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background: Soluble HLA-G (sHLA-G) has been suggested as a non-invasive marker for embryo selection to improve pregnancy rates after assisted reproduction technique (ART). Our study aimed at the identification of parameters influencing the detection of sHLA-G in embryo cultures (ECs) and at the prognostic relevance of sHLA-G in a multi-centre study. methods: In total 4212 EC from 2364 cycles were randomly collected from 29 German ART centres and analysed for sHLA-G by Luminex w -based technology. results: Among test and culture conditions, only the cleavage stage of the embryo was identified as an independent factor for sHLA-G detection (P , 0.001). Overall, sHLA-G was significantly associated with pregnancy after ART [P , 0.001; odds ratio: 2.0 (95% CI: 1.7– 2.4)], suggesting that sHLA-G testing might improve the pregnancy rate from 30 to 40%. Importantly, the sHLA-G status of embryos could be associated with pregnancy after single embryo transfer [P ¼ 0.002; odds ratio: 3.3 (95% CI: 1.5 –6.8)] doubling the probability of pregnancy rate to 26% after sHLA-G testing. The patient’s age, number of transferred embryos, morphological grading [EXP(B): 4.3 (95% CI: 2.1– 8.9)] of embryos and sHLA-G status [EXP(B): 2.3 (95% CI: 1.8 –3.1)] were independent predictors of pregnancy, with the latter two being most powerful. conclusions: This study provides significant evidence that the morphological scoring system is still the best strategy for the selection of embryos but that sHLA-G might be considered as a second parameter if a choice has to be made between embryos of morphologically equal quality.

Soluble or membrane-anchored ligands of NKG2D and their receptor have a critical role in the elimination of tumor cells and disease progression. Plasma samples of 98 patients with B-cell chronic lymphocytic leukemia (CLL) were analyzed with specific ELISA systems for soluble major histocompatibility complex class I-related chains (sMICA and sMICB) and UL-16-binding proteins (ULBP1, 2, and 3). The flow cytometric analysis of MICA on CLL cells and natural killer group 2 member D (NKG2D) receptors on NK cells was performed after thawing of frozen peripheral blood lymphocytes of CLL patients (N=51). Levels of sMICA, sMICB, and sULBP2 were significantly increased (P0.001) compared with 48 controls, whereas sULBP1 3 were not detectable in patients and controls. Levels of sMICA990 pg/ml (P=0.014), sMICB200 pg/ml (P=0.0001), and sULBP2105 pg/ml (P0.0001) were associated with poor treatment-free survival (TFS). Neither MICA nor NKG2D expression could be related to clinical parameters. In multivariate analysis Binet stage (P=0.002), sULBP2 (P=0.002) and ZAP-70 (P=0.002) were independent predictive factors for TFS. In patients with Binet stage A, sULBP2 levels105 pg/ml were strongly associated (P=0.0025) with poor TFS. Our data show that soluble but not membrane-anchored NKG2D ligands or receptors are of prognostic significance in CLL. Moreover, sULBP2 seems to be useful to identify early-stage patients with risk of disease progression.

The expression of human leukocyte antigen (HLA)-G, a nonclassical HLA class I molecule, and its soluble forms (sHLA-G) are found to improve graft acceptance. In this study we investigated whether sHLA-G is the most biologically relevant molecule among all types of soluble HLA class I molecules for graft acceptance. We addressed this question in kidney-transplanted (n = 32) and kidney/pancreas-transplanted patients (n = 29). To this end we analyzed the levels of total soluble HLA class I (sHLA-I) in comparison to sHLA-G in 488 plasma samples procured before and serial after transplantation by specific enzyme-linked immunoabsorbent assay. Samples from 126 healthy individuals served as controls. Pretransplantation sHLA-I levels were significantly increased in patients (p < 0.001), whereas sHLA-G levels were in the range of those of healthy controls. Importantly, pretransplantation sHLA-I and sHLA-G levels did not differ between the two groups. Patients with biopsy-proven rejection (n = 15) revealed significantly lower sHLA-G levels before transplantation (mean +/- standard error of the mean, 12.9 +/- 1.8 vs. 20.1 +/- 1.9, p = 0.013) and after transplantation (p = 0.006, two-way analysis of variance) than patients without rejection (n = 46). In contrast, sHLA-I was slightly increased after but not before transplantation in patients with rejection (p < 0.05, two-way analysis of variance). Nonparametric determination analysis showed that pretransplantation levels of sHLA-G < 11.5 ng/ml (sensitivity, 60%; specificity, 80.4%) were related to rejection. Regarding antibody status, retransplantation, number of HLA mismatches, recipient age, and recipient body mass index, multivariate analysis showed that sHLA-G but not sHLA-I is an independent risk factor for graft rejection. Thus high levels of sHLA-G but not of sHLA-I seem to contribute to better graft acceptance after kidney or kidney/pancreas transplantation.

This study examined the performance of a tuberculosis (TB)-specific enzyme-linked immunospot assay in 48 patients awaiting liver transplantation. They were tested with T-SPOT.TB, tuberculin skin test (TST), and lymphocyte transformation test (LTT) using tuberculin as a stimulus. A questionnaire was used to gain information on TB exposure. Four patients were defined as positive by T-SPOT.TB, 6 by TST, and 28 by LTT. The patients displaying positive results to T-SPOT.TB were also positive in the TST and LTT. We considered them to have latent TB because they were repeatedly (two or three times) positive to the T-SPOT.TB and reported TB exposure. Active TB was excluded by negative multislice computed tomography, negative culture, and absence of symptoms. In 1 patient, T-cell reactivity toward TB peptides was lost 1 and 2 months posttransplantation. Another patient, however, tested 8 and 13 months posttransplantation, displayed measurable cellular TB immune responses. This finding suggests that the measurement of cellular TB immune responses shortly after transplantation may fail. If possible, patients with end-stage liver disease should be screened for TB prior to transplantation. Our data are the first evidence that T-SPOT.TB may be useful to diagnose latent TB in patients awaiting liver transplantation.

Human amniotic fluid cells, known to express HLA-A, -B, and -C antigens, were tested for the presence of lymphocyte-stimulating antigens (LD or HLA-D) using modifications of the mixed lymphocyte culture (MLC) and primed lymphocyte typing (PLT) tests. Peripheral blood lymphocytes were co-cultured with various concentrations of allogeneic amniotic fluid cells, either growing as a monolayer culture in microtiter plates or suspended in medium following treatment with trypsin. The kinetics of such mixed lymphocyte amniotic fluid cell culture (MLAC) reactions were followed during days 3 to 8. Under none of these conditions did amniotic fluid cells significantly stimulate allogeneic lymphocytes, even after lymphocytes were specifically primed in the PLT assay to the HLA-D antigens segregating in the family of the amniotic fluid cell donor. Furthermore, in three-cell experiments, amniotic fluid cells failed to inhibit an ongoing MLC reaction, indicating that the absence of proliferative response to amniotic fluid cells is not due to active suppression. Taken together, these data strongly suggest that amniotic fluid cells either do not express HLA-D antigens or do not express them in a form that is detectable in either primary or secondary MLC.

Sixty-two unrelated Caucasian patients from the Munich area who had had paralytic poliomyelitis in the 1950s and the early 1960s were analyzed. HLA—A and —B typing was performed for 26 antigens. HLA—D typing was done using six different established homozygous typing cells defining the specificities Dw1-Dw5 and Dw11, plus one locally defined typing cell. None of the HLA—A, —B or —D determinants showed a significant deviation in frequency from control populations. Of interest may be a decrease of B8 and an increase of Bw16 (Bw38/39) and B27, but these deviations are not significant if their P values are corrected for the number of comparisons made.

The genetics of the MLR of the dog are studied using LD typing cells. The four new homozygous reference cells described bring the total number of defined LD specificities in this species to 11. In families, LD specificities generally segregate en bloc with the SD specificities of the DLA complex. The recombination frequency between the SD and LD loci is provisionally estimated to be 0.007. A slight but significant stimulation between two SD identical LD typed siblings could not be explained on the basis of a single recombination between the SD loci and one LD locus and therefore suggested the existence of an additional LD system. Unrelated SD different individuals with two different LD determinants in common frequently stimulate each other in MLR, but low responses are also encountered. The results described are compatible with a hypothesis of two segregant series of alleles for the genetic control of the MLR and linkage disequilibrium between certain alleles of these series. More conclusive evidence of the validity of this hypothesis and further knowledge of other factors which determine the response in the MLR are required before a reliable estimate of the importance of LD structures in histocompatibility can be made.

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23–35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68–100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed towards cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.

CD4+CD25+ regulatory T (Treg) cells play a major role in controlling autoimmunity by suppressing self-reactive T cells. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system, where T cells are of major importance. Treg cell frequency and function are potential therapeutic targets of MS treatment. Mitoxantrone (MX) is a potent immunosuppressant for the treatment of active MS. We investigated the long-term effects of MX on the suppressive function of Treg after mitogen and myelin basic protein (MBP) stimulation in 20 MX-treated patients. MX therapy did not restore the reduced suppressive activity of a mixture of CD25intermediate or CD25high expressing Treg (healthy controls, MBP: 37.3%; MS patients, MBP: –1.9 vs. 6.6% suppression after MX treatment for 6 months, p > 0.2). The frequency of Treg cells was not affected by MX. A single infusion of MX (10 mg/m2 body surface) induced a selective and persistent reduction of approximately 30% of absolute and relative counts of B lymphocytes (p < 0.05). MX therapy does not influence Treg cell frequency or function. MX seems to exhibit its efficacy in MS mainly by a suppression of humoral immunity.

Summary Background The standard assay for the detection of chromium sensitization, the patch test, does not allow discrimination between patients with and without clinical symptoms of allergy. Objective The aim of this study was to prove whether cellular in vitro tests are predictive of chromium allergy. Methods Chromium-sensitized volunteers with and without clinically manifest allergy and non-sensitized healthy controls (n=37, 19, and 26, respectively) were analysed by cellular in vitro methods using tri- and hexavalent chromium (chromium chloride and potassium dichromate) as stimuli. The results were correlated with clinical and anamnestic data. Results Sensitized individuals with an allergy displayed significantly higher lymphocyte transformation test (LTT) responses than sensitized volunteers without allergy and controls (P

Healthcare workers have an increased risk of tuberculosis infection compared with the general population. There have been few attempts to quantify the prevalence of latent tuberculosis infection amongst German healthcare workers, due to inadequacy of the current tuberculin skin test (TST). Therefore, it was our aim to investigate the prevalence of latent tuberculosis in this cohort using a tuberculosis-specific ELISpot (T-SPOT.TB) test and to compare the performance of this test to that of the TST. Ninety-five healthy participants working in departments of radiology were examined by ELISpot, lymphocyte transformation test and TST. For cellular in-vitro tests, tuberculosis-specific peptides and purified protein derivate (PPD) were used as antigens. These tests were combined with a questionnaire on prior tuberculosis exposure. Out of 95 healthcare workers, only one (1%) was defined as positive by T-SPOT.TB, 92 (97%) by PPD-ELISpot, 78 (82%) by PPD-lymphocyte transformation test and 32 (34%) by TST. Multivariate analysis showed that the TST was significantly affected (P

Twenty-six bone marrow transplantations were performed at the West Germany Tumour Centre at Essen between December 1975 and December 1981. Three patients had aplastic anaemia, 23 acute leukaemia. Of the leukaemia patients 13 had a recurrence and ten were in full remission at the time of the transplantation. Two of the patients with a recurrence received deep-frozen autologous marrow from the remission phase. One patient with aplastic anaemia had an identical twin as donour, while all other patients received allogeneic marrow from histocompatible family members. Eleven patients are still alive, all three of those with aplastic anaemia, one of those with leukaemia in a recurrence, and seven of those with acute leukaemia in full remission. Two of the survivors have a chronic graft-host reaction, but the others are symptom-free, after an observation period of up to four years. Main causes of death were leukemic recurrence (6), infection (5), and graft-host reaction (2). It would seem that at present bone marrow transplantation offers the best chance of a cure for severe aplastic anaemic and acute leukaemia in adults, but only if transplantation is undertaken early and not in the end-stage of the disease.

Bone-marrow transplantations were performed in 71 patients, 11 with panmyelopathy, 17 with recurrence of acute leukaemia, 25 with acute leukaemia and remission, 18 with chronic myeloid leukaemia. The transplantation was allogenic in 67, autologous in 2, isologous in 2. Eight patients each survived in the panmyelopathy and chronic myeloid leukaemia groups. In the group of patients with acute leukaemia only one patient of those in a recurrence survived the transplantation for several years, but after 6 years there was another recurrence. Of the 20 patients with acute myeloid leukaemia who received the transplantation during their first remission, 11 are still alive. Retransplantation because of the recurrence was employed in one case each of acute and chronic myeloid leukaemia. Main cause of death was interstitial pneumonia with an overall risk of 23%. Only 5% of patients developed severe acute graft-versus-host reaction, grades III-IV. The low incidence of this reaction is possibly due to the strict gnotobiotic measures which in most of the patients led to decontamination of the intestinal tract.

Over a 10-year period 120 patients (58 women, 62 men; median age 33 [14-53] years) with acute myeloid leukaemia were treated by allogenic (n = 90) or autologous bone marrow transplantation to maintain remission. After a median observation time of 41 (11-126) months 64 patients (53%) remain alive without recurrence of leukaemia. Ten years after allogenic transplantation performed during the first complete remission the probability of disease-free survival is 50 +/- 8%, as compared with 50 +/- 9% at 4.5 years after autologous transplantation. Significant factors influencing disease-free survival after allogenic transplantation during the first complete remission were the time interval up to the onset of remission and the length of the remission before transplantation. The chance of disease-free survival after allogenic transplantation in the second complete remission does not so far differ from the results achieved by transplantation in the first complete remission. The risk of recurrence after autologous transplantation in the first complete remission (47 +/- 10%) is significantly higher than that following allogenic transplantation (18 +/- 10%, P less than 0.0001). Acute graft versus host reactions occurred in 16% and chronic reactions in 36% of patients after allogenic transplantation. The mortality was 38% after allogenic transplantation and 7% after autologous transplantation.

HLA-G appears to be involved in regulatory functions counteracting the cellular immune response of T and NK cells by several pathways. We here summarize the HLA-G expression patterns in leukaemia with emphasis on the clinical relevance of this expression for disease progression. Especially in patients with B-chronic lymphocytic leukaemia (B-CLL) the HLA-G expression on B-CLL cells was strongly associated with a reduced treatment-free survival. The corresponding immunological parameters point to a broad immunosuppression in these patients. Thus, HLA-G seems to contribute to the impaired immune response in B-CLL supporting disease progression.

Human leukocyte antigen G (HLA-G) molecules are crucial for the maternal tolerance against the fetus during pregnancy. Thus, the presence of soluble HLA-G (sHLA-G) in embryo cultures is thought to be correlated to a successful pregnancy after assisted reproductive techniques (ART). Here, we established a rapid detection assay based on Luminex technology, which can be integrated into ART proceedings, allowing sHLA-G quantification in sample volumes of only 10 microl within 1.5 hours. Using this method, sHLA-G levels of 526 single-embryo cultures, 47 two-embryo cultures, and 15 three-embryo cultures were analyzed corresponding to 313 ART cycles. In 117 embryo cultures, sHLA-G was detectable. In single-embryo cultures, the sHLA-G levels were positively correlated to embryo quality (p = 0.048, r = 0.20, n = 100). The presence of sHLA-G in embryo cultures was significantly (p0.0001) associated with clinical pregnancy after intracytoplasmatic sperm injections (ICSI), especially in couples with male factor infertility, but not after in vitro fertilization (IVF) or in couples with female infertility. Importantly, in sHLA-G negative embryos, the abortion rate was increased threefold (p = 0.04). In conclusion, the results obtained by our novel method support strongly the diagnostic relevance of sHLA-G for predicting pregnancy outcome after ART. The ultimate conditions for this prediction have to be further investigated in a multicenter study.

In order to clarify the diagnostic relevance of soluble human leukocyte antigen-G (sHLA-G) molecules, reliable methods for the measurement of sHLA-G in various body fluids are of interest. Therefore, the aims of the 'Wet-Workshop for Quantification of Soluble HLA-G' held in Essen, Germany (at the Institute of Immunology, 18-20 October 2004) were to select and to validate HLA-G-specific enzyme-linked immunosorbent assay (ELISA) formats and purified standard HLA-G proteins, which can be easily generated and used as consensual references. We chose two ELISA formats, one for the simultaneous determination of shed HLA-G1 + sHLA-G5 (sHLA-G1 + G5) and one for the exclusive detection of HLA-G5 molecules. The first ELISA uses the antibody pair monoclonal antibody (mAb) MEM-G/9 + anti-beta2-microglobulin (beta2m), whereas the latter uses mAbs 5A6G7 + W6/32. Purified and well-defined HLA-G5 protein derived from insect SF9 cells transfected with HLA-G5 + human beta2m served as standard reagent. Twenty-five members of 13 international laboratories participated in the 3-day Wet-Workshop. The workshop demonstrated that the HLA-G5 protein was equally detected by both ELISA formats allowing direct comparison of quantitative results obtained by these two ELISA formats, and that sHLA-G1 + G5 and HLA-G5 molecules, respectively, were specifically and reproducibly quantified by the two ELISA formats. The comparison of the two ELISA results obtained allows the conclusion that sHLA-G1 and HLA-G5 molecules can exist in the blood of healthy donors. Moreover, there was evidence for a novel soluble HLA-G structure recognized by the mAbs 5A6G7 + W6/32 antibody combination but not by the one of mAb MEM-G/9 + anti-beta2m.

Background. Recent reports suggest that high pretransplant serum levels of soluble CD30 (sCD30) are a risk factor for rejections after kidney transplantation. The aim of our study was to elucidate the predictive value of pretransplant sCD30 levels for kidney transplantation outcome in a single-center patient cohort that has been treated with modern immunosuppressive therapies after transplantation. Methods. We retrospectively analyzed sCD30 in multiple pretransplant sera from 206 patients, of whom 174 were transplanted with a cadaveric kidney and 32 patients received an allograft from a living donor. Renal function after transplantation was estimated by measuring serum creatinine and by rejection diagnosis. Results. We could demonstrate a statistically significant association between increased pretransplant sCD30 values and graft failures (P= 0.005). Receiver operating curve analysis revealed a cutoff value of 124 U/mL pretransplant sCD30. A multivariate analysis confirmed pretransplant sCD30 values > 124 U/mL (P=0.011) and rejection episodes (P

Objective In the hematopoietic stem cell transplantation setting, granulocyte colony-stimulating factor (G-CSF) administration can reduce donor cell reactivity in vitro, but the clinical significance of this phenomenon was only sparsely defined. Methods We performed lymphocyte transformation tests in 28 related stem cell donors pre and 5 days post G-CSF treatment, respectively, and correlated proliferative responses of donor peripheral blood mononuclear cells with clinical parameters in the corresponding recipients. Results In vitro reactions towards 4 mitogens and 12 recall antigens at day 5 post G-CSF administration were predictive for the occurrence of chronic graft-vs-host disease (cGVHD). Here, proliferative responses towards the mitogen anti-CD3 monoclonal antibody (OKT3) above median were most informative; this threshold could be determined by discrimination and receiver operating curve (ROC) analyses. In the whole cohort (18 human leukocyte antigen [HLA]-identical and 10 partially mismatched donor-recipient pairs), OKT3 responses predicted cGVHD with an odds ratio of 33.0, a sensitivity of 79%, and a specificity of 90%. A subgroup analysis of HLA-identical pairs even yielded an odds ratio of 85.0. Furthermore, bivariate analysis defined HLA compatibility and responses towards OKT3 as independent risk factors for cGVHD ( p = 0.02 and p = 0.0007, respectively). Conclusion The proliferative capacity of G-CSF-mobilized donor cells appears as a graft factor that determines the future incidence of cGVHD in the corresponding recipient.

We investigated potential correlations between soluble HLA-G (sHLA-G) and soluble HLA class I (sHLA-I) levels, respectively, and parameters of disease activity or genetic factors determined by HLA-DRB1 and HLA-DQB1 in patients with rheumatoid arthritis (RA). SHLA-G plasma concentrations from 106 RA patients (mean age 59.8 years, 80 women) were assessed by a sensitive enzyme-linked immunosorbent assay format. The mean sHLA-G levels were lower and sHLA-I levels higher in the RA patients than in healthy controls. Correlation coefficients of 0.248 to 0.344 (p < 0.01) between sHLA-G and rheumatoid factor, CRP, and EULAR joint swelling score were found. Patients with disease-associated HLA epitopes had higher sHLA-G levels than those without. Significantly lower sHLA-G was observed in groups of patients having HLA-DRB1*03 or HLA-DQB1*02 compared to groups without these genotypes. In contrast, HLA-DQB1*03 or disease-associated epitopes combined with HLA-DQB1*03 were associated with higher sHLA-G levels, whereas the inverse was observed in the combined presence of HLA-DRB1*03 and HLA-DQB1*02. SHLA-G as a percentage of sHLA-I was lower in patients positive for HLA-DQB1*02 and higher in patients positive for HLA-DQB1*03 and in its combined presence with disease-associated epitopes or with HLA-DRB1*07. As especially sHLA-G strongly inhibits T and natural killer (NK) cell functions, low sHLA-G suggests that T and NK cell activities are not efficiently restricted by sHLA-G molecules in rheumatoid arthritis. The sHLA-G levels, however, increase in correlation with parameters of disease activity and appear to be affected by the presence of disease-predisposing epitopes and other HLA-DRB1, DQB1 genotypes.

Previously we have reported on the development of antibodies against MICA alleles in kidney transplant recipients. These alloantibodies have now been determined using a new assay using Luminex beads bound to soluble recombinant MICA antigens produced in insect cells. In the present study we have analyzed sera from 85 kidney transplant recipients on the waiting list and 66 patients transplanted within the last 4 years and 59 acid eluates obtained from allograft nephrectomy specimens. Many of the patients in those groups were sensitized and some had previous transplants (waiting list: 15%; post-tx: 7.6%; eluates 22%) and their sera were found to contain anti-human leukocyte antigen (HLA) and anti-MICA antibodies. Anti-MICA antibodies were detected in 21/85 (24.7%) of the waiting list patients and in 15/66 (22.7%) of the transplanted recipients; 11 of the eluates (18.6%) were found to have MICA-specific antibodies (6 of them also had anti-HLA antibodies and 5 did not). These data suggested that immunization against mismatched MICA alleles induces development of anti-MICA antibodies. The finding of MICA allele-specific antibodies in eluates of kidney transplants suggests that anti-MICA antibodies can be involved in the pathogenesis of kidney allograft rejection. Further studies will be required to determine whether patients who produce alloantibodies against MICA alleles are at risk for transplant rejection even when no HLA antibodies are detected.

Membrane-anchored and soluble human leukocyte antigen HLA-G (sHLA-G) molecules exert strong inhibiting signals after interaction with their cognate receptors ILT2 (CD85j), ILT4 (CD85d), and KIR2DL4 (CD158d) that are differentially expressed by natural killer cells, T cells, and antigen-presenting cells. These inhibitory functions can become operative in conditions in which such immune cells try to attack viral infected or tumor cells. Recently, clinical studies showed that sHLA-G molecules are also relevant in the prediction of allograft acceptance after heart transplantation, liver-kidney cotransplantation, and the successful implantation and development of embryos after in vitro fertilization. In view of this diagnostic potential, reliable methods for the measurement of sHLA-G molecules in various body fluids are of interest. Thus, the aims of the Wet Workshop for measurement of sHLA-G held in Essen, Germany (at the Institute of Immunology October 18–20, 2004) were to select and validate HLA-G–specific enzyme-linked immunosorbent assay (ELISA) formats and purified standard HLA-G proteins, which can be easily generated and used as consensual references. To this end, the antibody combinations monoclonal antibody (mAb) MEM-G/9 (capture) + anti-β2m (detection) and the mAb 5A6G7 (capture) + mAb W6/32 (detection) were chosen in an ELISA format for the simultaneous determination of shed HLA-G1 + soluble HLA-G5 (sHLA-G1 + HLA-G5) and for the exclusive detection of HLA-G5 molecules, respectively. As standard, protein HLA-G5 molecules were purified from insect SF9 cells coinfected by HLA-G5 + human β2m and characterized for their antigenic determinants. A total of 24 members in 13 teams participated in the 3-day sHLA-G Wet Workshop. All workshop materials, protocols, standard reagents, and samples were provided to each team by the organizers. The Wet-Workshop results clearly demonstrated that (1) the HLA-G5 standard reagent was equally detected by both ELISA formats; (2) sHLA-G1 + G5 and HLA-G5 molecules, respectively, were specifically detected by the two ELISA formats; and (3) both ELISA formats measure reproducibly the amounts of sHLA-G. The comparison of the two ELISA results obtained evidenced that in healthy donors sHLA-G1 molecules can exist in body fluids besides HLA-G5. Moreover, a novel soluble HLA-G structure can be predicted that is recognized by the mAb 5A6G7 + mAb W6/32 antibody combination, but not by the one of mAb MEM-G/9 + anti-β2m.

Background: Although Behcet’s disease (BD) is classified among the vasculitides laboratory diagnostic does not include regularly autoantibodies associated with vascular manifestations of systemic autoimmune diseases. Patients and methods: Twelve consecutive BD patients were studied for autoantibodies associated with vascular manifestations of systemic autoimmune diseases, HLA frequencies, and possible neurological involvement using neurophysiological methods and MRI. Results: HLA-C*15 and C*16 frequencies were significantly (p < 0.05) higher in the patients compared with a reference population. Immunoglobulin G concentrations of antiphosphatidylserine and antiribosomal phosphoprotein antibodies were significantly elevated in BD patients when compared with healthy controls. Conclusions: The increased frequencies of HLA-C alleles in BD patients may stress the role of NK cells in the pathogenesis of this disease. Antiphosphatidylserine autoantibodies may in view of their role in apoptosis be involved in the development of vasculitis in BD. Because concentrations of antiphosphatidylserine and antiribosomal phosphoprotein antibodies were increased in BD diagnostic tools of this disease should be extended with humoral parameters associated with vascular manifestations of systemic autoimmune diseases.

Background. To examine how killer-cell immunoglobulin-like receptor (KIR) ligand incompatibilities effect molecular relapse (MR), we compared the occurrence of bcr-abl-positive reverse-transcriptase polymerase chain reaction (RT-PCR) results in 236 CML patients (pts) after human leukocyte antigen (HLA)-identical (n= 158) (group 1), HLA class I antigen mismatched and KIR-ligand compatible (n=49) (group 2), and HLA class I antigen mismatched and KIR-ligand incompatible (n=29) (group 3) hematopoietic stem-cell transplantation. Methods. We performed a retrospective single-center study. MR was evaluated using the real-time RT-PCR method for the detection of bcr-abl transcripts. Results. In the first group, 133 of 158 (84%) pts were in the first chronic phase of CML, and the corresponding figures were 33 of 49 (67%) pts in group 2 and 19 of 29 (64%) in group 3 (P

It remains controversial whether alloreactive donor-derived natural killer (NK) cells display graft-versus-leukemia reactions after unmodified allogeneic hematopoietic stem cell transplantation (HSCT). The present study evaluated the role of inhibitory killer immunoglobulin–like receptor (KIR) ligand incompatibility using a well-defined and uniform setting of unmodified allogeneic HSCT in 374 patients with myeloid leukemias. The most striking finding was a significant heterogeneity in the 5-year estimates of hematologic leukemic relapse after human leukocyte antigen (HLA)–identical (n = 237; 22%), HLA class I–disparate (n = 89; 18%), and KIR ligand–incompatible transplantations (n = 48; 5%) (P < .04). Multivariate analysis confirmed that the relative relapse risk (RR) was influenced by HLA class I disparity alone (RR 0.49), but was lowest after HLA class I–disparate, KIR ligand–incompatible transplantations (RR 0.24) (P < .008). The primary graft failure rates, however, increased from 0.4% after HLA class I–identical to 2.3% after HLA class I–disparate, and to 6.3% after KIR ligand–incompatible transplantations, respectively (P < .02). Unlike some other reports, no beneficial effect of KIR ligand incompatibility on other major endpoints of allogeneic HSCT (transplantation-related mortality, and overall and event-free survival) was detectable in the present study. In conclusion, unmodified allogeneic HSCT from KIR ligand–incompatible donors provides a superior long-term antileukemic efficacy in patients with myeloid malignancies.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a cytokine widely used in the procurement of peripheral blood stem cells (PBSC) from donors for allogeneic hematopoietic cell transplantation. Therefore, we were interested in its immediate and long-term effects on cellular and soluble factors known to be involved in the immune response.We studied 35 PBSC donors by mixed lymphocyte culture (MLC) and lymphocyte transformation test (LTT), and 41 for soluble plasma factors (soluble human leukocyte antigen [sHLA]-G, -class I, -DR, and interleukin [IL]-10) pre and 5 days post initial rhG-CSF administration, respectively. In addition, 10 donors were reexamined at an average of 2 months (3-16 weeks) post-rhG-CSF.At 5 days post-rhG-CSF the donors presented a significant (p0.05) decrease of MLC, LTT mitogen, and recall antigen reactions. Plasma levels of sHLA-G, -class I, -DR, and IL-10 (p0.005 each) were significantly increased. The changes in IL-10 but not in sHLA were significantly (p0.05) correlated with LTT responses. In the 2-month follow-up there was no significant difference in alloreactivity and LTT reactions as compared to the pre-rhG-CSF results. The results generated after 3 to 16 weeks did not depend on the time point of investigation. Consistently, soluble factors decreased to pre-rhG-CSF levels.rhG-CSF administration suppresses cellular immune functions within 5 days and increases sHLA and IL-10 plasma levels. These immunomodulatory effects appear to be short-term only and vanished at an average of 2 months after rhG-CSF application.

Anterior uveitis (AU) is an autoimmune disease frequently associated with HLA-B27 antigen. Because of the immune regulatory properties of soluble human leukocyte antigen (sHLA) molecules, we quantified sHLA class I (sHLA-I) and sHLA-DR plasma levels in HLA-typed AU patients (n = 60). Randomly selected healthy individuals (n = 128) and HLA-B27 antigen-positive individuals (n = 24) with HLA phenotype frequencies similar to the HLA-B27 antigen-positive AU patients served as control panels. As expected, HLA-B27 phenotype was significantly increased in AU patients (n = 60), compared to healthy controls. Mean sHLA-I levels in AU patients were slightly higher than in randomly selected healthy controls. Regarding AU subgroups, elevated sHLA-I levels were only found in HLA-B27 antigen-negative patients. Compared to controls, sHLA-DR levels were significantly increased in AU patients and the subgroups of HLA-B27 antigen-negative and -positive patients but not Fuchs' heterochromic cyclitis (FHC). AU patients negative for HLA-B27 antigen with a chronic course had higher sHLA-DR levels than those with an acute course. The presence of associated systemic diseases in AU patients was related to elevated sHLA-DR levels. Secretion of sHLA-DR in blood differs among the various forms of AU. Systemic immune activation was present in AU but not in FHC.

Antiphospholipid syndrome (APS) is a severe complication in pregnancy that can lead to fetal death in the second or third trimester. As soluble HLA-DR (sHLA-DR) molecules are reported to be implicated in the etiology of pregnancy disorders and of autoimmune diseases, we studied sHLA-DR plasma levels in pregnant women with APS (n = 14) and in women with normal pregnancy (n = 15), in women with high-risk pregnancies such as preeclampsia (PE; n = 20) and intrauterine growth retardation (IUGR; n = 10) and in fertile non-pregnant women (n = 29). The sHLA-DR levels of pregnant women were assessed during the third trimester, at labor, in the first week, and in the third month of puerperium. The results obtained were compared with soluble CD95 ligand (sCD95L), an important signal molecule in the apoptosis pathway. The sHLA-DR levels in pregnant women with APS were approximately three times higher (mean 1.48 +/- 0.15 microg/ml) during the whole observation period than in fertile non-pregnant women (0.54 +/-.06 microg/ml) and nearly double in women with high risk (PE, 0.91 +/- 14 microg/ml; IUGR, 0.94 +/-.21 microg/ml) and in normal pregnancies (0.74 +/- 0.13 microg/ml). Furthermore, sHLA-DR levels of pregnant women with APS were positively correlated with the serum concentration of anti-anticardiolipin immunoglobulin G antibodies. For sCD95L plasma levels, no substantial variations were found among the different groups above. In pregnant women with APS, however, sHLA-DR levels were positively correlated with sCD95L levels. Further studies should clarify the functional involvement of sHLA-DR molecules in the induction of CD95/CD95L-mediated apoptosis pathway that may play a crucial role in the pathology of pregnancies complicated by APS.

Our clinical studies revealed significantly increased soluble HLA-G (sHLA-G) plasma levels in patients suffering from malignant melanoma, glioma, breast and ovarian cancer. Specific ELISpot assays demonstrate that sHLA-G molecules expressing intron-4 sequences are preferentially secreted by peripheral blood monocytes. In vitro, the sHLA-G secretion of monocytes and tumor cells was strongly enhanced by TH1 cytokines like IFN-alpha, -beta, -gamma whereas TH2 cytokines (e.g. IL-4, -10) had minor effects. As sHLA-G can inhibit the functions of T and NK cells high concentration of these molecules should systemically or at the tumor side reduce the immune surveillance and thus favour the progression of cancer.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a proven curative therapy for many hematologic malignancies. HSCT from HLA-identical sibling donors (ISDs) is still the golden standard. For the remaining 70% of the patients lacking an ISD, alternative (partially) HLA-matched family donors (MFDs) and HLA-matched unrelated donors (MUDs) are now widely accepted. However, it is presently unclear whether outcome after HSCT from an MFD or an MUD is superior. Thus, the classical clinical end points after HSCT from an ISD (n = 138), MFD (n = 86), and MUD (n = 101) were compared by means of univariate and multivariate statistical analyses. MFD transplantations with HLA class II (DRB1 ± DQB1) mismatches in graft-versus-host (GVH) direction showed an increased risk of grades II to IV graft-versus-host disease, and MFD transplantations with more than a single HLA class I (A ± B ± C) mismatch in host-versus-graft (HVG) direction were associated with a higher risk of graft failure. However, no significant difference in overall survival was detectable among the 3 study groups after adjustment for the main predictors of transplantation outcome. Thus, for patients lacking an ISD, an already identified MFD with an HLA-DRB1 ± DQB1 mismatch in GVH or a combined HLA-A ± B ± C mismatch in HVG direction should be accepted only in clinically urgent settings that leave no time to identify an MUD.

Only three anti-HLA-A monoclonal antibodies (mAbs) have been described in the literature. Two of them recognize determinants shared by only a few HLA-A allospecificities. The third one, mAb 3G11, recognizes a determinant shared by most HLA-A allospecificities. Being an IgM, the latter mAb is not likely to be a useful probe in immunohistochemical reactions and in functional assays. Therefore, in the present study we have characterized the specificity of the mAbs LGIII-147.4.1 and LGIII-220.6.2. The two mAbs that do not share idiotypic determinants recognize distinct but spatially close antigenic determinants expressed on most of the gene products of the HLA-A locus. Specifically, the determinant recognized by mAb LGIII-220.6.2 is expressed on HLA-A1, -A2, -A3, -A26, -A28, -A29, -A30, -A33, -A36, -A74 and -A80 allospecificities. The determinant recognized by mAb LGIII-147.4.1, which appears to be located on the amino-acid residues 79–83 of the heavy chain, is expressed on all HLA-A allospecificities but HLA-A23, -A24, -A25 and -A32. Because of its broad reactivity, the mAb LGIII-147.4.1 was characterized in a number of assays. It was found to be a useful probe to measure the HLA-A antigen level in serum, to assess the HLA-A restriction of cytotoxic T lymphocytes (CTL) and to monitor HLA-A antigen expression in normal and malignant lesions.

Summary Background The standard assay for the clinical detection of contact allergy against nickel is the patch test. For research purposes, the in vitro lymphocyte transformation test (LTT) is mostly applied. However, the in vivo application of allergens includes the potential of sensitization, whereas the LTT demands 6 days of cell culture and radioactive labelling procedures. Objective The purpose of this study was to validate an enzyme-linked immunospot (ELISpot) assay to specifically detect cellular response against nickel sulphate in correlation with patch test, anamnestic data, and LTT results. Methods In probands with positive (n 5 60) and negative patch test results (n 5 19), cytokine ELISpot and LTT were performed in parallel using various concentrations of nickel sulphate, various numbers of responding peripheral blood mononuclear cells (PBMC), and various durations of cell cultures. Results Concentrations of 50 and 75mM nickel sulphate were found to be optimal to stimulate in the ELISpot 4 10 5 and in the LTT 2 10 5 PBMC, respectively. An increase in ELISpot sensitivity was reached by pre-incubation with nickel sulphate for 24 h prior to transfer to ELISpot plates. In nickel-sensitive probands, an average precursor cell frequency of 19 10 5 , 1.7 10 5 , and 0.7 10 5 could be defined for IFN-g, IL-2, and IL-4 producing PBMC, respectively. In non-sensitive probands IFN-g producing cells were detectable, but with significantly lower frequency (2 10 5 ; P 5 0.004). The prior performance of patch tests had no significant effect on cytokine production or lymphocyte proliferation. Overall, the parameters patch test, anamnestic nickel allergy, ELISpot, and LTT results were positively correlated (Po0.05). Conclusions Based on these experiments, the quantitative in vitro detection of cell-mediated reactions towards nickel as well as other heavy metal ions should be possible utilizing the abovedescribed ELISpot assay.

The woodchuck (Marmota monax) is an animal model that is used in the study of human hepatitis B virus ( HBV ) infection. A knowledge of woodchuck MHC class I (Mamo-I) genes and gene products is therefore essential for understanding the antigen-specific T-cell responses in this animal model. A number of Mamo-I genes have been identified by molecular cloning and sequencing. However, the allelic nature of these genes has not been proven by classical genetics like the segregation analysis in families. In this study, we analyzed the allelic diversity of Mamo-I in two three-generation woodchuck families including 15 members by sequencing of Mamo-I genes and immunoblotting of Mamo-I proteins after one-dimensional isoelectric focusing (1D-IEF). In addition to four published Mamo-I alleles, six new alleles that belonged to the same locus as the known Mamo-I alleles (Mamo-A) were found within the two woodchuck families. A typical Mendelian segregation of Mamo-I gene and antigens was observed in the families studied. For simple and rapid detection of allelic variability of Mamo-I gene, a typing method based on the detection of PCR products amplified by sequence specific primers (SSP) has been developed and tested in 41 unrelated animals. The most prevalent allele was Mamo-A*01 with a frequency of 21.9% followed by Mamo-A*07 (12.2%). Our study established Mamo-A as a classical MHC class I locus by the polymorphic and allelic nature of Mamo-I gene in the woodchuck.

The lack of phenotype/genotype association in X-linked adrenoleukodystrophy (X-ALD) has prompted the search for disease modifying factors. We previously demonstrated increased serum antibody responses against myelin oligodendrocyte glycoprotein (MOG) in various clinical phenotypes of X-ALD allowing speculations that myelin specific humoral immune responses might be involved in phenotype generation of X-ALD. In the present study, we investigated the possible association of (1) a naturally occurring variable number tandem repeat (vntr) polymorphism (C allele) in the 3' flanking region of the interleukin-6 gene (IL-6), previously demonstrated to modify the course of Alzheimer's disease, systemic lupus erythematodes and Multiple Sclerosis (MS), (2) a tetranucleotide repeat polymorphism (TAAA)(n) in the 3' flanking region of the MOG gene and (3) HLA class II alleles with adult clinical phenotypes and serum antibody responses to MOG in 70 adult X-ALD patients. HLA class II alleles, (TAAA)(n) MOG gene polymorphisms, and IL-6 C allele were not associated with clinical phenotypes. Anti-MOG antibodies were detectable in 17/54 X-ALD patients (31.5%). Anti-MOG antibodies were associated with the 226 bp (TAAA)(n) MOG gene polymorphism but not with distinct clinical phenotypes.

Outcomes of highly purified CD34+ peripheral blood stem cell transplantation (PBSCT) for chronic phase chronic myeloid leukemia (CML) (n = 32) were compared with those of PBSCT (n = 19) and of bone marrow transplantation (BMT) (n = 22) in the HLA-compatible sibling donor setting. Median follow-up was 18 months after CD34+-PBSCT and unmanipulated PBSCT and 20 months after BMT. CD34+-PBSCT was associated with delayed T-cell immune reconstitution at 3 months and 12 months after transplantation compared with PBSCT (P

During pregnancy the fetus represents a semi-allograft. Both membrane-bound and soluble forms of the nonclassic human leukocyte antigen (HLA)-G protect the fetus from maternal immune attack. To assess the relevance of soluble HLA-G (sHLA-G) levels in the maternal circulation for the occurrence of characteristic pregnancy disorders, we analyzed sHLA-G plasma levels of women with normal and pathological pregnancies. Compared to normal pregnancy, significantly increased sHLA-G levels were detected in women delivered preterm because of intrauterine activation (uncontrollable labor, rupture of fetal membranes, cervical insufficiency) and women with Hemolysis, Elevated Liver enzymes, Low Platelet count (HELLP) syndrome. Contrary to these disorders, the sHLA-G levels in women with placental abruption were more than three times lower than in normal pregnancy (p < 0.0001). Nonparametric discriminant analysis showed that women with sHLA-G levels below 9.95 ng/mL had a relative risk of 7.12 for the development of placental abruption during further course of pregnancy. These results suggest that the occurrence of pregnancy-associated diseases is strongly influenced by maternal sHLA-G plasma levels.

One major materno-fetal interface in the human placenta is constituted by the syncytiotrophoblast, in contact with maternal blood of the intervillous space, which derives from differentiation and fusion of the villous cytotrophoblast (vct). In the present work, we purified vct from term placenta by depleting HLA class I- and class II-positive cells. We found by RT-PCR that both soluble intron 4-retaining HLA-G1 (sHLA-G1) and HLA-G2 isoforms were transcribed in purified vct. Using different HLA-G-specific mAb, we demonstrated by intracellular flow cytometry, Western blotting and ELISA, that sHLA-G1 but no soluble HLA class Ia molecule was secreted by vct. We then purified sHLA-G1 from vct culture supernatant and found that it exhibited an unusual glycosylation pattern. Finally, we showed that such trophoblast-derived sHLA-G1 triggered specific apoptosis of activated CD8+ T cells. Taken together, these results demonstrated that vct did secrete functional sHLA-G1 in primary culture and suggested that, in vivo, sHLA-G1 might be an important immunomodulatory molecule controlling the activity of maternal immune effector CD8+ cells circulating in the blood that immerses chorionic villi.

Despite numerous therapeutic options, the prognosis of malignant melanoma, once metastasized, is still poor. Thus, the search for reliable methods to identify patients with high risk of disease progression as early as possible is of major importance. In our study, we analyzed the predictive value of soluble HLA-DR (sHLA-DR) in comparison to S100-beta in serum from 183 melanoma patients of different stages of disease and with or without current therapy using immunosorbent assays. sHLA-DR serum levels of 121 healthy individuals served as controls. We found significantly (p0.0005) reduced sHLA-DR serum levels in melanoma patients (0.70 +/- 0.08 SEM microg/ml) compared to controls (1.49 +/- 0.10 SEM microg/ml). Reduced sHLA-DR and increased S100-beta levels were associated with advanced disease stages and tumor load. S100-beta was increased under cytostatic therapy (p0.0005), whereas sHLA-DR was not influenced by therapy modalities. Univariate analysis showed an association of sHLA-DR0.3 microg/ml and S100-beta0.12 microg/l with poor overall (p = 0.021 and p = 0.0009) and progression-free survival (p0.0005 and p = 0.0025). Multivariate analysis revealed disease stage (p = 0.0093) and tumor burden (p0.0005) as independent predictive factors for overall survival, and sHLA-DR (p = 0.0007) and tumor burden (p = 0.0015) for progression-free survival. In contrast to S100-beta, sHLA-DR serum concentrations0.3 microg/ml were strongly associated (p = 0.0001) with poor progression-free survival in a subgroup of 60 nonmetastasized patients. In conclusion, our results suggest sHLA-DR as a potent prognostic serum marker in melanoma patients superior to S100-beta in helping to identify early-stage patients at high risk of disease progression.

Outcomes after peripheral blood stem cell transplantation (PBSCT) for chronic phase chronic myeloid leukemia (n = 37) were compared with outcomes after bone marrow transplantation (BMT) (n = 54) in the HLA-compatible unrelated donor setting. Median follow-up was 17 months after PBSCT and 29 months after BMT. Both neutrophil and platelet recovery were faster after PBSCT (P

Allogeneic peripheral stem cell transplantation in six children with non-malignant hematologic or metabolic diseases which are eventually fatal was carried out with parental donors. Given three to five HLA mismatches, all grafts underwent CD34+ cell selection as graft-versus-host prophylaxis. The patients received median doses of 16.7 x 10(6) CD34+ cells/kg and 1.2 x 10(4) CD3+ cells/kg. All transplants engrafted. Neutrophils >0.5/nl were reached on day 11 (9-19) and platelets >50/nl on day 13 (10-25). Acute GVHD responding to steriods occured in three of six patients; it was restricted to the skin and overall did not exceed grade I. Two patients died of viral infections and four are alive with stable blood counts for 13, 15, 25 and 26 months. For children with non-malignant diseases which will eventually be fatal and which can be cured or ameliorated by allogeneic BMT, CD34+-selected stem cell transplants from mismatched or even haploidentical parents can be used if no other suitable donor is available. With high CD34+ cell doses and low CD3+ cell numbers, engraftment and avoidance of acute GVHD can be expected. Infections after transplantation remain the primary threat to survival.

Background: The etiology of thrombangitis obliterans is still unclear. Although cellular infiltration of the vessel wall is known, no studies on peripheral blood mononuclear cells are reported. Therefore, we assessed leucocyte subpopulations and circulating immune complexes in patients with thrombangitis obliterans and a control group of normal people. Patients and methods: 31 patients (40 ± 2 years, 24 male, 7 female) with thrombangitis obliterans were included, based on the following criteria: age of manifestation, acral ischemia in legs and arms, previous thrombophlebitis or phlebitis saltans. Manifestation of atherosclerosis or other vasculitic manifestations were excluded. Leucocyte subpopulations, levels of C-reactive protein (CRP) and circulating immune complexes (CIC) were investigated. An age-matched control group (n = 25) was recruited from voluntary blood donors. Results: Leucocyte counts in the thrombangitis group (mean ± SD: 10839 ± 782/nl) were significantly different from the control group (6205 ± 414/nl, p < 0.0001). The same was true for absolute counts of granulocytes, monocytes and lymphocytes. The results were independent from CRP, which was elevated only in 6 patients. Relative counts of naive helper T-cells were significantly lower in the patient group. HLA-DR expression on B-cells was lower on the patients’ lymphocytes. The concentrations of IgA, IgG and IgM in CIC were higher in the thrombangitis patients compared to the control group. C1q-binding capacity and phosphatidylserine antibodies showed no differences. Conclusions: Patients suffering from thrombangitis obliterans show alterations of leucocyte counts and their subpopulations as well as alterations of the humoral (IgCIC) immune system.

The G protein L3 subunit (GNB3) 825T allele is predictive of enhanced Gi protein activation. Studying the influence of C825T allele status on cellular in vitro immune responses towards recall antigens and interleukin-2 stimulation we observed a 2^4-fold, significantly increased proliferation in homozygous 825T (TT) vs. C825 allele (CC) carriers. Further- more, lymphocyte chemotaxis and CD4 + T cell counts of individuals with TT+TC genotypes were significantly enhanced compared to the CC genotype. In summary, it appears that C825T allele status is highly predictive of immunocompetence and could be a candidate gene in disorders associated with inadequate immune response. fl 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Soluble HLA-G (sHLA-G) molecules are found in the peripheral blood of healthy females and males, in cord blood and in amniotic fluids and discussed to be a mediator in maternal-fetal tolerance. In this study we investigated whether there are allele-specific differences in expression of sHLA-G molecules. For this, the sHLA-G plasma concentrations of 94 healthy unrelated individuals were measured by ELISA and correlated to their HLA-G genotypes, as determined by sequence analysis of exon 2 and 3 of the HLA-G gene. Mean sHLA-G levels in individuals with the most common HLA-G alleles G*01011 (27.0+/-2.1 SEM ng/ml, n=66), G*01012 (28.4+/-3.2 SEM ng/ml, n=34) were very similar. In contrast, individuals carrying the HLA-G*01013 (8.1+/-1.7 SEM ng/ml, n=17) or the "null" allele HLA-G*0105N (8.2+/-3.2 SEM ng/ml, n=7) presented significantly (P(c)=0.001 and P(c)

BACKGROUND The nonclassic human major histocompatibility complex class I antigens human leukocyte antigen (HLA)–G are proposed to protect tumor cells from natural killer cell lysis. In the current study, the authors measured soluble HLA-G molecules (sHLA-G) in serum from patients with malignant melanoma. METHODS Soluble HLA-G was determined in serum samples of 190 melanoma patients with various stages of disease, with or without current therapy including interferon (IFN)–α and different cytostatics in comparison to 126 healthy controls by using a two-step enzyme-linked immunoadsorbent assay. RESULTS Serum sHLA-G was significantly (P < 0.0005) elevated in melanoma patients (mean ± standard error of the mean [SEM] = 41.95 ± 2.15 ng/mL) compared with healthy controls (mean ± SEM = 22.92 ± 1.51 ng/mL). Univariate analysis revealed a correlation of sHLA-G serum level with advanced stages of disease (P < 0.001) and tumor load (P < 0.05). Patients undergoing immunotherapy with IFN-α (n = 31) showed an increased serum sHLA-G (mean ± SEM = 62.05 ± 7.58 ng/mL; P < 0.0005), whereas other treatment regimens (n = 24) did not influence sHLA-G serum concentrations. Multivariate analysis revealed treatment with IFN-α as the only impact factor for elevated serum sHLA-G, lacking any correlation with stage of disease or tumor burden. Furthermore, IFN-α was found to upregulate HLA-G cell surface expression on circulating monocytes. sHLA-G serum level was not associated with recurrence free or overall survival. CONCLUSIONS This study shows increased sHLA-G serum concentrations in melanoma patients and additional enhancement upon treatment with IFN-α. The level of serum sHLA-G, however, had no negative impact on patients' prognosis. Cancer 2001;92:369–76. © 2001 American Cancer Society.

Acknowledgments: This study was supported by a grant from the Belgian Red Cross. The authors thank Prof. W. Duquet for valuable help with the statistical analysis of the results. Abstract: We investigated correlations between soluble HLA-DR (sHLA-DR) molecules and several clinical, biological and genetic parameters associated with rheumatoid arthritis (RA) disease activity. Serum sHLA-DR concentrations were determined in 146 samples from 89 RA patients by an ELISA format, using an antibody combination of mouse and rat monoclonal anti-human HLA-DR antibodies. The mean sHLA-DR serum level in RA patients was significantly increased with 277±19 ng/ml compared to 142±13 ng/ml of 80 healthy controls (P

The association of HLA class I heavy chains with β2-microglobulin (β2m) changes their antigenic profile. As a result, Abs react with either β2m-free or β2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both β2m-associated and β2m-free HLA class I heavy chains. The reactivity with β2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194–198 in the α3 domain. The reactivity with β2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on

Objective: To examine the influence of natural killer (NK) cells and HLA-DR molecules on B cells in the development of severe sepsis after injury. Design. Prospective study. Setting: Medical school, Germany. Subjects: 46 severely injured (Injury Severity Score >16) patients. Interventions: Blood samples were taken immediately after admission and subsequently for 14 days. Main Outcome Measures: HLA-DR expression on B cells and counts of B and NK cells measured by flow cytometry, and morphological estimation of large granular lymphocytes by microscopy. Results: HLA-DR expression on circulating B cells was significantly reduced from days 6-14 after admission in 13 patients with subsequent severe sepsis compared with 33 patients who did not develop sepsis. In septic patients NK cell counts were significantly decreased from day 4 onwards (p < 0.05). CD16+/CD56+ cells correlated with the morphology of large granular lymphocytes. Conclusion: In severely injured patients reduced counts of NK cells and HLA-DR molecules on B lymphocytes seem to be part of an immune deviation that is associated with the development of severe sepsis.

Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P

The clinical results, cellular immune reconstitution, and hematopoietic chimerism obtained after transplantation of recombinant human granulocyte colony-stimulating factor mobilized allogeneic peripheral blood stem cells (PBSCs) from genotypically human leukocyte antigen (HLA)-identical sibling (n = 36) or alternative family donors (n = 24) were prospectively compared in patients with hematologic malignancies. Thirty-two of 34 evaluable patients with HLA-identical sibling donors and all patients with alternative family donors achieved trilineage engraftment. The median time intervals to reach peripheral neutrophil counts

□ The destruction of hematopoiesis and lympopoiesis by total body irradiation or high dose chemotherapy for the treatment of malignancy can be reversed by the transplantation of allogeneic or autologous hematopoietic stem cells. In primary disorders of bone marrow or immune system, allogeneic stem cells replace deficient cells. Acute leukemias can be cured, with in 50 to 80% disease free survival after 5 to 8 years. The allogeneic graft versus leukemia effect by immunoreactive cells reduces the relapse rate in myeloid and lymphoid malignancies. 40 to 70% of patients with chronic myeloid leukemia remain disease free after more than 5 years. Patients with malignant lymphoma have a 40 to 70% chance of cure with autologous transplantation, which is not increased by allogeneic cells, because of a higher incidence of severe complications. An increasing number of patients without option for cure is treated with the aim of prolonging remission or retarding disease progression, such as in chronic myeloid leukemia, multiple myeloma and certain solid tumors. New studies suggest in breast cancer with axillary lymph node metastases, that adjuvant high dose chemotherapy with autologous stem cell support will significantly improve disease free survival from 30 to over 60% after 3 to 5 years. In congenital metabolic and storage diseases deficient enzymes are substituted by the allogeneic cells. Clinical trials explore the use of stem cell transplantation after myeloablative therapy in autoimmune disorders as well as in gene therapy with transfected hematopoietic stem cells.