves as a reliable and unique parameter of the cellular immune function. We conclude that the ImmuKnow assay, together with existing clinical tools, may allow for the immune monitoring of pediatric liver recipients.

Immunogenetics of HLA class II in Israeli patients with adult-onset type 1 diabetes mellitus.

The distribution of HLA class II alleles and genotypes in Israelis of different ethnic origin with adult-onset type 1 diabetes (T1D) was examined. The results were compared with published findings in healthy Israelis and childhood-onset T1D Israelis. An additional comparison was made between subgroups of patients with rapidly and slowly progressive adult-onset T1D (LADA). A DNA-based low-resolution analysis was performed for DRB1* and DQB1* alleles and a high-resolution analysis for DRB1*04 and DQB1*1 alleles. In all, 87% of the study group was positive for DRB1*03 or DRB1*04 compared with 36% of the healthy controls. The main alleles accounting for susceptibility to T1D were DRB1*0402, found in 77.9% of carriers of DRB1*04 and DQB1*0302, found in 74.6% of carriers of DQB1*03. The DQB1*0602 was not detected in any patient. The distribution was similar to that reported in Israeli children with T1D and significantly different from healthy Israelis. There was no significant difference in the distribution of HLA class II alleles between patients with rapidly progressive T1D or LADA. It may be concluded that the different ages of onset of T1D and its different forms of development in Israeli patients are apparently not caused by a different prevalence of HLA class II alleles.

Preceeding the rejection: in search for a comprehensive post-transplant immune monitoring platform.

The survival of a transplanted organ is dependent on maintenance of continuous immunosuppression. However, even the strictest adherence to the recommended drug levels does not prevent the occurrence of numerous complications associated with immunosuppression. The efficacy of immunosuppression therapy protocols would be enhanced greatly by the availability of biotechnologies capable of identifying and predicting immunological events prior to the manifestation of clinical parameters indicating graft failure. The aim of the study was to evaluate the potential contribution of some modern tools for post-transplantation monitoring, and to propose a method for combining them into a comprehensive mechanism for this purpose. The technologies utilized in this study are among a group of 'cutting edge' diagnostic methods at the initial steps of evaluation for their potential contribution for post-transplantation immune monitoring. This study was a pioneering opportunity to combine and utilize these tools jointly. The method of research was based on monitoring 13 adult kidney transplant recipients. The Immuknow assay determined cellular immunity status by quantitative measurement of intracellular ATP level in CD4(+) lymphocytes after PHA stimulation. Sera were analyzed for concentration of soluble CD30 reflecting primary allo-stimulation and for donor specific anti-HLA antibodies responsible for accelerated and refractory rejection. The results were correlated with clinical and pathological parameters and appraisal of predictive value was attempted. In Immuknow assay analysis ATP incremental changes indicative of rejection or infection were found in 75% and in 50% incidences, respectively. In stable patients, the ATP deviation from the preoperative baseline, indicative of stable engraftment, was much less pronounced than in other habitual clinical tests. CD30 concentrations were measured greatly above normal values prior to biopsy-proven rejection episodes, both before and after the transplant operation. Anti-HLA antibodies were elevated at a later stage, concurrently with clinical manifestation of graft failure and rejection. Anti-HLA antibody level remained negligible in patients going through a stable post-transplant clinical course. Overall, the utilization of the platform of combined biotechnologies could serve as a valuable tool for immune monitoring in organ transplantation, allowing for therapeutic intervention that can favorably affect the clinical outcome.

Frontiers of stem cell transplantation monitoring: capturing graft dynamics through routine longitudinal chimerism analysis.

Quantitative chimerism testing has become an indispensable tool for following the course and success of allogeneic hematopoietic stem cell transplants. In this paper, we describe the current laboratory approach to quantitative chimerism testing based on an analysis of short tandem repeats, and explain why performing this analysis longitudinally is important and feasible. Longitudinal analysis focuses on relative changes appearing in the course of sequential samples, and as such exploits the ultimate potential of this intrinsically semi-quantitative platform. Such an analysis is more informative than single static values, less likely to be confused with platform artifacts, and is individualized to the particular patient. It is particularly useful with non-myeloablative conditioning, where mixed chimerism is common. When longitudinal chimerism analysis is performed on lineage-specific subpopulations, the sensitivity, specificity and mechanistic implications of the data are augmented. Importantly, longitudinal monitoring is a routinely feasible laboratory option because multiplex STR-PCR kits are available commercially, and modern software can be used to perform computation, reliability testing, and longitudinal tracking in a rapid, easy to use format. The ChimerTrack application, a shareware program developed in our laboratory for this purpose, produces a report that automatically summarizes and illustrates the quantitative temporal course of the patient's chimeric status. Such a longitudinal perspective enhances the value of quantitative chimerism monitoring for decisions regarding immunomodulatory post-transplant therapy. This information also provides unique insights into the biological dynamics of engraftment underlying the fluctuations in the temporal course of a patient's chimeric status.

Platelet-derived growth factor gene polymorphism in recurrent hepatitis C infection after liver transplantation.

BACKGROUND: Recurrent hepatitis C virus (HCV) infection is particularly aggressive in the post liver transplantation setting, with rapid progression of liver fibrosis. Platelet-derived growth factor (PDGF) is reportedly involved in the pathogenesis of liver fibrosis. The aim of this study was to evaluate the possible contribution of molecular variants of the PDGF-B gene to recurrent HCV infection after liver transplantation. METHODS: DNA was extracted from peripheral blood mononuclear cells of 40 patients who underwent liver transplantation for chronic HCV infection and genotyped for polymorphisms in PDGF-B at positions +1135 (A to C) and +286 (A to G). Intrahepatic PDGF-B expression was detected by immunohistochemistry and assessed semiquantitatively. Forty-seven healthy individuals served as controls. RESULTS: Recurrent HCV infection occurred in 34 patients (85%) after a median interval of 10.5 months (range 1.5-60.0). A statistically significant difference was observed in the distribution of the PDGF-B gene polymorphism at position +1135, but not +286 between patients and controls (P=0.05). The A/A genotype occurred at a highly significantly increased rate in patients with recurrent HCV infection than in those without (64.7% vs. 16.67%, P=0.0001), and in patients with severe than in those with nonsevere recurrence (100% vs. 53.85%, P=0.05). The expression level of intrahepatic PDGF-B was found to be highly correlated with the fibrosis stage (P<0.0001). Further analysis yielded a highly statistically significant relationship between the PDGF-B gene polymorphism at position +1135 and clinical parameters of disease severity. CONCLUSIONS: PDGF-B gene polymorphism appears to be associated with severe recurrent HCV infection after liver transplantation. PDGF-B may play an essential role in the development and progression of hepatic fibrosis. These findings, if confirmed, may have important therapeutic implications.