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Twelve novel human leukocyte antigen class II alleles are described; eight DRB1 alleles and four DQB1 alleles. Nine of the variants are single nucleotide substitutions from their most homologous allele, of which six result in amino acid changes (DRB1*0459, *1156 and *1522; DQB1*0205, *0320 and *0321) and three are silent substitutions (DRB1*030105 and *040304, and DQB1*030104). The remaining alleles (DRB1*0906, *1464 and *1468) differ from their most similar alleles by two to three nucleotide substitutions which alter one to two amino acids.

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PURPOSE: To define the magnetic resonance (MR) imaging appearance of shell osteochondral allografts of the knee and compare the MR findings with antibody responses. MATERIALS AND METHODS: Thirty-six grafts were evaluated with a 1.5-T unit with T1-, intermediate-, and T2-weighted, and three-dimensional spoiled gradient-recalled MR imaging at 3, 6, 12, 24, and/or 36 months after surgery. Nineteen patients underwent imaging serially. Two osteoradiologists scored by consensus host marrow edema, thickness of graft-host interface, signal intensity of graft marrow, cyst formation, joint effusion, articular cartilage defects, and surface collapse. Patients were divided into antibody-positive (AP) (n = 11) and antibody-negative (AN) (n = 25) groups evenly distributed across the different time points on the basis of results of anti-human leukocyte antigen antibody screening. MR findings for the two groups were compared. RESULTS: AP patients demonstrated greater mean edema (P<.002), thicker interface (P<.03), and more abnormal graft marrow (P<.04) than AN patients, and they had a higher proportion of surface collapse (P<.03). CONCLUSION: Humoral immune responses were associated with more inflammation and less complete incorporation after allograft placement. MR imaging shows promise as a surrogate biomarker for success of shell osteochondral allograft implantation.

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A new assay using flow cytometry has been established to assess natural killer (NK) lytic activity with common bench top instrumentation. This assay uses a cyanine membrane dye to stain live K562 target cells and an iodide nuclear dye to evaluate dead cells, and provides a method of reliably separating target and effector cell populations. Effector cells remain unstained (fluorescent negative) throughout the procedure. The damaged pre-labeled targets appear doubly stained as their membranes become permeable to the nuclear stain during incubation. Percent cytotoxicity of various effector:target cell ratios is discerned using flow cytometric analysis after a 2 h incubation in this new assay, as compared to 4 h with the 51Cr-release 'gold standard' assay for cell-mediated cytotoxicity. Comparisons of normal individuals tested in parallel with the fluorescent dyes and the 51Cr-release assay have shown direct correlations. This new two-color flow cytometric technique has proven to be uncomplicated and reproducible when used in the clinical setting.

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Antithymocyte serum (ATS), a polyclonal antibody preparation raised in rabbits, has been used as rescue therapy for severe rejection and induction of immune prophylaxis in our pediatric patients with heart transplants. To evaluate the customized pediatric ATS dosages, circulating plasma levels of unbound ATS were measured by an indirect flow cytometric analysis. ATS blood levels and their effects on in vitro lymphocyte function (mixed lymphocyte culture), peripheral blood lymphocyte subsets (immunophenotyping), and in vivo response, as measured by echocardiographic or biopsy data, were studied in three pediatric transplant patient groups. Detectable levels of circulating ATS were present 24 hours after infusion and correlated with the decrease in CD2+ peripheral blood lymphocytes. As expected, detectable ATS levels were measured only in the ATS treatment groups. Significant differences in lymphocyte subsets were seen between patients receiving ATS and those never receiving ATS (p < 0.01), with the non-ATS patients having normal lymphocyte subset percentages (CD2 = 60% +/- 29%). The mixed lymphocyte culture response was suppressed to a greater degree in the ATS therapy groups (86% vs 75%, p < 0.02), although these results were confounded by the use of high-dose steroids in all groups, which inhibit allogeneic responses. We conclude that effective immunologic monitoring of ATS therapy can be accomplished by peripheral blood lymphocyte subset determinations and ATS serum levels.

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Ten healthy males (mean age 22.3 +/- 0.8 yr) pedaled with maximal effort for 30 s against a workload adjusted prior to the start of the test to 0.98 N.kg body mass-1. Blood samples were collected before, and 3 min and 1 h following exercise. Peak and average power mean values were 1020 +/- 51 and 738 +/- 34 W, respectively. Total leukocytes increased 40% in response to the exercise bout, but were 16% below pretest levels after 1 h of recovery (F = 123, P < 0.001). Neutrophils and lymphocytes represented approximately 60% and 30% of the leukocytosis, respectively. Lymphocytes increased 30% following exercise, but were 36% below pretest levels after 1 h recovery (F = 56.4, P < 0.001). The post-test lymphocytosis can be explained primarily from the 176% increase in natural killer cells (NK) and 28% increase in cytotoxic/suppressor T cells, while the 1-h recovery lymphopenia occurred because of a sharp decrease in total T cells and a moderate decrease in NK cells. No significant changes in lymphocyte proliferative response or serum immunoglobulin levels were found when appropriate adjustments for changes in plasma volume or lymphocyte subset changes were made. Plasma epinephrine increased 300% in response to the exercise bout, and best explains the measured changes in circulating levels of lymphocyte subsets. These results demonstrate that changes in circulating levels of leukocyte and lymphocyte subsets, especially NK cells, occur rapidly in response to 30 s of brief, heavy exertion.

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The discovery of immunoglobulin E (IgE) is considered the most important contribution, to date, in the field of clinical allergy. Studies in rodents and humans have suggested that IgE production could be regulated by antigen-specific helper and suppressor T cells and by isotype-specific factors having affinity for IgE. In recent years, the synthesis of IgE has been shown to be regulated, in part, by a cytokine network. This review summarizes the cytokines that up-regulate (interleukins-4, 5, and 6) and down-regulate (interferon-gamma and interleukin-2) the production of IgE. Emphasis is placed on IL-4 and IFN-gamma, two lymphokines known to play a major, but reciprocal, role in IgE synthesis. Increased insight into the various mechanisms of IgE control by cytokines and their receptors will eventually lead to improved treatment strategies in the clinical management of IgE-mediated allergy.

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Recovering bone marrow transplant (BMT) recipients have 20-70% circulating lymphocytes which co-express CD8 and CD11b (normals = 5-15%). These CD8/11b cells comprise at least two subpopulations distinguished by their expression of CD3. The CD3+ CD8/11b cells are T lymphocytes which exhibit in vitro suppressor activity (Ts); the CD3- CD8/11b cells express CD16 and are natural killer (NK) cells. In this study, we investigated whether such cells influenced circulating levels of IgA and IgM in 18 BMT recipients who each had greater than 30% circulating CD8/11b cells. We observed that in all patients whose CD8/11b cells were Ts lymphocytes (7/7) IgM and IgA levels were less than 10% of normal. Among those patients whose CD8/11b cells were NK cells (n = 11), two groups were distinguished. In one group (n = 5), less than 35% of patient NK cells expressed CD57 and serum levels of IgM and IgA were less than 10% of normal. In the second group (n = 6) greater than 60% of the NK cells expressed CD57 and serum levels of IgM and IgA were normal. In summary, our data indicate that BMT recipients have at least three distinct subsets of CD8/11b lymphocyte populations which may differentially regulate IgM and IgA production in vivo.

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The immunobiology of human immunodeficiency virus (HIV) and the role of laboratory testing in the diagnosis and management of HIV infection are reviewed. HIV is one of a family of RNA viruses called retroviruses. HIV has three structural genes (one of which codes for reverse transcriptase) and six regulatory and maturation genes. Upon infection in humans, HIV commandeers the immune system by infecting and lysing T-helper lymphocytes. Since these cells are key to directing the body's immune defenses, the person becomes susceptible to a variety of opportunistic infections, neoplasias, and neurologic disorders. Laboratory tests for HIV are used for three purposes: screening of large populations (such blood donors), diagnosis of current or latent infection, and monitoring of disease progression. Diagnosis of HIV infection relies on HIV antibody detection, viral cultures, antigen detection, or polymerase chain reaction viral genome detection. Disease progression can be estimated using immunophenotyping with flow cytometry or using other immunologic markers. The immunologic variables associated with HIV infection disclose a growing spectrum of immune deficits. New tests for diagnosing and monitoring patients infected with HIV have been quickly incorporated into clinical practice.

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Several lymphocyte crossmatch procedures used in clinical histocompatibility laboratories, including complement dependent (CDC, AHG-CDC) and complement independent (flow cytometric, chromium release) techniques, are used to assess the likelihood of allograft rejection due to preformed donor specific antibody. Crossmatch assays can be extremely sensitive and detect very low levels of donor reactive antibody present in the potential recipient. Since positive crossmatches are usually associated with allograft rejection, a positive crossmatch is generally a contraindication to organ transplantation. Recent data, however, suggests that not all positive crossmatches lead to graft rejection, particularly those due to autoantibodies. This underscores the need to critically evaluate any positive crossmatch to determine if the antibodies involved in the reaction are relevant to allograft rejection.

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The flow cytometric crossmatch (FCXM) has become an increasingly utilized method to detect low levels of anti-donor antibodies (e.g., anti-HLA) in potential renal allograft recipients. Anti-donor antibodies not apparent in the standard complement-dependent crossmatch, but detectable by the FCXM, are often associated with increased episodes of graft rejection and early graft failure. In this study we examined several parameters of the FCXM in order to establish a standardized methodology. First, we observed that optimal staining results were obtained when the secondary antibody was an Fc-specific F(ab'), anti-human IgG. In contrast to an anti-whole immunoglobulin antibody, the anti-Fc specific reagent did not react with surface immunoglobulin on B cells but was reactive with cytophilic immunoglobulin present on CD16+ cells. Next we determined that dualcolor analysis was superior to single-color analysis both for the evaluation of T cell reactivities and for the discrimination of T cell from B cell reactivities. Additionally, dual-color analysis revealed that the density of class I histocompatibility antigens on B cells is greater than on T cells, indicating that B cells may be a more sensitive target for detecting low levels of anti-class I antibodies. Finally, we determined that a shift in the mean fluorescence intensity of greater than 10 channels on a 256-channel, 3-decade log scale was indicative of a positive FCXM. The data presented in these studies provide the basis for performing standardized dual-color FCXM with increased sensitivity and specificity.

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The mechanism of OKT3 therapy is complex and may include depletion of circulating CD3 cells, modulation of the CD3 molecule, and/or functional inactivation of T cells. Although the absolute number of circulating CD3 cells in OKT3-treated patients is used to monitor therapy, many laboratories assign CD3 numbers based on reactivity with OKT3. These CD3 numbers could be artificially low since the epitope recognized by OKT3 may already be occupied. Using a monoclonal antibody against a different CD3 epitope, we detected CD3 expression on T lymphocytes from 18/18 OKT3-treated patients. Nonetheless, OKT3 therapy in these patients was clinically successful, suggesting that monitoring patients solely for CD3 is uninformative. Since CD3 is associated with the T-cell receptor (TcR), we also evaluated alpha-TcR-1, a monoclonal antibody which detects a conformational determinant of the CD3/TcR alpha/beta complex, and found that less than 1% of the CD3 cells from OKT3-treated patients reacted. Furthermore, these cells were unresponsive to allogeneic stimulation. However, when patient cells were cultured overnight in the absence of OKT3, both alpha-TcR 1 binding and responsiveness to allogeneic stimulation became detectable. Thus, the monitoring of patients treated with OKT3 can be more informative if lymphocytes are tested for reactivity with alpha-TcR-1 and an alpha-CD3 antibody other than OKT3.

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The Ss sialoglycoprotein (glycophorin B) and its antigens in Rhnull erythrocytes, which lack the Rhesus blood group antigens, due to apparently silent (amorphic type) or independent suppressor (regulator type) genes, were investigated. The quantity of the molecule in amorphic and in regulator type red cell membranes was found to be decreased by about 60%-70%, as judged from sodium-dodecylsulfate polyacrylamide gel electrophoresis. The Ss glycoprotein content in the erythrocytes from heterozygotes (regulator type) was diminished to an extent of about 30%. Confirming and extending previous studies, the S, s, Ux, Uz and 'N' antigens were slightly weakened in Rhnull erythrocytes. The U and Duclos receptors were only slightly or not depressed in amorphic Rhnull cells, but almost absent from or not detectable in those of the regulator type. This demonstrates that an additional alteration, apart from the decreased Ss glycoprotein content of the membranes, accounts for the weakness of these receptors in regulator type cells. We propose the hypothesis that (a) protein(s) encoded by the Rhesus locus form(s) a complex with the Ss glycoprotein. Thus, it (they) might facilitate the incorporation of the Ss glycoprotein into the membrane and also contribute to the complete expression of the U and Duclos antigens in normal cells.

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Eight general formulas are presented for estimation of the dizygotic/monozygotic ratio (DMR) for twins of the same sex and blood groups. The derivation of these formulas and example applications for each are given in plain English without complex statistical symbols. The formulas cover all blood group systems including multiallele systems with silent gene(s) as well as the HLA system. Predetermined DMR values for commonly used genetic markers (except HLA) are provided for U.S. white and black persons.

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The isolation and identification of three major alpha-keto end products (glyoxylate, pyruvate, alpha-ketoglutarate) of the isocitrate lyase reaction in 18-day chick embryo liver have been described. This was accomplished by the separation of these alpha-keto acids as their 2,4-dinitrophenylhydrazones (DNPHs) by high-performance liquid chromatography (HPLC). The DNPHs of alpha-keto acids were eluted with an isocratic solvent system of methanol-water-acetic acid (60:38.5:1.5) containing 5 mM tetrabutylammonium phosphate from a reversed-phase ultrasphere C18 (IP) and from a radial compression C18 column. The separation can be completed on the radial compression column within 15-20 min as compared to 30-40 min with a conventional reversed-phase column. Retention times and peak areas were integrated for both the assay samples and reference compounds. A relative measure of alpha-keto acid in the peak was calculated by comparison with the standard. The identification of each peak was done on the basis of retention time matching, co-chromatography with authentic compounds, and stopped flow UV-VIS scanning between 240 and 440 nm. Glyoxylate represented 5% of the total product of the isocitrate lyase reaction. Day 18 parallels the peak period of embryonic hepatic glycogenesis which occurs at a time when the original egg glucose reserve has been depleted.

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On the basis of the existence of incompatible phenotypes between the child and the alleged father, exclusion of paternity can be obtained even when the mother is not available for testing. Incompatible phenotypes in each genetic marker system of common erythrocyte antigens and enzymes, serum proteins, and major histocompatibility antigens are compiled. The efficiencies of each system and groups of systems in the exclusion of paternity are estimated; the cumulative chance is about 88% if all the common genetic marker systems are utilized.

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A procedure is described for estimating the chance of paternity from HLA-typing results. Phenotypes consisting of HLA-A and HLA-B antigens are divided into four groups. For each group, formulas have been derived to calculate the chance of passing a specific haplotype; those for common phenotypes have been predetermined and compiled into concise tables. Consequently, the paternity index can be derived in four simple steps. In reporting, paternity index is best expressed in percentages as relative chance of paternity and nonpaternity. In this way, the positive and negative aspects of estimation are both spelled out.

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