The putative role of HLA-C recognition in graft versus host disease (GVHD) and graft rejection after unrelated bone marrow transplantation (BMT).

We assessed cytotoxic activity of large granular lymphocytes (LGLs) derived from 10 patients transplanted from molecular HLA-C mismatched (5) and matched (5) unrelated donors and compared it to the cytotoxic activity of 10 patients transplanted from HLA-identical siblings. In addition, we correlated clinical outcome with the level of molecular HLA-C disparity in a cohort of 22 patients who underwent unrelated BMT. Cells obtained from patients transplanted (related or unrelated) from fully matched donors did not generate allospecific lysis of patient (pre-BMT) or donor PHA blasts. Five of nine patients who received BMT from HLA-C mismatched unrelated donors developed > grade II graft-vs.-host disease (GVHD), and four developed graft rejection. Cells derived from three of three patients with GVHD lysed patients' pre-BMT PHA blasts. In the patients with GVHD grade III-IV, cytotoxicity was higher (60-70%) than in the patient with grade II GVHD (20%) (p < 0.05). Cytotoxic cells derived from one patient who rejected his graft lysed donor PHA blasts. In one remaining patient who had graft rejection followed by autologous rescue, no in vitro allospecificity was observed. In summary, cytotoxic cells from patients transplanted with marrow mismatched at locus C demonstrated in vitro cytolysis of PHA blasts, and this phenomenon showed positive correlation with the clinical outcome of the BMT. These findings may indicate specific allorecognition. A mismatch at locus C leading to alloreactivity should be considered a risk factor in determining an appropriate match for allogeneic BMT, especially when the donor is unrelated.

Enhanced lymphoid cell recovery after bone marrow transplantation: correlation with the presence of costimulatory antibodies in serum.

We describe a patient with T cell deficiency who underwent bone marrow transplantation (BMT) from an HLA-identical brother. The patient's white blood cell count recovered with exceptional rapidity post-BMT: after 7 to 9 days it rose sharply to 98x10(9) cells/L, 76% of which were mononuclear leukocytes. It then decreased, and a second peak was observed 250 days post-BMT. Lymphocytes from both peaks displayed a phenotype of mature T cells together with characteristics of a constitutively activated state; that is, they 1) exhibited high levels of tyrosine-phosphorylated T cell receptor (TCR) zeta chain, 2) spontaneously secreted IL-2, 3) expressed activation specific cell surface markers, and 4) were unresponsive to in vitro stimuli. The increased cell counts in both peaks correlated with the presence of anti-lymphocytic antibodies in the patient's serum, which reacted with peripheral blood lymphocytes (PBLs) both from the donor and from unrelated individuals. These antibodies were present before BMT and reappeared post-BMT. Variable number tandem repeats analysis revealed that the patient's PBLs were chimeras for up to 2 years post-BMT. This finding could explain the newly synthesized post-BMT anti-lymphocytic antibodies and the appearance of the second WBC peak during that period. The patient's anti-lymphocytic antibodies displayed costimulatory activity, enhancing the in vitro proliferation of normal T cells suboptimally activated via the TCR. The unique characteristics of these antibodies could explain the enhanced T cell recovery observed post-BMT as well as the constitutive activation state of these cells. Furthermore, such antibodies may eventually facilitate development of a therapeutic method for inducing enhanced post-BMT recovery.

Variation in Cost and Quality in Kidney Transplantation.

BACKGROUND: Bending the cost curve in medical expenses is a high national priority. The relationship between cost and kidney allograft failure has not been fully investigated in the United States. METHODS: Using Medicare claims from the United States Renal Data System, we determined costs for all adults with Medicare coverage who underwent kidney transplant January 1, 2007, to June 30, 2009. We compared relative cost (observed/expected payment) for year 1 after transplantation for all transplant centers, adjusting for recipient, donor, and transplant characteristics, region, and local wage index. Using program-specific reports from the Scientific Registry of Transplant Recipients, we correlated relative cost with observed/expected allograft failure between centers, excluding small centers. RESULTS: Among 19,603 transplants at 166 centers, mean observed cost per patient per center was $65,366 (interquartile range, $55,094-$71,624). Mean relative cost was 0.99 (± 0.20); mean observed/expected allograft failure was 1.03 (± 0.46). Overall, there was no correlation between relative cost and observed/expected allograft failure (r = 0.096, P = 0.22). Comparing centers with higher than expected costs and allograft failure rates (lower performing) and centers with lower than expected costs and failure rates (higher-performing) showed differences in donor and recipient characteristics. As these characteristics were accounted for in the adjusted cost and allograft failure models, they are unlikely to explain the differences between higher- and lower-performing centers. CONCLUSIONS: Further investigations are needed to determine specific cost-effective practices of higher- and lower-performing centers to reduce costs and incidence of allograft failure.

A sensitive and specific ELISA using monoclonal capture antibodies for the detection of HLA antigens in blood stains.

A double antibody ELISA technique is described to detect HLA antigens in extracts of blood stains. The assay involves capture of free HLA determinants using an immobilized monoclonal antibody directed against monomorphic regions of HLA class I and HLA class II antigens. The captured antigens are then detected using alloantisera directed against the polymorphic regions of the captured HLA entities. The technique is able to detect specific HLA-A, B, and DR antigens in extracts prepared from blood smears as well as from dried and freshly thawed lymphocytes. The assay may be of potential use in forensic medicine, particularly in instances where extraction of nucleic acids for fingerprinting is not feasible.

Enzyme-linked immunosorbent assay for HLA determination on fresh and dried lymphocytes.

HLA-A and -B antigens were detected on fresh and dried peripheral blood lymphocytes by an enzyme-linked immunosorbent assay. Intact cells fixed to plates with glutaraldehyde were used as antigen and anti-HLA alloantisera as a source of antibodies. Determination of HLA antigens by the ELISA technique was comparable with the complement-dependent cytotoxicity test. The relative stability of HLA antigens as shown in this report and the extensive polymorphism of the HLA system make the ELISA technique a promising tool for the analysis of HLA antigens on non-living cells including, for example, medicolegal investigation of blood stains.

Cytokine production in human mixed leukocyte reactions performed in serum-free media.

The mixed leukocyte reaction (MLR) is an in vitro test commonly performed in a serum-containing medium (SCM), and used to study allorecognition and cellular immunity accompanied by cytokine release. We investigated the possibility of performing the MLR test in serum-free media (SFM) by comparing human leukocyte proliferation and cytokine release in MLRs performed in SFM and SCM. Of the four SFM tested, only Biotarget- was as effective as SCM in supporting leukocyte proliferation and IL-2 secretion. Both phenomena were observed only in allogeneic combinations. The levels of IL-1, IL-6, and TNFalpha in allogeneic MLR combinations in SFM were half those in SCM cultures; the kinetics of their release were the same. With the exception of IL-2, a high degree of spontaneous release of the other three cytokines analyzed was observed in responder cells, in irradiated stimulator cells, and in autologous combinations cultured in both SCM and SFM. It appears that unlike IL-2, the cytokines IL-1, IL-6, and TNFalpha are nonspecifically produced in MLR and cannot serve as sensitive indices of HLA disparity.

A short human and mouse MLR assay utilizing lymphokine (IL-2, IL-3) secretion as an early activation event.

The mixed leukocyte reaction is the only functional in vitro assay currently employed for confirmation of MHC matching between bone marrow recipients and their prospective donors and for MHC class II (HLA-Dw) typing. This assay is, however, time-consuming (6 days for human MLR), whereas for clinical purposes results are often required much earlier. In an attempt to shorten the MLR incubation period, we tested IL-2 (in human MLR) and IL-2/IL-3 (in mouse MLR) production as an indication of early stages of T cell activation. We here describe a shorter assay in which IL-2 and IL-3 secretion during MLR was assessed by adding the respective lymphokine-dependent cell lines either to the MLR supernatants or directly to the original MLR cultures, using the colorimetric (3-[4,5 Dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) (MTT) technique or the 3H-thymidine incorporation assay. In both human and mouse MLR systems, lymphokine production peaked at 24-48 hr after culture initiation, allowing tests to be completed within 48 to 72 hr. Weak MLR responses, as detected by lymphokine production, could be considerably amplified by irradiating (250-1000 cGy) the responder cells and by adding heparin (1-10 U/ml) to the cultures. The results obtained by this novel procedure correlated with those obtained by the standard 6-day human MLR assay in over 250 combinations tested thus far, and therefore it may replace the standard MLR procedure.

Prediction by a modified mixed leukocyte reaction assay of graft-versus-host disease and graft rejection after allogeneic bone marrow transplantation.

In this report we describe a modified, sensitive MLR test that appears to detect fine antigenic disparities between HLA-identical siblings confirmed as such by serology and the standard MLR test. In a group of 40 consecutive allogeneic bone marrow transplants, reactivity detected by the modified MLR test correlated with the development of rejection of matched marrow grafts and onset of acute graft vs. host disease (aGVHD). Thus, 13/15 positively reacting patient/donor pairs developed one of these complications (P < 0.001), while only 2/25 developed aGVHD in the negatively reacting group. This test may be useful for selecting the most compatible donor when several potential donors are available.

Positivity in a modified mixed leukocyte reaction test correlates with molecular HLA-C disparity in prediction of unrelated bone marrow transplantation outcome.

The modified mixed leukocyte reaction (MMLR) test consists of the standard MLR (SMLR) test to which interleukin-4 (IL-4) has been added. It is a sensitive procedure capable of detecting alloreactivity not detected by the SMLR. In the present study we applied the MMLR test to unrelated bone marrow transplantation (BMT) in an attempt to predict graft versus host disease (GVHD) and graft rejection (GR) by detecting alloreactivity between recipient/donor pairs otherwise found to be fully matched (HLA class I A and B tested by serology; class II DRB1 and DQB1 by sequence specific oligonucleotide probes [SSOP]) and by studying the relationship of MMLR alloreactivity and HLA-C disparity in the prediction of transplant related complications. Thirty-five patients transplanted from unrelated donors were included in the study. The MMLR test was seen to correlate with the incidence of transplant related complications, as of the 19 positive, cases 12 (63%) developed acute GVHD and 7 (37%) GR, while of the 16 negative cases only 5 (31%) developed GVHD (4 acute, 1 chronic) (p = 0.0001) and 2 (12.5%) GR. No such correlation was seen between the SMLR and the incidence of transplant related complications: the SMLR test was positive in only 4 (11%) cases (all of which developed GVHD or GR) but of the 31 negative cases 22 (71%) also developed GVHD or GR. Reactivity in the MMLR also correlated with molecular HLA-C disparity (p = 0.015): While of the 19 positive cases 10 (53%) had molecular HLA-C disparity, of the 16 cases with negative MMLR, 14 (87.5%) were matched for molecular HLA-C. Two-way analysis confirmed that patients with positive MMLR transplanted from HLA-C mismatched donors were more likely to develop post BMT complications, including GVHD and GR, than patients with negative MMLR transplanted from HLA-C matched donors (r = +0.70) (p = 0.001). We conclude that the MMLR test may be a useful tool in the prediction of transplant related complications such as GVHD and GR, post unrelated BMT. Moreover, the MMLR test, in conjunction with molecular HLA-C typing, may improve unrelated donor selection.

Alloreactivity of umbilical cord blood mononuclear cells: specific hyporesponse to noninherited maternal antigens.

Earlier studies noted that patients who underwent cord blood (CB) transplantation had a lower incidence of graft-versus-host disease (GVHD) than those who underwent bone marrow transplantation (BMT). The premise that the immune reactivity of CB mononuclear cells (CB-MNC) to HLA mismatched combinations and to noninherited maternal antigens (NIMA) may be one of the factors involved in this phenomenon is still debatable. In this study we have attempted to evaluate the alloresponse and alloreactivity induced by CB-MNC by means of the standard mixed lymphocyte reaction test (SMLR) and the more sensitive, modified mixed lymphocyte reaction test (MMLR). Both techniques were used to test CB-MNC (n = 28) against HLA class II mismatched MNC from mothers (n = 26), fathers (n = 12), and unrelated individuals (n = 60) who served as controls. Alloresponse capabilities and stimulation capacities of CB-MNC in the SMLR were similar to those of control MNC: relative response (RR) = 73 vs. 65 and 58 vs. 65, respectively. Similar results were obtained in the MMLR. CB-MNC responded weakly to the maternal MNC in comparison with control MNC (RR = 47 vs. 73 [p = 0.0099]), while a stronger response was noted to the paternal than the maternal MNC (RR = 72 vs. 47 [p = 0.045]). Our results demonstrate that CB-MNC both respond to and induce alloresponse in HLA mismatched combinations. Moreover, the hyporesponse of CB-MNC to maternal cells that we observed suggests a form of tolerance to NIMA, which is probably due to the fetus's exposure to these antigens in its intrauterine life.

HLA and graft-versus-host disease: a population-based study of HLA phenotypes of Jewish and Arabic bone marrow transplanted patients in Israel.

The relationship between the incidence of graft-versus-host disease (GVHD) and human leukocyte antigens (HLA) was analyzed in HLA-identical sibling bone marrow transplantation donor/recipient pairs from the two major populations in Israel: Jewish and Arabic. HLA phenotypes were significantly different, in both groups, between patients and healthy controls. Among Jews, a higher incidence of GVHD was found with A28, CW3, CW6 of class I and DR7, DQ2 of class II; a lower incidence was found with A2, A26, B14. Among Arabs, a higher incidence of GVHD was found with B41, CW7 of class I, and DR2, DR13 of class II. The development of GVHD is apparently associated with HLA phenotypes specific to each population.

Differential expression of HLA class-I antigens on B and T lymphocytes obtained from human lymphoid tissues.

The amount of HLA class-I antigens was determined on the surface of enriched populations of B and T lymphocytes obtained from human peripheral blood, lymph nodes and spleens. The assays were performed using monoclonal antibodies that recognize different determinants on HLA class-I antigens and utilizing a simple and sensitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that B lymphocytes obtained from human peripheral blood, lymph nodes and spleens express at least twice as many HLA class-I antigens as T lymphocytes obtained from the same organs.

Bone marrow transplantation using unrelated and family related donors: the impact of HLA-C disparity.

The clinical outcomes of unrelated and of family related allogeneic bone marrow transplantation (BMT) were correlated with HLA disparity in a study in which molecular phenotyping of HLA-C was performed for unrelated donor-recipient pairs. The study included 30 patients who underwent BMT from unrelated donors and 43 patients who underwent BMT from family related donors. The unrelated group included 14 full match pairs, 12 class-I-C (molecular HLA-C) mismatched pairs, and 4 haploidentical pairs. The family related group included 9 full match pairs, 19 class-I mismatched pairs, and 15 haploidentical pairs. In the unrelated BMT group, the transplant-related complications of mortality, graft-versus-host disease (GVHD), and graft rejection, were significantly higher in molecular HLA-C mismatched than matched patients (67% vs 14%, P < 0.02; 50% vs 7%, P < 0.02; 42% vs 0, P < 0.02). The actuarial survival and the disease free survival (DFS) at 18 months was better for molecular HLA-C matched than for HLA-C mismatched unrelated transplants: 33% vs 18% (P = 0.065, NS), and 29% vs 16%, respectively. For the family related BMT group, the actuarial survival at 18 months was significantly higher for patients who were fully matched compared to those who were class-I mismatched, or those who were haploidentical pairs: 67%, 37%, and 13% respectively (P < 0.02). The actuarial DFS at 18 months of patients who were fully matched and of those who were class-I mismatched was similar, and better than that of haploidentical patients 45%, 37% (NS) and 13% respectively (P < 0.045). A lower incidence of transplant-related mortality occurred in class-I mismatched, family related (37%) than in locus-C mismatched unrelated patients (67%), and no difference was observed in the fully matched family related and unrelated patients. We conclude that a mismatch in locus C may be detrimental to BMT outcome and should therefore be included as a risk factor in the routine pre-BMT HLA phenotyping.

Human leukocyte antigens (HLA) class I and class II on sperm cells studied at the serological, cellular, and genomic levels.

The expression of human leukocyte antigens (HLA) on highly purified human ejaculated sperm cells was studied using the sensitive enzyme-linked immunosorbent assay (ELISA) technique and a wide panel of monoclonal antibodies to class I and class II HLA. In addition, the stimulatory capacity of these cells was tested in mixed cultures of lymphocytes and spermatozoa, and the levels of RNA homologous to the HLA class I and class II genes were determined. The results obtained using the ELISA indicate that the class I and class II HLA serologically defined antigens are weakly expressed on the cell surface of the mature spermatozoa. Highly purified sperm cells consistently stimulated heterologous lymphocytes but not when HLA-DR compatibility was observed between stimulator and responder. The proliferative response of lymphocytes induced by sperm cells was lower than the response obtained in a lymphocyte-lymphocyte combination, though the kinetics of the response were similar in both cases. In addition, it was found that spermatozoa contained RNA species homologous to HLA class II DR beta and DQ beta genes sequences but not to HLA class I sequences. The levels of these RNA species were significantly reduced after interferon stimulation. Lymphocytes that served as positive control were found to contain RNA complementary to both HLA class I and class II genes.

Zonal variation of HLA expression on human cornea epithelium.

Seven human corneas were studied, using the enzyme-linked immunosorbent assay (ELISA) technique, for expression of human leukocyte antigens (HLA) class-I and class-II antigens on the epithelial cells of the central 8-mm corneal button and the remaining corneal periphery and limbus. The antigenicity was compared with that of Raji and Daudi cell lines, which served as controls. It was demonstrated that the epithelium of the two corneal parts shows a high expression of HLA class-I antigens, albeit not as strong as that found in the control cell lines. On the other hand, HLA class-II antigens were found only in the limbal-peripheral area. Expression of the HLA class-I antigens was weaker in the central portion of the cornea. It is proposed that the absence of HLA class-II antigens expression in the central portion of the cornea offers an additional explanation for the greater success of small corneal grafts, since the recognition step necessary for the immunological response is lacking.

Detection and identification of HLA antigens in dried spleens by means of enzyme linked immunosorbent assay (ELISA).

HLA class-I and class-II antigens were detected in extracts of dry spleen tissue allowed to age from 1 to 17 months, using monomorphic anti HLA monoclonal antibodies and the ELISA technique. Using polymorphic HLA alloantisera samples of spleen tissue could be characterized and differentiated. The potential use of dried tissues and blood stains is discussed.

Regular radon activity concentration and effective dose measurements inside the great pyramid with passive nuclear track detectors.

Radon activity concentrations and equilibrium factors inside the great pyramid of "Cheops" were measured with passive nuclear track detectors. The variation of these concentrations in location was investigated. Seasonal variation of radon activity concentrations with winter maximum and summer minimum were observed inside the pyramid. The 1-y average radon activity concentration ranged from a minimum of 20 to a maximum of 170 Bq m(-3). Results show that the yearly average equilibrium factor between radon and its progeny was assessed as 0.16 and 0.36 inside the pyramid and near entrance, respectively. Moreover, the estimated annual effective dose was 0.05 mSv to tour guides and varied from 0.19 to 0.36 mSv for the pyramid guards; for visitors the average effective dose was 0.15 microSv per visit. These are lower than the 3-10 mSv y(-1) dose limit recommend by ICRP 65.