Risk associations between HLA-DPB1 T-cell epitope matching and outcome of unrelated hematopoietic cell transplantation are independent of HLA-DPA1.

HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T-cell epitope (TCE) groups are associated with increased mortality after hematopoietic SCT (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of TRM compared to permissive mismatches (hazard ratio (HR) 1.30, confidence interval (CI) 1.06-1.53; P=0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37-0.80; P=0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence.

Hematopoietic stem cell donor registry strategies for assigning search determinants and matching relationships.

Registries and cord blood banks around the world collect and store the HLA types of volunteers in order to identify matched unrelated donors for patients requiring hematopoietic stem cell transplantation. This task is complicated by the many formats in which HLA types are provided by the testing laboratories (types obtained by serology vs by DNA-based methods; high vs intermediate vs low resolution) and by the need to identify which of these diverse types are most likely to match the HLA assignments of a searching patient as closely as possible. Conversion of the assignments to 'search determinants' may be included within the algorithm used to select and prioritize a list of potentially suitable donors, either as an aid to matching or as a tool to optimize the performance of comparisons within large data files. The strategies used by registries to create search determinants are described. A set of search determinants, utilized by the National Marrow Donor Program, is provided as an example and is intended to initiate further discussion aimed at understanding the process used by each registry with the possibility of developing a standard process among registries worldwide.

Phase II study of a moderate-intensity preparative regimen with allogeneic peripheral blood stem cell transplantation for hematologic diseases: the Texas Transplant Consortium experience.

Conventional preparative regimens for allogeneic stem cell transplantation are associated with excessive regimen-related toxicity (RRT) in some patients because of underlying comorbidities, advanced age, or prior treatment. We studied a preparative regimen designed to reduce RRT, yet allow for adequate engraftment and development of a graft-versus-malignancy effect. Thirty patients (median age, 57 years) were entered on study. Twenty-nine patientsreceived stem cells from HLA-identical siblings and 1 from a sibling mismatched for 1 antigen at the A locus. Sixteen patients had received previous stem cell transplants (6 allogeneic and 10 autologous). The preparative regimen consisted of fludarabine 30 mg/M2 per day IV on day -10 to day -5, busulfan 1 mg/kg per dose PO (n = 6) or 0.8 mg/kg per dose IV (n = 24) for 8 doses every 6 hours on day -6 to day -5, and horse-derived antithymocyte globulin 5 mg/kg per day IV (n = 12) or 15 mg/kg per day IV (n = 18) on day -4 to day -1. GVHD prophylaxis consisted of cyclosporine (CYA) 3 mg/kg BID PO starting on day -3 (n = 13) or CYA and methotrexate 15 mg/m2 IV on day +1 and 10 mg/m2 IV on day +3 and day +6 (n = 17). The median number of CD34 cells transplanted was 3.19 x 10(6)/kg. All patients demonstrated recovery of hematopoietic function. Twenty-six (89%) of 29 evaluable patients achieved greater than 90% donor cell chimerism before day 100. Three patients never achieved greater than 90% donor chimerism, and another 3 patients subsequently lost donor chimerism. All 6 of these patients had autologous reconstitution with progressive disease. RRT was minimal; 7 patients had greater than grade II nonhematologic toxicity and there were no toxic deaths attributable to the conditioning regimen. Transplantation-related mortality was 7% (95% confidence interval [CI], 6%-8%) at 3 months and 28% (95% CI, 23%-34%) at 12 months after transplantation. Non-relapse-related mortality was most often due to infection. Grade II or greater GVHD developed in 56% of evaluable patients, and all patients with disease response developed GVHD. Actuarial estimates of overall and disease-free survival at 12 months were 52% (95% CI, 43%-63%) and 30% (95% CI, 24%-37%), respectively. Although this preparative regimen allowed adequate engraftment with minimal RRT, GVHD and infectious complications caused significant morbidity and mortality. Further study to define appropriate patient populations for this regimen, while limiting GVHD and infection risks, is needed.

Improved accuracy in differentiating malignant from benign mammographic abnormalities: a simple, improved magnetic resonance imaging method.

BACKGROUND: Although several refinements have been reported for breast magnetic resonance imaging (MRI), there has been no uniform agreement by researchers on the optimal method. The authors report a simple and effective MRI method that incorporated the best qualities of other breast MRI methods yet eliminated the complexity of dynamic sequences and computer subtraction. This new method used fat-suppression, a 3D technique, a dedicated breast coil, and quantitation of lesion enhancement. METHODS: Sixty-one mammographically suspicious lesions were evaluated with a fat-suppressed T1-weighted 3D FLASH sequence before and after administration of Gd-DTPA. Abnormalities were evaluated primarily by the degree of lesional enhancement; lesional morphology was assessed as a secondary criterion. For small or multiple lesions, the authors reformatted images to produce MRI findings that corresponded to the mammographic abnormality. To allow accurate pathologic correlation, all subjects underwent stereotactic or excisional biopsy of the suspicious lesions. RESULTS: Using this new method, all 15 breast carcinomas were enhanced with a signal intensity (SI) increase of > or = 180% (mean = 337%). No benign lesions enhanced at a SI of > 180%. The difference in degree of enhancement between malignant and benign lesions was statistically significant (P < 0.05). There were overlapping degrees of postcontrast enhancement among fibroadenomas (n = 13; mean SI = 70%) and atypical hyperplasias (n =; 11; mean SI = 82%), but morphologic characteristics allowed for discrimination between these two entities. In the remaining benign breast disease lesions, there was minimal enhancement. CONCLUSIONS: 3D fat-suppressed sequencing using this new MRI method accurately discriminated between benign and malignant mammographic abnormalities and eliminated the time-intensive and complex MRI methods without sacrificing accuracy.

Induction of determinant spreading and of Th1 responses by in vitro stimulation with HER-2 peptides.

Immunization with tumor antigens induces cellular and humoral immune responses. These responses by T cells are specific for defined epitopes (determinants) in the molecule of the immunizing tumor antigen. Extension of such responses to self-antigens requires induction of autoimmunity to the tumor. As with systems of autoimmune disease, expression of T cell autoimmunity is charaterized by diversification of responses from the inducer determinant to other responder (cryptic) determinants. Since similar strategies may be useful for therapy of human cancers, we investigated whether the induction of response to a HER-2 peptide F7 (776-789) induces enhanced reactivity of other HER-2 peptides. We found that stimulation with F7 can expand a response to another epitope F13 (884-899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased interferon gamma secretion, and proliferation, but was not observed with another donor who shared HLA-DR14 and HLA-DQ5 with the patient. Since repeated vaccination with the same epitope may lead to a decline of primary cell reactivity caused by apoptosis spreading the response to other epitopes, the tumor antigen may provide an approach for maintaining an inflammatory Th1 response during cancer vaccination.

Shared cadaver donor-husband HLA class I mismatches as a risk factor for renal graft rejection in previously pregnant women.

During the last few years, we have observed four cases in which accelerated rejection of a cadaver donor kidney in a previously pregnant woman could be clearly attributed to the rapid emergence of anti-human leukocyte antigen (HLA) antibodies that had been stimulated by mismatched paternal antigens but were completely undetectable at the time of transplantation. In addition to reviewing those cases, we also reviewed data on 19 other women with a history of at least one pregnancy who underwent transplantation with a first cadaveric kidney since 1991 and were followed for at least six months. The HLA antigens of the husbands had to have been determined and all accelerated rejection or early graft losses due to confirmed or presumed immunological causes were considered. Of the 19 additional women meeting these inclusion criteria, three suffered early immunological graft loss. As in our index cases, two of these women had also received kidneys from donors who shared at least one major immunogenic mismatched antigen with the respective husband for a total of six of seven women with early immunological graft loss. Only one of the 16 women without accelerated rejection or early immunological graft loss had a donor who shared a mismatched antigen with her husband. The difference between the two groups is statistically significant (p = 0.0005). These findings, considered with individual cases reported by other groups, indicate that transplantation from a cadaver donor with immunogenic mismatched class I HLA antigen(s) shared with the husband should be avoided in women with a previous history of pregnancy even when anti-HLA antibodies are not currently detected.

Presence of HLA-C-restricted cytotoxic T-lymphocyte responses in long-term nonprogressors infected with human immunodeficiency virus.

Approximately 5% of people with human immunodeficiency virus type 1 (HIV-1) infection remain free of disease for 10 or more years. These long-term nonprogressors (LTNPs) exhibit lower viral loads and stable CD4+ lymphocyte counts. The immunologic basis for this disease-free condition is not known. Because cytotoxic T lymphocytes (CTLs) constitute a major immune defense mechanism for sustained recovery from viral infections, we analyzed HIV-specific CTL responses in three asymptomatic LTNPs. We observed the presence of HIV-1 envelope-specific CTL responses mediated by HLA class I C-restricted CD8+ cells in these individuals. Using autologous target cells and a panel of HLA-matching and -mismatching B-cell lines as targets, we determined that HLA-Cw7 is the restricting element for the observed CTL activity. Additionally, we identified three peptides, one previously not reported, from conserved regions in the envelope protein as CTL epitopes. We previously reported these peptides to be efficient in inducing HIV-specific cellular immune responses in murine and nonhuman primate models. Our results support the role of the HLA-C locus in generating CTL responses and constitute the first report of an HLA-Cw7-restricted HIV-1 envelope-specific CTL response in HIV+ LTNPs, which may be important in the control of HIV replication in vivo.

Multicenter evaluation of the flow cytometry T-cell crossmatch: results from the American Society of Histocompatibility and Immunogenetics-College of American Pathologists proficiency testing program.

BACKGROUND: The performance characteristics and interlaboratory comparisons of the T-cell flow cytometry crossmatch remain largely unknown. METHODS: This study was performed using data from the ASHI-CAP proficiency testing program. Four unknown sera and two unknown cells were sent to participating laboratories twice a year for 4 years. RESULTS: In one survey in which different crossmatch techniques were compared, flow cytometry was slightly more sensitive than the antiglobulin method and considerably more sensitive than direct cytotoxicity. However, the proportion of participants in any given survey detecting antibodies in all sera expected to be positive was 50-60% and has not changed over the years. Failure to detect antibodies correlated with low antibody concentration, diluting the unknown serum by the testing laboratory, and with the instrument used. False positive results with normal sera were infrequent. Fluorescence intensity values were not standardized and were highly variable, but when fluorescence units reported by individual laboratories were divided by their own positive-negative cutoff values, results from different centers were more comparable. In general, fluorescence-to-cutoff ratios >5 correlated with complement binding activity, whereas values <5 denoted concentrations below those required to fix complement. CONCLUSIONS: Flow cytometry, as used by most centers, is highly sensitive and allows relative antibody quantitation. Furthermore, the data define objective parameters that may help to standardize the test and improve its predictive value in clinical transplantation.

Genetic regulation of the capacity to make immunoglobulin G to pneumococcal capsular polysaccharides.

BACKGROUND: Genetic regulation of immunoglobulin G(IgG) responses to pneumococcal capsular polysaccharides (PPS), has been demonstrated in mice but not in humans. Earlier studies from this laboratory showed that healthy adults have a varying capacity to generate IgG antibody to PPS; this study sought to determine whether this capacity is genetically controlled. METHODS: A 23-valent pneumococcal vaccine was administered to 72 unrelated White adults, 4 nuclear families, and 61 members of an extended Ashkenazic Jewish family. Selected individuals later received one or more doses of the vaccine and/or a single dose of protein-conjugated PPS. Four to six weeks after each vaccination, IgG to PPS was measured by ELISA. Immunoglobulin allotypes and HLA types were determined by standard techniques. RESULTS: After vaccination, 53% of the 72 unrelated White adults had measurable levels of IgG antibody to all of 10 PPS studied (high-level responders), 36% had IgG to 6-9 PPS, and 11% had IgG to < or = 5 of 10 PPS (low-level responders). Persons who did not make IgG to an individual PPS also failed to make IgM or IgA to that antigen. Low-level responders had reduced mean IgG levels to PPS to which they did make IgG; nevertheless, their total serum concentrations of IgG, IgG2, IgA, and IgM were normal, and each made IgG2 to at least one PPS, all indicating that a global defect in Ig production was not responsible. The responder status of offspring was highly associated with that of their parents. Segregation analysis of 61 Ashkenazic family members revealed that the capacity to generate anti-PPS IgG was inherited in a mixed, codominant fashion. Repeated vaccination or administration of protein-conjugated PPS did not elicit measurable IgG in nonresponders. The HLA type was not associated with antibody responses. An association between IgG level and Gm(23)+ allotype was observed in unrelated Whites but not in Ashkenazic Jews. CONCLUSIONS: Thus, humans exhibit a variable capacity to respond to PPS. This response is hereditable in a mixed, codominant fashion. The absence of IgG to a PPS, even after antigen is presented in a protein-conjugate form, may reflect a genetically mediated failure to recognize polysaccharide antigens. Since persons who respond to fewer PPS also have lower levels of IgG to PPS to which they do respond, genetically determined deficiencies in events that involve proliferation of committed B lymphocytes may also play a role.

Characterization of T-cell receptor V beta repertoire in ovarian tumour-reacting CD3+ CD8+ CD4- CTL lines.

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumor responses. To characterize the T-cell antigen receptor (TCR) V beta expression in autologous cytotoxic effectors we isolated CD3+ CD8+ CD4- cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR V beta repertoire of CD3+ CD8+ CD4- lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cell-sensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8+ CTL lines expressed a broad distribution of TCR V beta repertoire which was dominated by particular groups of V beta families in each CTL line. However, no predominant expression of one or the same V beta segment in all CTL lines was observed although statistical correlations between V beta family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR V beta families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR V beta repertoire of human ovarian tumour-reactive CD3+ CD8+ CD4- CTL from different individuals of known HLA types.

Oligopeptide induction of a cytotoxic T lymphocyte response to HER-2/neu proto-oncogene in vitro.

The HER-2/neu proto-oncogene (HER-2) encodes a transmembrane protein whose expression is enhanced in a number of breast and ovarian tumors and correlates with tumor aggressiveness. Because of its expression on normal epithelial cells, HER-2 can be defined as a tumor associated antigen and is of interest as a target of a therapeutic anti-tumor T cell response. To investigate whether oligopeptides analogs of HER-2 isolated from a likely target area of T cells can induce an anti-tumor CTL response, peripheral blood mononuclear cells were stimulated in vitro with HER-2 synthetic peptides. CTL cultures generated recognized peptides used as immunogen. A CD3+CD8+CD4- line isolated from these cultures lysed HLA-A2+, HER-2+ ovarian tumors but not natural killer target K562 cells, and showed significantly higher lysis of HER-2high than of HER-2low ovarian tumors. This lysis was inhibited by HER-2 peptide-pulsed HLA-A2+ targets, suggesting that similar epitopes are presented on tumor cells associated with HLA-A2. The observation that peptide analogs of a proto-oncogene can induce CTL in vitro which express tumor lysis dependent on the levels of expression of HER-2 is novel for human tumor systems. Targeting by T cells of HER-2 may prove useful for understanding the mechanisms of recognition, tolerance, and therapeutic use of human tumor reactive T cells.

Relationship of increased levels of circulating intercellular adhesion molecule 1 after heart transplantation to rejection: human leukocyte antigen mismatch and survival.

Noninvasive methods to assess immune activation would be helpful in optimizing therapy after heart transplantation to reduce rejection (acute and chronic) and complications caused by excessive immunosuppressive therapy. Intercellular adhesion molecule 1 has been shown to play an important role in T-cell activation and allograft rejection. A soluble form of intercellular adhesion molecule 1 has been discovered to be circulating in plasma. To test the hypothesis that increased levels of circulating intercellular adhesion molecule 1 may have prognostic value as a marker of immune activation, we examined whether levels of circulating intercellular adhesion molecule 1 during the early postoperative period correlated with endomyocardial biopsy scores, soluble interleukin-2 receptor levels, human leukocyte antigen mismatch, and survival. For the first 3 weeks after surgery, serum was obtained once weekly on the same day as endomyocardial biopsy samples from 52 patients who survived more than 30 days after heart transplantation. A sandwich enzyme-linked immunosorbent assay was used to measure circulating intercellular adhesion molecule 1 and soluble interleukin-2 receptor. Increased circulating intercellular adhesion molecule 1 levels did not correlate with endomyocardial biopsy scores but were associated with greater mismatch at the human leukocyte antigen-B and -DR loci (p = 0.02). A significant correlation was found (p = 0.002) between circulating intercellular adhesion molecule 1 levels and soluble interleukin-2 receptor, albeit with a low r value of 0.27. Survival was reduced in patients with high levels of circulating intercellular adhesion molecule 1 (p = 0.006) or soluble interleukin-2 receptor (p = 0.001) with the greatest reduction in survival when both were elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Strategies for prenatal HLA typing for potential cord blood donor evaluation.

The reasons for prenatal HLA typing for potential cord blood stem cell donor evaluation are reviewed and the strategies for doing so are summarized. Cultured fetal cells are class I HLA typed by modified serological techniques. When indicated, the fetal cells are also class II HLA typed by one or another DNA typing method. The same procedures can be used for the prenatal identification of a potential donor for an affected fetus.

Tuberculous epididymitis and epididymo-orchitis: sonographic findings.

The findings at scrotal sonography in 10 patients with tuberculous epididymitis and in 2 with nontuberculous epididymitis are presented. In 6 patients with tuberculous epididymitis the testes were also involved (epididymo-orchitis). The most notable sonographic findings of tuberculous epididymitis were an enlarged epididymis, predominantly in the tail portion, and marked heterogeneity of the echo texture of the involved epididymis. Sonographic findings of associated testicular involvement consisted of a diffusely enlarged hypoechoic testis or ill defined focal intratesticular hypoechoic areas, or an irregular margin between the testis and epididymis. The sonographic findings encountered in patients with tuberculous epididymitis appear to be different from those encountered in nontuberculous epididymitis. Sonography might prove helpful in aiding the clinical distinction between these 2 forms of epididymitis and in demonstrating associated testicular involvement in tuberculous epididymitis.

Cytotoxic T-cell clones isolated from ovarian tumour infiltrating lymphocytes recognize common determinants on non-ovarian tumour clones.

CTL-TIL lines have been developed from tumour infiltrating lymphocytes (TIL) from the ascites of patients with ovarian carcinoma, and used to investigate whether common tumour antigens are expressed on allogeneic ovarian tumours epithelial tumour lines derived from colon and pancreatic carcinoma. Three CTL lines expressed preferential cytolytic activity against autologous tumour cells and against certain allogeneic ovarian tumour cells that shared HLA-A2 molecules. Analysis of the target specificity of these CTL lines indicated that they also lysed human colon and pancreatic tumour lines sharing HLA-A2. CTL-TIL clones isolated from these lines were found to lyse HLA-A2+ ovarian, colon and pancreatic tumours, and to recognize clonally distributed common epitopes on pancreas and colon tumour clones. These results indicate that shared tumour antigens can be found among tumours of common epithelial cell origin. These results indicate a novel class of T-cell-definable tumour antigens recognized by tumour-reactive CTL on human tumours and may be significant for understanding of cellular immunity in ovarian cancer, identification of CTL-defined tumour antigens and future adoptive specific immunotherapeutic approaches in ovarian cancer.

Congenital 21-hydroxylase deficiency as a new deletion mutation. Detection in a proband during subsequent prenatal diagnosis by HLA typing and DNA analysis.

A child with 21-OH-def whose 9 weeks' pregnant mother was referred for prenatal diagnosis was found upon very careful histocompatibility testing to lack expression of any of his father's HLA antigens on his peripheral blood lymphocytes. The possibility of alternative paternity was considered to be extremely unlikely after additional genetic marker tests. The conclusion that the affected child's disease resulted from inheritance of a maternal CYP21B (21-OH) deletion and a de novo deletion in the paternal chromosome 6 segment that includes both the CYP21B (21-OH) and HLA genes was confirmed by subsequent DNA analysis using 21-OH, C4, DPB, and PCH6 probes. The presence of a heterozygous RFLP for DPB, the absence of a deletion for either CYP21B (21-OH) or C4 genes, and the presence of a paternal HLA antigen haplotype on the fetal cells additionally indicated that the fetus lacked the same deletion and could be predicted to be completely normal.

Multiple large calculi in a continent urinary reservoir: a case report.

Continent urinary reservoirs have increasingly become popular during the last decade but they are associated with long-term complications. We report a case of huge multiple stones in an ileocecal reservoir, which formed as a result of noncompliance with intermittent self-catheterization. An open method was required for removal of the stones to prevent recurrent urosepsis.

A familial lymphoproliferative disorder presenting with primary pulmonary manifestations.

A familial lymphoproliferative disorder presented in three male siblings with primary pulmonary involvement manifested as either lymphoid interstitial pneumonia or an angiodestructive polymorphous infiltrate morphologically resembling lymphomatoid granulomatosis. The polymorphous infiltrate consisted chiefly of mature T-cells with a few B-cells and plasma cells, and gene rearrangement studies failed to show clonality. Epstein-Barr virus, frequently associated with proliferative lesions in males in the X-linked lymphoproliferative syndrome, was not demonstrated in any of the pulmonary lesions. An HLA haplotype shared among the affected siblings was A1, B8, DR4. The unusual clinical presentation plus the lack of involvement by EBV in the pulmonary lesions suggests that this is a previously undescribed familial lymphoproliferative disorder.

Tight linkage of the gene for spinocerebellar ataxia to D6S89 on the short arm of chromosome 6 in a kindred for which close linkage to both HLA and F13A1 is excluded.

A locus for an autosomal dominant form of spinocerebellar ataxia (SCA1) has been assigned to the short arm of chromosome 6 on the basis of linkage to the major histocompatibility system (HLA). In this study of a five-generation American black family, close linkage between the disease locus and both HLA and the coagulation factor XIIIA (F13A1) locus was excluded, and lod scores for all locations of the disease locus between HLA and F13A1 were less than -1.4. These results suggest that the locus causing spinocerebellar ataxia in this family is not in this region. However, the disease locus was found to be closely linked to a microsatellite polymorphism, D6S89, which is between HLA and F13A1. The maximum lod score for SCA1 and D6S89 is 4.90 at a recombination fraction of 0, both in males and in females. These data show that exclusion of close linkage to the HLA complex and F13A1 in a kindred with spinocerebellar ataxia does not rule out the possibility that the disease locus in that family is on 6p. Accordingly, all families segregating a dominantly inherited ataxia should be evaluated for linkage to D6S89, to determine whether the locus causing the disease is SCA1.