Search PubMed⌕ Search

Biomedical subjects

R J Winchester

Publications and source records attributed to R J Winchester.

At least 19 recordsLinked to original sources
PubMed1994

Human immunodeficiency virus type 1 strains in the lungs of infected individuals evolve independently from those in peripheral blood and are highly conserved in the C-terminal region of the envelope V3 loop.

To determine whether human immunodeficiency virus type 1 (HIV-1) strains in the lungs of infected individuals are derived from proviral forms contemporaneously present in the peripheral blood or whether they evolve independently as an autonomous pool of viral quasispecies, HIV-1 envelope V3 domain structures at these sites were analyzed and compared. The V3 loop proviral nucleotide and inferred amino acid sequences from lung bronchoalveolar lavage, where HIV-1 is primarily found in macrophages, were more homogeneous within individuals than those from unseparated peripheral blood mononuclear cells, where virus is predominantly in T cells. Comparison between individuals revealed that strains from bronchoalveolar lavage, but not from peripheral blood mononuclear cells, contained V3 domain nucleotide sequences with a great degree of homogeneity in the C-terminal region and a highly conserved, negatively charged amino acid motif. This V3 loop C-terminal structure could be important in the ability of HIV-1 to infect alveolar macrophages. Phylogenetic analyses of V3 domain nucleotide sequences in cells of monocyte/macrophage lineage at both sites revealed the strains in lung macrophages to have evolved further from a presumed ancestral species than those in blood monocytes and to differ considerably in the inferred V3 loop amino acid structures. These results show that, as disease progression occurs, viral strains in monocyte/macrophage lineage cells within the lung and blood microenvironments are not in a state of unrestricted bidirectional traffic but, instead, evolve independently.

Amino Acid Sequence↗
PubMed1994

Dynamic state of beta 2 integrin phosphorylation: regulation of neutrophil aggregation involves a phosphatase-dependent pathway.

The role of phosphorylation and dephosphorylation events in homotypic neutrophil aggregation mediated by CD11b/CD18 molecules was investigated using okadaic acid, an inhibitor of serine and threonine phosphatases. In the absence of exogenous stimuli the addition of okadaic acid to neutrophils resulted in a dose-dependent increase in phosphorylation of the CD18 beta chain that was further augmented by PMA but unaffected by FMLP. Phosphorylation induced by okadaic acid was reversed by staurosporine and minimally decreased by the less selective PKA/PKC inhibitors, H-7 and H-8. This suggests the existence of constitutive phosphatase and kinase activity emphasizing the dynamic state of phosphorylation and dephosphorylation of the beta 2 integrins. Unlike PMA, okadaic acid did not promote homotypic neutrophil aggregation. Furthermore, both the PMA-induced pathway of irreversible aggregation, blocked by staurosporine, as well as the FMLP-induced pathway of reversible aggregation, augmented by staurosporine, were inhibited by okadaic acid in a dose- and time-dependent manner. These results provide evidence that a phosphatase-dependent step is involved in each of these two distinct pathways that regulate neutrophil aggregation mediated by beta 2 integrin activation.

Adult↗
PubMed1993

Identification of mothers at risk for congenital heart block and other neonatal lupus syndromes in their children. Comparison of enzyme-linked immunosorbent assay and immunoblot for measurement of anti-SS-A/Ro and anti-SS-B/La antibodies.

OBJECTIVE: To identify the fine specificity patterns of maternal anti-SS-A/Ro and anti-SS-B/La antibodies that are associated with the birth of a child with transient or permanent manifestations of neonatal lupus syndromes, and to suggest a predictor algorithm for use in counseling. METHODS: Sera were obtained from 4 groups of mothers: 57 whose children had congenital heart block, 12 whose children had transient dermatologic or hepatic manifestations of neonatal lupus but no detectable cardiac involvement, 152 with systemic lupus erythematosus and related autoimmune diseases, who gave birth to healthy infants, and 30 with autoimmune diseases whose pregnancy resulted in miscarriage, fetal death, or early postpartum death unrelated to neonatal lupus. Antibodies to SS-A/Ro and SS-B/La were assessed by enzyme-linked immunosorbent assay (ELISA) and by sodium dodecyl sulfate (SDS)-immunoblot. RESULTS: Anti-SS-A/Ro antibodies were identified by ELISA in 100%, 91%, 47%, and 43% of the mothers of infants with heart block, with transient neonatal lupus, healthy infants, and fetal death, respectively. High titers of anti-SS-A/Ro antibodies were present more often in mothers of children with cardiac disease or transient neonatal lupus than in either of the other 2 groups. Maternal antibodies to SS-B/La were detected by ELISA in 76% of the heart block group, 73% of the cutaneous neonatal lupus group, 15% of the group with healthy children, and 7% of the fetal death group. On SDS-immunoblot, sera from 91% of the heart block group mothers who had antibodies to SS-A/Ro but not to SS-B/La recognized at least 1 SS-A/Ro antigen, with significantly greater reactivity against the 52-kd component. In contrast, only 62% of the anti-SS-A/Ro positive, anti-SS-B/La negative responders in the healthy group recognized the 52-kd and/or the 60-kd component. Although there was no profile of anti-SS-A/Ro response unique to the mothers of children with heart block or cutaneous manifestations of neonatal lupus, only 1% of the healthy infants were born to mothers with antibodies directed to both the 52-kd SS-A/Ro and 48-kd SS-B/La antigens and not to the 60-kd SS-A/Ro antigen. CONCLUSION: Women with antibodies to both SS-A/Ro and SS-B/La have an increased risk of giving birth to children with neonatal lupus, especially if the anti-SS-A/Ro response identifies the 52-kd component on SDS-immunoblot. Women whose sera contain only anti-SS-A/Ro antibodies in low titer and only recognize determinants that are altered by conditions of SDS-immunoblot have a low risk for giving birth to a child with neonatal lupus. Specific antibody profiles do not distinguish among the manifestations of the neonatal lupus syndromes.

Animals↗
PubMed1991

Engagement of CD14 on human monocytes terminates T cell proliferation by delivering a negative signal to T cells.

We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti-CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative signal to T cells, rather than from disruption of a costimulatory monocyte-derived signal, because incubation of monocytes with anti-CD14 mAb also inhibited monocyte-independent T cell proliferation induced by PMA and ionophore. These results, together, point to a role of CD14 in the monocyte-dependent regulation of T cell proliferation.

Adult↗
PubMed1991

Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18).

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.

Alkaloids↗
PubMed1990

Further DNA sequence microheterogeneity of the HLA-DR4/Dw13 haplotype group: importance of amino acid position 86 of the DR beta 1 chain for T-cell recognition.

Using the polymerase chain reaction we have isolated and sequenced cDNA clones corresponding to the polymorphic first domain of the DR beta 1 chain from the DR4, "Dw13" cell line, JHa. We have found that the JHa DR beta 1 allele differs from previously reported Dw13 alleles by a single amino acid substitution at position 86. The functional relevance of this polymorphism is supported by the reactivity pattern of a T-cell clone, E38. E38 is an alloreactive T-cell clone which reacts with all Dw14 stimulator cells and all Dw13-positive cells tested except the "Dw13"-positive homozygous typing cell line JHa. Inhibition studies with monoclonal antibodies revealed the stimulating determinant to be on DR and not on DQ or DP molecules. These data indicate that position 86 of the DR beta 1 chain can play an important role in the formation of determinants recognized by T cells.

Amino Acid Sequence↗
PubMed1989

Class II major histocompatibility complex gene sequences in rheumatoid arthritis. The third diversity regions of both DR beta 1 genes in two DR1, DRw10-positive individuals specify the same inferred amino acid sequence as the DR beta 1 and DR beta 2 genes of a DR4 (Dw14) haplotype.

The DR1 and DRw10 beta 1 chain genes were isolated from each of 2 individuals with rheumatoid arthritis who were heterozygous for these class II major histocompatibility complex specificities. The sequences of the DR1 beta 1 chains from both patients were identical, differing from previously reported DR beta 1 chains of individuals without RA by 2 amino acid substitutions, at positions 85 (Val-Ala) and 86 (Gly-Val), and by a silent mutation at the last nucleotide of codon 78 (C-T), resulting in the loss of a Pst I restriction endonuclease site. Identical DRw10 beta 1 chain genes were found in both patients. These were shown to encode the epitope recognized by monoclonal antibody 109d6. This antibody also recognizes an epitope on the DRw53 beta 2 chain of the DR4 haplotype. The third diversity regions of the DR1 beta (amino acids 67-74) and the DRw10 beta 1 chains (amino acids 67-73) were identical, respectively, with those of the DR4 (Dw14) beta 1 and beta 2 chains, raising the possibility that in these patients, the third diversity regions of the two DR beta 1 chain genes present in trans are conformationally equivalent to the cis-encoded third diversity regions of the DR4 (Dw14), DR beta 1, and beta 2 chains. The nucleotide sequences of the DQ beta complementary DNA clones were identical to that of the DQw1 beta chain, and no DR beta 2 complementary DNA clones were identified.

Amino Acid Sequence↗
PubMed1989

Potential mechanisms for coordinate gene activation in the rheumatoid synoviocyte: implications and hypotheses.

Evidence is reviewed to support the concept that synovial cells in rheumatoid arthritis have undergone distinctive alterations at the cellular and subcellular level that result in their taking on some of the characteristics that are also manifest by transformed cells. These phenotypic modulations could be indirectly driven by cytokines in a paracrine or autocrine fashion. Specific regional patterns of cell phenotype modulation were used to argue against a simple widely diffusing direct inductive effect to cytokines and in favor of microenvironmental determinants. It is hypothesized that these extracellular factors induce novel activation in a coordinate manner by acting through master regulatory genes operating in cells with specific microenvironmental interactions. Two of these regulatory genes, fos and jun, are discussed in detail because of their induction by growth factors and their central role in the transactivation of genes which have been implicated in rheumatoid synovitis. A model for gene activation in the rheumatoid synovium is proposed based on the premise that fos and jun are an important link in the intracellular transduction pathways used by cytokines to induce cellular phenotypic changes.

Animals↗
PubMed1988

Genetic susceptibility to rheumatoid arthritis and human leukocyte antigen class II polymorphism. The role of shared conformational determinants.

Genetic susceptibility for rheumatoid arthritis has been associated with both human leukocyte antigen (HLA)-DR4 and HLA-DR1, depending on the ethnic origin of the population under study. Furthermore, various subtypes of DR4 exist, only some of which appear to be associated with rheumatoid arthritis. DNA sequence analysis of the DR-beta chain genes encoding the DR4 subtypes as well as DR1 has led to the observation that similar third hypervariable region sequences are found on rheumatoid arthritis-associated DR-beta chain alleles. The data indicate that third hypervariable region sequence polymorphisms strongly influence T cell recognition as well as risk for rheumatoid arthritis. This has led to the hypothesis that genetic susceptibility for rheumatoid arthritis is due to a group of similar third hypervariable region sequences that may share conformational determinants important in antigen presentation and/or immune regulation.

Alleles↗
PubMed1988

Anti-IgM-mediated B cell signaling. Molecular analysis of ligand binding requisites for human B cell clonal expansion and tolerance.

The ligand binding requisites for membrane IgM-mediated signaling of human B lymphocyte clonal expansion and B cell tolerance were investigated with a well-characterized set of soluble murine anti-human IgM mAbs. Evaluation of the impact of mu chain domain specificity, affinity, and binding stoichiometry for membrane IgM on antibody-induced regulation of normal and leukemic B cell DNA synthesis revealed that the ligand binding requisites for inducing or, alternatively, suppressing B cell DNA synthesis are significantly different. First, while the induction of S phase entry required micrograms/ml concentrations of ligand, orders of magnitude lower concentrations of ligand sufficed for inhibitory signaling. Second, while an upper affinity threshold for achieving maximal stimulation of B cell DNA synthesis was never detected, inhibitory signaling by bivalent ligands appeared to become relatively affinity independent at Fab binding affinities greater than 7.0 x 10(6) M-1. Third, while a C mu 1-specific mAb with an enhanced incidence of monogamous binding to mIgM was ineffective at inducing B cell DNA synthesis, the antibody was not significantly compromised in ability to initiate inhibitory signals. These differences could be observed in a clonal B cell population which positively or negatively responded to mIgM ligation depending upon its state of activation. The accumulated observations indicate that the ligand binding requisites for inhibitory signal transduction in human B lymphocytes are much less rigorous than those for stimulatory signal transduction and suggest that many physiologically relevant anti-Ig antibodies are more likely to function in the negative feedback regulation of B cell responses than in the direct triggering of human B cell clonal expansion.

Animals↗
PubMed1988

Dissociation between increased surface expression of gp165/95 and homotypic neutrophil aggregation.

Whether homotypic neutrophil aggregation depends on the quantitative increase of gp165/95 molecules (Mac 1, CR3) recruited to the cell surface during activation was studied using mAb of the CD11b group that recognize distinct epitopes encoded by the alpha-subunit of this glycoprotein. After the addition of antibody MN41, neutrophils did not aggregate in response to a chemoattractant, FMLP. Blockade of preexisting surface gp165/95 by mAb MN41, followed by removal of the excess antibody from the mixture, was used to show that the molecules of gp165/95 newly expressed in response to stimulation by a chemoattractant were incapable of effectively mediating the induced cell-cell interactions of aggregation. Flow cytometry studies confirmed that binding of unlabeled antibody MN41 did not block further increases in surface expression of gp165/95 after stimulation with FMLP. These data suggest that molecules of gp165/95 exhibit two functionally distinct forms, one, present on the surface of freshly isolated neutrophils, that becomes competent to mediate the aggregation response upon activation by a stimulus and a second form that can be translocated to the cell surface by the stimulus but is greatly diminished if not lacking in the ability to participate in that aggregation event.

Antibodies, Monoclonal↗
PubMed1988

The molecular basis of susceptibility to rheumatoid arthritis: the conformational equivalence hypothesis.

This is an interpretive review of recent immunologic and molecular biologic data concerning the molecular basis of susceptibility to rheumatoid arthritis. The central point of view was taken that the major histocompatibility complex (MHC) class II molecules encoding disease susceptibility function in an immune recognition event involving an antigen "X" that currently eludes characterization. The problem of understanding the meaning of the association of susceptibility with diverse MHC alleles such as DR4 (Dw4 and Dw14), DR1, and DRw10 is approached by detailed biochemical analysis that led to the identification of common stretches of amino acid sequence, presumably encoding conformationally equivalent structures. Non-classic MHC polymorphisms related to disease susceptibility but not associated with particular alleles such as identified by Ab 109d6 proved especially valuable in suggesting new directions for attempting to understand the significance of these associations. Consideration is given to the possibility that a family of either slightly different or identical conformations encoded in either cis or trans cumulatively confer the liability to develop rheumatoid arthritis, and implying a highly non-classic mode of inheritance. The available data do not permit a distinction between the possibilities that an antigen "X" was being presented to T cells or whether the distinctive conformations of the MHC class II molecule serve the same role as antigen "X" but are directly recognized by T cells. However, with additional data, some limited insight should be able to be inferred about the nature of an antigen "X" that specifically binds to the MHC conformation with a complementary interaction. It seems reasonable to consider the pathogenesis of rheumatoid arthritis as a typical immune response resulting from a simple immune recognition event of a single antigenic molecule.

Arthritis, Rheumatoid↗
PubMed1988

Expression of Ia and monocyte-macrophage lineage antigens in giant cell tumor of bone and related lesions.

An immunohistochemical study of six giant cell tumors of bone and eight related lesions (aneurysmal bone cyst, fibrous histiocytoma, and giant cell tumor of tendon sheath) was performed using a panel of monoclonal antibodies directed to the Ia and monocyte-macrophage lineage antigens. In all types of lesion, osteoclastlike multinucleate giant cells were negative for both types of antigen, but a proportion of mononuclear cells gave positive reactions. While the possibility that these cells are reactive cannot be excluded, in giant cell tumor and malignant fibrous histiocytoma, their frequency and their morphologic similarity to the rest of the tissue suggest that they may be an intrinsic part of the neoplasm. This finding is consistent with the presumed fibrohistiocytic nature of these tumors.

Acid Phosphatase↗
PubMed1987

High frequency of clonal immunoglobulin or T cell receptor gene rearrangements in acute myelogenous leukemia expressing terminal deoxyribonucleotidyltransferase.

Ig and T cell receptor rearrangements have been used as irreversible markers of lineage and clonality in the study of B- and T-lymphoid populations. We have addressed the issue of lymphoid lineage specificity of these rearrangements by analyzing a panel of 25 TdT- acute myelogenous leukemias, 13 TdT+ AMLs, and 4 TdT+ undifferentiated leukemias. We report that while gene rearrangements represent extremely rare events in classical TdT- AML (less than 8%), rearrangements of either the Ig or T beta locus or both were detectable in the majority of the TdT+ AMLs (greater than 60%), and rearrangements of both loci were detectable in all of the TdT+ undifferentiated leukemias. These data demonstrate a significant association between TdT expression and Ig or T beta gene rearrangements even outside the lymphoid lineage, further supporting a role for TdT in Ig and T cell receptor gene assembly. These data also indicate that a coordinated program of lymphoid gene expression involving TdT-CD7-expression and Ig/T beta rearrangements can be activated before myeloid commitment. Whether the activation of this program represents a normal, albeit rare, event in early myelopoiesis or a transformation-related event present only in leukemic cells remains to be determined.

Cell Differentiation↗
PubMed1987

Intrauterine therapy for presumptive fetal myocarditis with acquired heart block due to systemic lupus erythematosus. Experience in a mother with a predominance of SS-B (La) antibodies.

An experimental therapeutic regimen for congenital lupus erythematosus, which consists of treating the mother with high doses of dexamethasone and performing plasmapheresis (3 times per week), was developed. This regimen was administered to a woman whose pregnancy was complicated by the abrupt onset of fetal pericarditis, myocarditis, and complete heart block in the twenty-fourth week of gestation. The effectiveness of plasmapheresis was evidenced by a decrease in the very high titer of SS-B (La) antibodies in the mother. After 5 weeks of therapy, the cardiac disease was ameliorated. The infant tolerated delivery at week 31, and the heart block has persisted. The rationale for this therapy for fetal disease is discussed, with emphasis on 2 goals: to diminish the quantity of maternal antibody that is transmitted to the fetus, and to suppress the manifestations of fetal carditis with the use of glucocorticoids that are not inactivated by the placenta. Tentative recommendations for more intensive prenatal monitoring of maternal and fetal status for those at risk are outlined.

Adult↗
PubMed1987

Occurrence of specific phytohaemagglutinin-reactive immunoglobulins in the sera of certain individuals. Relevance to the detection of cell surface antigens on phytohaemagglutinin-stimulated cells.

While investigating sera for possible antibodies to TL-like determinants, multiparous sera were selected that had specific cytotoxic reactivity against phytohaemagglutinin (PHA)-activated lymphocytes of certain individuals and were negative against unstimulated T or B cells from the same donor. The reactivity patterns were not correlated to any HLA specificities. However, in an indirect immunofluorescence assay with flow cytometry, these same sera had strong specific monomorphic reactivity to PHA-activated lymphocytes from all the individuals in the panel. Unstimulated cells remained negative. In contrast, other human sera lacked reactivity with PHA-stimulated lymphocytes. The addition of PHA to fresh lymphocytes followed by incubation at 4 degrees C for 30 min resulted in the same pattern of reactive and non-reactive sera. When incubated with PHA, different cell types, including 0- erythrocytes and a murine lymphoid cell line, reacted similarly with the sera. Using plates coated directly with PHA-E, L, and P in a cell-free ELISA system. PHA-specific sera reacted specifically with PHA-coated wells. The anti-PHA activity was removed by absorption with 0- erythrocytes coated with PHA without affecting the titre of the anti-HLA antibody in the same serum. These findings suggested that IgG molecules in certain sera react directly with residual PHA bound to the cell surface and not necessarily with molecules of cellular origin induced by exposure to PHA, complicating the search for antibodies specific for T-cell activation antigens.

Antibody Specificity↗