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Characterization of the novel HLA B*18:79 allele is described., (© 2012 John Wiley & Sons A/S.)

Characterization of the novel HLA alleles B*35:11:03, B*42:01:03, and B*51:01:35 is described., (© 2012 John Wiley & Sons A/S.)

Characterization of the novel HLA alleles A*02:330, A*11:108, B*40:175, and B*40:176 is described., (© 2011 John Wiley & Sons A/S.)

B*15:228 differs from B*15:01:01:01 at three nucleotides in exon 4., (© 2011 John Wiley & Sons A/S.)

A*03:132 differs from A*03:01:01:01 at nucleotide 853 (codon 261) in exon 4., (© 2011 John Wiley & Sons A/S.)

Background: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related deaths. Donor HLA antibodies have been implicated in TRALI cases. Blood centers are implementing TRALI risk reduction strategies based on HLA antibody screening of some subpopulations of ever-pregnant apheresis platelet (PLT) donors. However, if screening assay cutoffs are too sensitive, donation loss may adversely impact blood availability., Study Design and Methods: Pregnancy history and HLA antibody screening and single-antigen bead data from blood donors in the Retrovirus Epidemiology Donor Study-II Leukocyte Antibody Prevalence Study were evaluated for correlations between assay screening values, HLA antibody titer, and number of HLA antigen specificities. The probabilities of matching a cognate antigen in a recipient were calculated and examined in association with total number of specificities observed and screening values. The relative impact of imposing various screening assay cutoffs or pregnancy stratification was examined in relation to detection of HLA antibody-reactive donations and loss of donors and donations., Results: We provide evidence that higher HLA antibody screening assay values are associated with maintaining higher screening signals upon dilution and an increased breadth of specificities compared with lower screening values; the latter correlated with an increased risk of a cognate antigen match in potential recipients. Depending on the TRALI risk reduction strategy used, the potential loss of donations ranged between 0.9 and 6.0%., Conclusion: This analysis should enable blood centers to decide upon a TRALI risk reduction strategy for apheresis PLTs that is consistent with how much donation loss the blood center can tolerate., (© 2011 American Association of Blood Banks.)

Background: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Blood centers are implementing TRALI risk reduction strategies based on screening apheresis donors for antibodies to human leukocyte antigens (HLA)., Study Design and Methods: HLA antibody screening was performed on 7920 blood donors from the Leukocyte Antibody Prevalence Study (LAPS) using Luminex-based normalized background (NBG) cutoff ratios of 10.8 (Class I) and 6.9 (Class II). Single antigen bead (SAB) assay cutoffs of 2500 median fluorescence intensity units (Class I) and 1500 (Class II) were established based on results of two subpopulations of LAPS donors. Antibody frequencies against HLA A, B, C, DR, DQ, and DP antigens were determined for screen-reactive donors with prior pregnancies., Results: SAB reactivity for samples above our multiantigen bead NBG cutoffs was 78% for Class I and 79% for Class II. The SAB-positive rate increased among women with zero to four or more pregnancies (0.3%-15.6% Class I and 0.4%-18% Class II; p < 0.00001). The highest frequency antibodies were DR11 and B15 (4.4% of women with prior pregnancies). The majority of Class I positives contained more than five specificities. For Class II, antibody-positive women segregated into two groups: a single specificity or more than five specificities., Conclusions: Identification of HLA antigen specificities supports pregnancy associations previously found with screening assays. The significance of particular HLA specificities for inducing TRALI is currently being evaluated in a large lookback study of recipients of high-plasma-volume components from this donor cohort., (© 2010 American Association of Blood Banks.)

Cw*0774 differs from Cw*070201 by one nucleotide within the coding sequence of exons 2-4. DQB1*060105 differs from DQB1*060101 by one nucleotide within the coding sequence of exons 2-3.

This report describes the discovery and characterization of the human leukocyte antigen-B*4466 allele.

This report describes the discovery and characterization of the HLA-Cw*0817 allele.

This report describes the discovery and characterization of the HLA-Cw*0517 allele.

This report describes the discovery and characterization of the novel Cw*0346 allele.

Discovery of the novel HLA-B*5149 allele in a North American Caucasian individual is described. It differs from B*510101 by one nucleotide within the coding sequence of exons 1-6. A substitution at nucleotide position 488 in exon 3 changes alanine to glycine in amino acid position 139.

Discovery of the novel DRB1*0464 allele is described. This allele contains a nucleotide substitution in codon 13 that changes the amino acid histidine coded for in all other DRB1*04 alleles to tyrosine. This allele was found in a parent and one child of a North American Caucasian family with the haplotype: A*03, B*07, DRB1*0464, DRB4*0103, DQB1*0301.

Type II collagen (CII) is a potent arthritogen in the BB rat. To determine whether a restricted group of T cells is involved in the pathogenesis of collagen-induced arthritis, lymphocytes from synovium, peripheral blood, and lymph nodes of arthritic rats were studied for T cell receptor (TCR) V beta gene usage using polymerase chain reaction (PCR). Oligoclonal TCR V beta usage was found only in synovium recovered day 2 post-arthritis onset, but not day 7; lymph node and peripheral blood T cells showed diverse TCR usage at both times. To determine whether T cell local clonal expansion occurred in synovium at day 2 of arthritis, cDNA for four TCR beta families was sequenced through VDJ regions. Strong selective expansion of TCR V beta 8.2, 4, and 17 was noted. Importantly, the dominant clonotype of V beta 8.2 was identical to that of a lymph node-derived T hybridoma specific for the immunodominant epitope in CII(181-210). Cells from synovium (day 2 postonset) analyzed by flow cytometry also showed V beta 8.2+ cell enrichment. These observations, plus finding that T cells from inflamed synovium respond to CII(181-201) in vitro, suggest the local recruitment and clonal expansion of some T cells families, possibly driven by autologous CII released during cartilage degradation.

This study shows the induction of HLA-DR (DR) in fibroblasts by IFN-gamma and investigates the molecular mechanisms involved in the further DR down-regulation by TGF-beta 1. Kinetics of DR induction on human dermal fibroblasts by IFN-gamma showed that 1 hr of exposure was required to induce detectable levels of DR, and maximal DR expression was achieved only after 2 days of exposure to IFN-gamma. TGF-beta 1 inhibited DR induction by IFN-gamma, although complete inhibition never could be achieved, even with high concentrations of TGF-beta 1 and low concentrations of IFN-gamma. Inhibition was not accounted for by reduction in cell numbers, as TGF-beta 1 stimulated growth of the fibroblasts. Inhibition of DR induction was seen only if TGF-beta 1 was added during the first 24 hr of IFN-gamma treatment. TGF-beta 1 inhibited equally well if the cells were pretreated for as little as 1 hr and then washed before addition of IFN-gamma. TGF-beta 1 did not cause an overall suppression of protein synthesis. Northern blot analysis revealed that TGF-beta 1 greatly reduced the steady-state level of DR beta mRNA induced by IFN-gamma at 24 hr, and then DRP transcripts became undetectable at later stages. It is concluded that early intracellular signals must build up to stimulate maximum DR synthesis, which, later on, are inactivated or degraded by the action of TGF-beta 1. We suggest that these mechanisms regulating DR gene transcription involve the action of genes coding for specific IFN-gamma-inducible transcriptional factors that are turned on and off in an expeditious manner.

Purified Escherichia coli type 1 fimbriae have been shown previously to stimulate T-cell-independent proliferation of human B lymphocytes. The response is mediated by the mannose-specific, lectin-like adhesin protein FimH. Here we show that type 1 fimbriae also stimulate immunoglobulin (Ig) secretion by B cells. The response was maximal at three days of culture and consisted predominantly of the IgM isotype. It was independent of serum components, T lymphocytes, monocytes, and natural killer cells. Highly purified resting B cells were induced to proliferate and secrete Ig in response to the fimbriae. The role of FimH in the response was shown by the failure of FimH- type 1 fimbriae to stimulate and by inhibition of the response with alpha-methyl mannoside. In light of the fact that carbohydrate-binding adhesins have been found on a wide variety of microorganisms, these studies suggest the possibility that responses of other cell types to other microbial adhesins will be discovered.

Cells of the gram-negative bacterium Escherichia coli are able to attach to various host cells by means of a mannose-specific adhesin associated with type 1 fimbriae. Here we show that fragmentation of type 1 fimbriae by freezing and thawing results in increased mannose-binding activity as demonstrated by increased hemagglutination, increased stimulation of human lymphocyte proliferation, and increased binding of the mannose-containing enzyme horseradish peroxidase. Increased activity in all three assays was mannose sensitive and was not exhibited by FimH- mutant type 1 fimbriae lacking the adhesin. Scatchard analysis of the data from peroxidase binding assays showed that unfrozen and frozen fimbriae contain binding sites displaying two classes of affinity. Frozen and thawed fimbriae expressed an increase in the number of high-affinity binding sites. These results show that fragmentation of the fimbrial structure exposes cryptic mannose-binding activity associated with type 1 fimbriae, presumably that of internally located adhesin molecules. Our data support earlier observations that adhesin moieties of type 1 fimbriae are located both at the tips and at intervals along the length of the fimbriae. In addition, our data suggest that only the adhesin moieties that are located at the fimbrial tips are functional in binding mannose. Adhesins located along the length of the fimbriae have their mannose-binding activity buried within the fimbrial structure and hence are not functional. We propose an updated model for the structure of type 1 fimbriae that is in agreement with the above observations.

In the accompanying report, suppressor T cells were demonstrated that did not recognize cross-reactivity between native and denatured ovalbumin (N-OVA and D-OVA). Here we show that the T cell tolerance induced by i.v. injections of antigen does detect cross-reactivity between N-OVA and D-OVA. Mice that had been immunized with either N-OVA or D-OVA in adjuvant could be rendered profoundly unresponsive if either N-OVA or D-OVA, but not an unrelated protein, were injected i.v. Cross-tolerance was observed in assays of antigen-induced T cell proliferation and helper T cell activity. Tolerance was distinguished from suppressor T cell activity by three criteria: 1) specificity for N-OVA and D-OVA, 2) sensitivity to abrogation by cyclophosphamide, 3) duration of effectiveness. These results confirm observations made by others that suggest that tolerance is mediated by an additional mechanism(s) other than suppressor T cells. Based on a hypothesis that cross-reactivity between native and denatured antigen is related to macrophage processing of antigen, these data also suggest a critical role for processed antigen in the induction of tolerance when antigen is administered i.v.

The T cell hybridoma FS7-20, produced by the fusion of normal B10.BR T cells to the AKR thymoma BW5147, was found when stimulated with concanavalin A (Con A) to produce the lymphokines: interleukin 2 (IL 2), interferon-gamma (IFN gamma), macrophage-activating factor (MAF), Ia induction factor IaIF), and the B cell helper factor interleukin X (IL X). The clones and subclones of FS7-20 varied dramatically in their ability to produce these lymphokines, presumably because of karyotypic variations. The ability to produce IL 2 segregated independently from the ability to produce the four other lymphokine activities; however, production of the latter activities showed a strong correlation. This coordinate production of IFN gamma, MAF, IaIF, and IL X was also observed with a cloned normal cytotoxic T cell line, cr15. These results suggest either that IFN gamma, MAF, IaIF, and IL X are all manifestations of a single molecular species or that, although these activities are different structurally, their production is controlled by a common genetic mechanism. In support of the first possibility, the IFN gamma, MAF, IaIF, and IL X activity produced by FS7-20 were all found to be equally sensitive to inactivation at pH 2. These results illustrate the usefulness of using T cell hybridomas for the study of lymphokines.

Lysosomal proteinases are increased in the tissue lesions of experimental allergic encephalomyelitis and have been implicated in the degradation of myelin proteins. The cellular origins of the increased proteinases are not known but reactive astrocytes found in areas of increased activity are candidate cells. To evaluate the potential of astrocytes as the source of these proteinases, cathepsin B (CB) and cathepsin D (CD) levels were measured in lysates of cultured astrocytes from neonatal rats. Because astrocytes are activated by inflammatory mediators in demyelinating lesions the effect of activation on proteinase levels was examined. Culture supernatants from mononuclear leukocytes stimulated with either concanavalin A or phytohemagglutinin (PHA) induced significant increases in the astrocytic proteinases. Neither PHA alone, interleukin-1, interleukin-2, nor gamma-interferon induced significant increases. Fractions of the supernatant from PHA stimulated mononuclear leukocytes were tested and activity was found in fractions corresponding to a molecular weight of 45-50,000. These studies demonstrate that astrocytes contain significant amounts of CB and CD activity which can be increased by a factor or factors released by activated mononuclear leukocytes.

In this communication, we have demonstrated that hydrolysis of the nucleotide sugar can cause errors in the detection of an ectoglycosyltransferase. Spleen cell suspensions can incorporate radioactivity when incubated with labeled UDP-galactose, but all the activity is due to decomposition of the nucleotide sugar and uptake of the free sugar. The fibroblast cell lines can incroporate carbohydrate directly from UDP-galactose. Several criteria are presented with can be used to demonstrate that a nucleotide sugar is the direct carbohydrate donor.

A self-reactive T cell hybridoma that secretes IL-2 in response to H-2d haplotype cells resulted from a fusion of BALB/cBy lymph node cells with the AKR thymoma BW5147. The lymph node cells used had been enriched for cells reactive to (TG)-A--L, but neither this antigen nor fetal calf serum were required for stimulation of the hybridoma designated 3DT52.5. The gene product responsible for stimulation mapped to the H-2D region. Allogeneic cells of the b, f, k, q, and s haplotypes failed to stimulate. Not all H-2d haplotype cells were effective stimulators of 3DT52.5. Peritoneal cells and splenic B cells were much more stimulatory than splenic T cells. Most tumor cell lines of H-2d derivation and of B cell or macrophage/monocyte lineage were stimulatory, whereas H-2d T cell lines were not. The capacity to stimulate 3DT52.5 did not correlate with the ability to stimulate I region-restricted hybridomas, or with the ability to be induced to stimulate such hybridomas. Stimulatory cell lines did not apparently produce a soluble factor required for stimulation, and negative cell lines were not inhibitory. The monoclonal antibody 27-11-13, which reacts with H-2D of the b, d, and q haplotypes, inhibited stimulation of 3DT52.5 but did not inhibit stimulation of the sibling hybridoma 3DT18.11, which responds to (TG)-A--L plus I-Ad. Conversely, the monoclonal anti-I-Ad antibody MK-D6 inhibited stimulation of 3DT18.11 but not 3DT52.5. Although it is clear that 3DT52.5 recognizes a class I antigen coded for in the H-2D region, the precise molecular nature of the antigen is unknown. The structure of the antigen receptor on this hybridoma may prove to be of interest when it can be compared with receptors found on T cell hybridomas restricted by class II histocompatibility antigens.

Escherichia coli type 1 fimbriae contain in association with the major structural protein a lectin-like adhesin moiety that mediates attachment of E. coli to mannose-containing receptors on the surface of host cells. We have investigated the lymphocyte mitogenic activity of this mannose-specific adhesin by comparing the ability of purified wild type type 1 fimbriae containing the adhesin and mutant type 1 fimbriae lacking the adhesin to stimulate proliferation in human lymphocytes. Both fimbriae stimulated a peak of proliferation at 8 days whereas only the wild type fimbriae stimulated an additional peak of proliferation occurring at 3 days. Proliferation at 3 days but not at 8 days could be blocked by the addition of alpha-methyl-D-mannoside. Neonatal lymphocytes from umbilical cord blood responded to both wild type and mutant fimbriae in a fashion similar to adult cells. Stimulation of separated T and non-T cell populations indicated that the proliferation seen at 3 days was solely due to non-T cells whereas the 8-day response was due to T cell proliferation. The addition of gamma-irradiated T cells did not appear to enhance the 3-day response of the non-T cells. However, the 8-day response by T cells was dependent on the presence of gamma-irradiated non-T cells. In cultures of unseparated cells, wild type fimbriae stimulated more than 75% of the B cells to enter the S and G2 phase at 3 days whereas at 8 days cycling T cells were present in both wild type and mutant fimbriae-stimulated cultures. Taken together, our observations suggest that the adhesin molecule stimulates a polyclonal mitogenic response in B cells that peaks at 3 days, and other structural components of the fimbriae are responsible for evoking an 8-day (probably immune) response in T cells.

Hyaluronic acid (HA) is believed to play a critical role in wound healing and in morphogenesis. Factors controlling the production of HA by fibroblasts in normal and pathological states are not completely understood. In this report we have observed that natural human interleukin (IL-1)1 beta and human recombinant (hrIL)-1 alpha and beta are potent stimulators of HA production by fibroblasts in vitro. Hyaluronic acid is the major species of glycosaminoglycan (GAG) stimulated by IL-1 in fibroblasts. PGE2 does not appear to be involved directly in this IL-1 effect on fibroblasts, but stimulation of HA production by IL-1 is dependent on protein synthesis. The synthetic human IL-1 beta peptide 163-171 (Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys), which has been previously shown to stimulate thymocyte proliferation but not fibroblast PGE2 production, is also able to stimulate fibroblast HA production. The synthesis and secretion of IL-1 by mononuclear phagocytes at sites of inflammation and immune reactions in vivo could potentially serve as a signal for fibroblasts to synthesize HA, which in turn could serve to facilitate and modulate reparative and immune processes by virtue of its ability to alter cell-cell, cell matrix, and cell-membrane receptor interactions.

Plaque-forming cell responses were assessed in unprimed mouse spleen cells after stimulation in vitro with SRBC or "T-independent" type 2 (DNP-Ficoll or DNP-liposomes) or type 1 (TNP-LPS or DNP-liposomes containing lipid A) antigens. Rigorously T cell depleted, Sephadex G-10 passed spleen cells were almost completely unresponsive to all these antigens. In every case, responsiveness could be restored by adding supernates from concanavalin A stimulated spleen cells (Con A SN). Experiments in which supernates containing interleukin 1 (IL-1) from the P388D1 cell line, interleukin 2 (IL-2), and a non-IL-2 T cell replacing factor (IL-X) were mixed showed that optimal responses to all the above antigens could only be obtained if both IL-2 and IL-X were present. IL-2 was the only factor that had any effect alone. IL-2 restored responsiveness to different degrees depending on the antigen. This ranged from about 3% (for SRBC) to about 70% (for TNP-LPS) of the optimal response. The P388D1 supernate enhanced responsiveness only if IL-2 and IL-X were present, and only with the T-independent type 2 antigens. These results demonstrate that at least some examples of both type 1 and 2 "T-independent" antigens actually require nonspecific factors one of which, IL-2, is clearly T cell derived, to stimulate antibody production by B cells.

Using murine splenic B cell preparations depleted of macrophages and rigorously depleted of T cells, we studied the role of nonspecific helper factors in in vitro antibody responses to T cell-independent (TI) type 1 and type 2 antigens. TNP-lipopolysaccharide, TNP-Brucella abortus, and DNP-liposomes containing lipid A were chosen as examples of TI type 1 antigens. DNP-Ficoll and DNP-liposomes without lipid A were chosen as TI type 2 antigens. Only the type 1 antigens were able to elicit significant, albeit very weak, responses without added helper factors. Both type 1 and 2 antigens required factors present in supernatants from concanavalin A-stimulated spleen cells (Con A SN) to stimulate optimum antibody responses. Interleukin 2- (IL 2) containing supernatant from the T cell hybridoma FS6-14.13 supported suboptimal responses to varying degrees with each TI antigen, in contrast to its lack of effect on responses to sheep red blood cells in the absence of additional factors. This activity of the FS6-14.13 supernatant was removed by absorption with the IL 2-dependent T cell line HT-2, suggesting that IL 2 was the active component. Another factor, IL-X, which is distinct from both IL 1 and IL 2 and is also found in Con A SN, was required in addition to IL 2 to achieve optimal responses with both types of TI antigens. These results clearly establish a role for factors derived from T cells in the activation of B cells by both type 1 and type 2 TI antigens.