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Biomedical subjects

H M Dosch

Publications and source records attributed to H M Dosch.

At least 55 records · Page 3Linked to original sources
PubMed1993

The growth transformation of human B cells involves superinduction of hsp70 and hsp90.

Epstein-Barr virus (EBV) is a latent human herpes virus associated with a range of malignant and non-malignant disorders. EBV binds to CD21 virus receptors on B lymphocytes and growth transforms these cells; in susceptible (e.g., immunodeficient) hosts such cells rapidly expand into fatal lymphomas. Virus binding and infection trigger a cascade of cellular events which are transformation prerequisite and analogous to non-oncogenic cell activation events but which differ in several quantitative or qualitative respects. Unique trans-membrane Ca2+ currents, Na+/H+ exchange, as well as tyrosine phosphorylation and p56lck-gene induction suggest that even early on the transformation process has oncogenic specificity. In this report we describe that two additional cellular gene families, the stress proteins hsp70 and hsp90, are coordinately induced at mRNA and protein levels and, quite different from hsp induction by thermal stress, this induction is dependent on EBV-induced trans-membrane Ca2+ currents. Blockade of hsp induction prevents transformation. The kinetics and induction prerequisites set this response well apart from reported responses to thermal or viral stress protein induction. Like p56lck-, hsp induction is purely a post-receptor binding event and not dependent on expression of any viral gene. The induction kinetics, with a peak at approximately 12-16 hr and subsequent decline to control levels, considerably extend the chronological map of elements in the CD21-dependent branch of the transformation pathway and suggest a specific role of induced hsp different from the cell cycle-related functions observed in other cell systems.

Actins↗
PubMed1993

Unexpected patterns of Epstein-Barr virus gene expression during early stages of B cell transformation.

Following Epstein-Barr virus (EBV) binding to its CD21 cell surface receptor, virus internalization, nuclear translocation, and circularization of the viral episome were found to occur within 30 min, immediately preceding the expression of EB nuclear antigen (EBNA)-1 and -2 and latent membrane protein (LMP)-1 and -2 genes. Early viral gene expression was unaffected by blockade of the virus induced, transformation-prerequisite cellular activation pathway (Ca2+ currents, tyrosine phosphorylation, induction of p56lck, hsp70, and hsp90). Despite life times of only 3 h, antisense (but not sense) oligonucleotides for the above latency genes prevented subsequent transformation. Any one antisense oligonucleotide dramatically depleted transcripts not only of the target gene, but of all other latency genes. The blocking effect of antisense oligonucleotides allowed us to identify a new transformation-prerequisite latency gene near the fused termini. The concerted regulation of EBV gene expression is highly unusual and unexplained but our results imply critical, perhaps regulatory roles for initial latency gene transcripts themselves.

Antigens, Viral↗
PubMed1993

Detection of viral sequences by internally calibrated gene amplification.

Inherent pitfalls of the polymerase chain reaction (PCR) can become serious difficulties when transferring research applications to high-volume routine procedures such as biofermentation process control and clinical diagnostics. Difficulties include 1) the danger of accidental sample contamination with positive control templates; 2) variable amplification due to positional effects in thermocycler blocks and unequal primer efficiency for sense/anti-sense strands; and 3) the need for reliable controls, which provide confidence for reporting negative reactions. Using the PCR detection system for Epstein-Barr virus as a model, we have developed a quick process to generate mutant internal co-amplification templates. These can be used for titration of amplification sensitivity. More importantly, single tube co-amplification without titrations allows determination of the minimum sensitivity achieved in each individual reaction; critical information when reporting negative diagnostic results. Mutant and native fragments are easy to distinguish by size, and sample cross contamination can be readily identified. The system should be easily adaptable to gene amplification procedures, which aim to routinely detect the presence of a given gene fragment in a controlled fashion.

Antigens, Viral↗
PubMed1992

A bovine albumin peptide as a possible trigger of insulin-dependent diabetes mellitus.

BACKGROUND: Cow's milk has been implicated as a possible trigger of the autoimmune response that destroys pancreatic beta cells in genetically susceptible hosts, thus causing diabetes mellitus. Studies in animals have suggested that bovine serum albumin (BSA) is the milk protein responsible, and an albumin peptide containing 17 amino acids (ABBOS) may be the reactive epitope. Antibodies to this peptide react with p69, a beta-cell surface protein that may represent the target antigen for milk-induced beta-cell--specific immunity. METHODS: We used immunoassays and Western blot analysis to analyze anti-BSA antibodies in the serum of 142 children with insulin-dependent diabetes mellitus, 79 healthy children, and 300 adult blood donors. Anti-ABBOS antibodies were measured in 44 diabetic patients at the time of diagnosis, three to four months later, and one to two years later. RESULTS: All the diabetic patients had elevated serum concentrations of IgG anti-BSA antibodies (but not of antibodies to other milk proteins), the bulk of which were specific for ABBOS: The mean (+/- SE) concentration was 8.5 +/- 0.2 kilofluorescence units (kfU) per microliter, as compared with 1.3 +/- 0.1 kfU per microliter in the healthy children. IgA antibodies were elevated as well, but not IgM antibodies. The antibody concentrations declined after diagnosis, reaching normal levels in most patients within one to two years. The initial decline involved anti-ABBOS--specific antibodies almost exclusively. Much lower serum concentrations of anti-BSA antibodies were found in all 379 control subjects, but only 2.5 percent of them had small amounts of ABBOS-specific IgG. CONCLUSIONS: Patients with insulin-dependent diabetes mellitus have immunity to cow's-milk albumin, with antibodies to an albumin peptide that are capable of reacting with a beta-cell--specific surface protein. Such antibodies could participate in the development of islet dysfunction.

Antibodies↗
PubMed1992

Diagnosis of nasopharyngeal carcinoma by DNA amplification of tissue obtained by fine-needle aspiration.

BACKGROUND: In nasopharyngeal carcinoma the primary lesion is often difficult to find. Metastatic lesions occur frequently but are difficult to distinguish from other head and neck tumors. The viral genome of the Epstein-Barr virus (EBV) can be identified in the cells of this carcinoma. METHODS: We used the polymerase chain reaction (PCR) to test for the presence of EBV genomes in 15 samples of metastatic squamous-cell carcinoma of the neck obtained by fine-needle aspiration and in 26 samples obtained by biopsy of lymph nodes. For controls we used disease-free lymph nodes from 10 patients with various head and neck tumors, tonsillar tissue from 46 subjects, blood from 59 EBV-seropositive blood donors, and mononuclear cells from 8 patients with fatal lymphoproliferative lesions. RESULTS: Of the 41 malignant lesions examined, only the nine nasopharyngeal carcinomas (one primary lesion and eight metastases) contained EBV genomes. None of the 20 nodes with other types of cancer, the 10 disease-free nodes, or any of the 105 normal control samples contained detectable EBV. In two patients with suspected metastases from occult primary tumors, the presence of EBV was predictive of nasopharyngeal carcinoma; in both cases overt nasopharyngeal carcinoma developed within one year. CONCLUSIONS: In patients with suspected nasopharyngeal carcinoma, fine-needle aspiration can provide tissue for diagnosis by DNA amplification of EBV genomes. The presence of EBV in metastases from an occult primary tumor is predictive of the development of overt nasopharyngeal carcinoma.

Base Sequence↗
PubMed1992

Disease-associated anti-bovine serum albumin antibodies in type 1 (insulin-dependent) diabetes mellitus are detected by particle concentration fluoroimmunoassay, and not by enzyme linked immunoassay.

We recently developed a particle concentration fluoroimmunoassay for the measurement of serum antibodies to bovine serum albumin in patients with Type 1 (insulin-dependent) diabetes mellitus. We observed elevated IgG-anti-bovine serum albumin antibodies in 100% of newly-diagnosed diabetic children and in 2.5% of matched control children. Here we compare the fluoroimmunoassay and the more commonly available enzyme linked immunoassay technique, exchanging coded serum samples from 40 newly-diagnosed diabetic children and 179 control children between two laboratories. Particle concentration fluoroimmunoassay detected elevated IgG-anti-bovine serum albumin antibodies in all diabetic children, enzyme immunoassay in 25% (p less than 0.0001). Fluoroimmunoassay detected elevated levels in 2.2% and enzyme immunoassay in 10% of control children (p less than 0.002). Elevated IgA-anti-bovine serum albumin antibodies in patients were slightly more often detected by fluoroimmunoassay than by enzyme immunoassay, while in control children enzyme immunoassays detected elevated levels three times more often (p less than 0.01). Values measured in either assay showed overall no correlation in either patient (IgG:rs = 0.28; IgA:rs = 0.11) or control sera (IgG:rs = 0.02; IgA:rs = -0.05). Fluoroimmunoassay for IgG was 100% disease-sensitive (enzyme immunoassay: 25%, p less than 0.0001) and more disease-specific (IgG; p less than 0.02). Our findings demonstrate that these assay techniques detected distinct subsets of anti-bovine serum albumin antibodies with little (IgG) or some (IgA) overlap. In fluoroimmunoassay procedures, antigen:antibody binding occurs within 1-2 min while hours are allowed in an enzyme immunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

A deletion map of the human immunoglobulin heavy chain variable region.

Analysis of VH gene segments deleted in the process of immunoglobulin heavy chain (IGH) variable region assembly in three series of monoclonal B cell lines has been used to determine the human VH region organization. A deletion map of the relative positions of 21 different VH gene segments has been determined. The characterization of B cell lines from three unrelated adults of two racial groups yielded the same relative VH gene segment order, suggesting that the overall order of VH genes in the normal population is constant. This VH gene segment order was consistent with what we had previously generated from physical mapping techniques. DH segments from the second DH cluster, distinct from the major DH locus 3' of the VH region, were not observed to be used in 32 different rearrangements. Approximately 77% of the VH-(D)JH rearrangements involved VH gene segments within 500 kb of the JH region, indicating that human B cell lines preferentially rearrange JH-proximal VH gene segments. The switch, observed in mice, from the fetal use of JH-proximal VH gene segments to an adult VH use dependent upon VH family size may therefore not occur in humans. This detailed map of the VH gene segments is a necessary prerequisite for understanding VH usage in development and disease.

Adult↗
PubMed1991

The tyrosine kinase lck is critically involved in the growth transformation of human B lymphocytes.

Epstein-Barr virus (EBV) exposure of human B lymphocytes induces rapid, Ca(2+)-dependent tyrosine phosphorylation of two cytosolic proteins, one likely the CD21 EBV receptor and another unknown species of 55-60 kDa. We now identify the latter protein as the tyrosine kinase lck (p56lck). In T cells many activation events reduce the high constitutive p56lck expression levels typical for that lineage, and they induce the appearance of a 60-kDa lck species. We now demonstrate that in B cells exposed to EBV the at best low constitutive p56lck expression levels are rapidly and transiently up-regulated without generation of 60-kDa lck. lck-specific antisense oligonucleotides block p56lck induction and prevent subsequent B cell activation and immortalization whereas B cell activation by nononcogenic agents was unaffected. We propose that p56lck superinduction is a transformation prerequisite which signals entry into the oncogenic growth transformation process.

B-Lymphocytes↗
PubMed1991

Cellular toxicity of sulfamethoxazole reactive metabolites--I. Inhibition of intracellular esterase activity prior to cell death.

Reactive metabolites produced by oxidative metabolism of the parent compound are considered responsible for the toxicity of a number of drugs, including idiosyncratic reactions to sulfonamide antibiotics. Using sulfamethoxazole hydroxylamine (SMX-HA) as a model compound, we report the use of a pH-sensitive fluorescent probe, 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), to identify early subcellular targets of chemically synthesized, toxic drug metabolites in peripheral blood mononuclear cells. When toxicity was assessed with this probe immediately after a 2-hr drug challenge, SMX-HA produced a concentration-dependent decrease in cellular fluorescence which was not accompanied by the development of compromised cell membrane integrity until 18 hr later. Dissipation of pH gradients across the cell membrane with nigericin and monensin demonstrated that decreased intracellular pH was only a small component of SMX-HA-induced toxicity. Loading cells with BCECF 30 min prior to SMX-HA challenge produced only a 3% decrease in cellular fluorescence at an SMX-HA concentration of 1 mM, whereas addition of BCECF after drug challenge resulted in a 71% decrease in fluorescence, consistent with a direct drug effect on cellular esterase activity. This was confirmed by monitoring BCECF cleavage in cell lysates in the presence and absence of SMX-HA. These studies demonstrate that inhibition of cellular esterase activity accounted for the observed loss of cellular fluorescence after drug exposure. Since changes in cellular fluorescence at 2 hr correlated well with cell death at 18 hr, we conclude that SMX-HA inhibition of intracellular esterase activity is an early event in the process that terminates in metabolite-induced cell death.

Cell Membrane↗
PubMed1991

Cellular toxicity of sulfamethoxazole reactive metabolites--II. Inhibition of natural killer activity in human peripheral blood mononuclear cells.

Based on the identification of intracellular esterase activity as one early target of sulfamethoxazole hydroxylamine (SMX-HA), we wished to determine if the metabolite affected immune functions which involve esterases. The natural killer (NK) activity of human peripheral blood mononuclear cells (PBMC) was assessed with a cell concentration fluorescence technique following exposure to SMX-HA. When K562 target cells were incubated (4 hr/37 degrees) with various ratios of untreated PBMC effector to K562 target cells (E:T), NK activity increased from 17.8 +/- 3.1% (mean +/- SEM; N = 12) at an E:T ratio of 5:1 to 46.2 +/- 2.0% at an E:T ratio of 40:1. Pretreatment of fresh PBMC with 0.1 to 1.0 mM SMX-HA produced a concentration-dependent inhibition of NK activity (E:T ratio 40:1) reaching approximately 80% at 1 mM SMX-HA. Maximum suppression of NK activity was completed within a 60-min pretreatment period with measurable inhibition detected within 30 min. The viability of effector cells was not affected by the metabolite during the pretreatment period. Therefore, the SMX-HA effects could not be directly attributed to decreased viability of the effector cells; they were irreversible and could be prevented by the inclusion of exogenous reduced glutathione (GSH) in a concentration-dependent manner. Given the important roles of NK cells in immune responsiveness and host resistance, our findings of rapid functional inactivation of the cytolytic effector function provide a possible link between idiosyncratic drug toxicity and drug effects directly on components of the immune system.

Cell Survival↗
PubMed1991

Exogenous but not endogenous EBV induces lymphomas in beige/nude/xid mice carrying human lymphoid xenografts.

Epstein--Barr virus (EBV) is a latent human herpes virus that growth-transforms EBV receptor/CD21+ B cells and is associated with several high-frequency malignancies. Reactivation of latent EBV occurs in approximately 1/3 of organ graft recipients and a majority of AIDS patients; EBV-positive B lymphoproliferative lesions represent often fatal complications in organ transplantation and late-stage AIDS. Although such lymphomas can arise from endogenous virus, the high tumor risk in EBV-seronegative transplant recipients implies de novo infection, in particular virus transmission with intra-graft B lymphocytes. Since SCID mice engrafted with human lymphocytes (SCIDhum) typically develop endogenous EBV+ (human) tumors in their graft it is difficult to study exogenous virus transmission in this model. We here demonstrate that beige/nude/xid mice engrafted with human lymphoid cells (BNXhum) selectively accept human B but not T cell grafts. Unexpectedly these mice fail to develop endogenous lymphomas observed in SCIDhum mice engrafted in parallel. However, injection of as few as less than 500 EBV particles produces rapidly fatal, polyclonal lymphomas in BNXhum animals. This virus sensitivity of BNXhum approaches conditions for EBV transmission with organ grafts.

Animals↗
PubMed1991

Milk proteins in the etiology of insulin-dependent diabetes mellitus (IDDM).

The etiology of insulin-dependent diabetes mellitus (IDDM) is multifactorial. The final cause of the disease, the specific destruction of the islet beta-cells, is the result of a cellular/humoral autoimmune process that operates in individuals with a particular genetic background in response to an external triggering factor(s). The most likely environmental triggers are virus infections and dietary factors. Among the latter group dietary proteins, mainly cow milk proteins, have been found to be important. Elimination of intact cow milk proteins from the diet significantly reduced the incidence of IDDM in the spontaneously diabetic BB rat, the elimination being most effective when it occurs during the pre-weaning period. Conversely, in newly discovered diabetics (both rats and children) increased levels of antibodies to cow milk proteins as compared with non-diabetic controls were found. These higher titres of antibodies were against beta-lactoglobulin and anti-bovine serum albumin. In further studies we found that antibodies to bovine serum albumin cross-react with a beta-cell membrane protein of Mr 69,000 and that this protein is likely induced by interferon. At the molecular level, a region of the bovine serum albumin has distinct homology to the beta-subunits of the MHC class II proteins Ia, DQ and DR, and antibodies raised against this bovine serum albumin region identified the same 69K beta cell membrane protein, in the same manner as antibodies to the third hypervariable region of DR-beta did. Our hypothesis is that bovine milk proteins (mainly bovine serum albumin) might be an important environmental factor providing specific peptides that share antigenic epitopes with host cell proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Expression of IgE from a nonrearranged epsilon locus in cloned B-lymphoblastoid cells that also express IgM.

During development, B lymphocytes have the ability to switch from synthesis of IgM to immunoglobulins of another isotype such as IgG, IgA, or IgE. This class switching mechanism has been shown to involve DNA rearrangement and concomitant deletion of intervening CH genes. In our report, an EBV-transformed B lymphoblastoid cloned cell line is described that simultaneously expressed and secreted both IgM and IgE. DNA analysis showed the (nonproductive) rearrangement of one allele to gamma and (productive) rearrangement of the other allele to mu. Germ-line arrangement of the C epsilon gene was preserved on both alleles.

B-Lymphocytes↗
PubMed1990

Interactions between N-acetyl-p-benzoquinone imine and fluorescent calcium probes: implications for mechanistic toxicology.

Intracellular free calcium ([Ca2+]i) homeostasis has been implicated as an early target in both cellular necrosis and apoptosis. In this study, we have used peripheral blood mononuclear cells (PBMC) as target cells to investigate the effects of several reactive metabolites associated with drug toxicity on [Ca2+]i in order to delineate further early events in cytotoxicity. Compounds implicated in both drug-induced necrosis (N-acetyl-p-benzoquinone imine; NAPQI) and drug hypersensitivity (sulfamethoxazole hydroxylamine; SMX-HA) were examined and their effects on [Ca2+]i compared with those of the T cell mitogen phytohemagglutinin (PHA; 1.5 micrograms/ml) and the calcium ionophore ionomycin (2.5 microM). PHA and ionomycin produced characteristic elevations in [Ca2+]i as monitored by an increase in the fluorescence of fluo-3-loaded cells. SMX-HA did not significantly affect [Ca2+]i at concentrations previously shown to be cytotoxic to PBMC (100 and 500 microM), suggesting that Ca2+ homeostasis is not an early target for SMX-HA toxicity. Addition of NAPQI (250 microM) to fluo-3-loaded cells produced a marked decrease in fluorescence which was not reversed by ionomycin. Conversely, addition of NAPQI to cells loaded with indo-1 resulted in a rapid increase in fluorescence. This effect, however, was found to be attributable to NAPQI addition per se rather than to an increase in [Ca2+]i. HPLC and fluorescence analysis of samples generated from the decomposition of NAPQI revealed the presence of several products which fluoresced intensely at the excitation/emission wavelength pairs of a number of fluorescent probes commonly used to monitor [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Aniline Compounds↗
PubMed1990

Expression of glycolipid receptors to Shiga-like toxin on human B lymphocytes: a mechanism for the failure of long-lived antibody response to dysenteric disease.

Fresh and transformed human B lineage cells were found to be sensitive to the cytotoxic action of Shiga-like toxin (SLT), a bacterial cytotoxin. The toxin was specifically bound by the glycolipids globotriosylceramide and galabiosylceramide expressed on the surface of sensitive cells. Mutant Daudi cells selected for resistance to SLT cytotoxicity (SLTR20) were deficient in SLT-binding glycolipids and failed to bind SLT to their surface, suggesting a role for these glycolipids in the mediation of SLT cytotoxicity. Of a number of normal and transformed lymphoid and myeloid cells screened for SLT sensitivity, only B lymphoid cells were susceptible to SLT action. Moreover, B lymphoid cells were the only cells expressing the SLT binding glycolipids. In vitro B cell activation studies with Epstein-Barr virus and pokeweed mitogen both indicated that the vast majority of SLT-sensitive B cells belong to the IgG and IgA committed subset, whereas most IgM and IgM/D producing cells were resistant to SLT toxicity. The selective elimination of IgG and IgA committed cells may explain the production of only IgM class anti-SLT antibodies in Shigella-infected humans leading to the failure of long-term immunity to dysenteric disease.

Antibodies, Bacterial↗