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Biomedical subjects

H M Friedman

Publications and source records attributed to H M Friedman.

At least 55 records · Page 3Linked to original sources
PubMed1990

Viral activation of the coagulation cascade: molecular interactions at the surface of infected endothelial cells.

Herpesviral infection of endothelial cells (ECs) induces arterial injury. We now demonstrate that such infection promoted enhanced monocyte-endothelial adhesion. Enhanced adhesion was blocked by monoclonal antibodies to the viral-encoded cell surface glycoprotein gC but not by antibodies to gD or gE. Adhesion was also blocked by treating ECs with specific thrombin inhibitors or by growing cells in prothrombin-depleted serum. We found that gC bound and promoted activation of factor X on infected ECs, thereby contributing to thrombin generation. Factor X also bound to transfected L cells that were induced to express gC. Cross-linking and immunoprecipitation studies demonstrated factor X-gC complex formation on the surface of these cells. We suggest that gC-dependent thrombin generation by herpes-infected endothelium may be an important mediator of vascular pathology during viral infection.

Blood Coagulation↗
PubMed1990

Glycoprotein C of herpes simplex virus type 1 prevents complement-mediated cell lysis and virus neutralization.

Glycoprotein gC1 of herpes simplex virus type 1 (HSV-1) binds complement component C3b. To determine if gC1 modifies the interaction of complement with virus-infected cells or cell-free virus, ns-1, a mutant HSV-1 strain that does not express gC1 at the cell surface and does not bind C3b, was compared with its parental strain, NS. Cells infected with the gC1 mutant were more susceptible to cytolysis mediated by antibody and complement or complement alone. The gC1 or gD1 genes were expressed in mammalian cells under the control of an inducible promoter. Cells induced to express gC1 resisted complement cytolysis, while cells expressing gD1 did not. gC1 modified cytolysis of virus-infected or -transfected cells by blocking alternative complement pathway activation. gC1 also modified complement-dependent virus neutralization, which was mediated by inhibiting the classical complement pathway. These results indicate a protective role for gC1 on the virion and at the cell surface.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1990

Cytotoxic T lymphocytes specific for herpes simplex virus (HSV) studied using adenovirus vectors expressing HSV glycoproteins.

In previous work, we observed that H-2k-restricted herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTLs) were effectively able to lyse transfected target cells expressing HSV glycoprotein C (gC), but not cells expressing gB, gD or gE. To confirm and extend our observations on the specificity of anti-HSV CTLs, recombinant adenovirus (Ad) vectors able to express HSV-1 gB or gC (AdgB2 or AdgC) were constructed. Syngeneic target cells infected with AdgB2 were efficiently lysed by primary H-2b and H-2d, but not by H-2k-restricted HSV-specific CTL. Limiting dilution studies indicated that 4 to 10% of H-2b-restricted HSV-specific CTLs recognize gB. H-2k, H-2b and H-2d-restricted anti-HSV-1 CTLs were unable to lyse AdgC-infected syngeneic target cells. To examine the apparent discrepancy between the previous results involving transfected H-2k cells expressing gC and the present results involving AdgC-infected cells, gC-expressing cell lines used in previous experiments were subcloned and retested in CTL assays. DC2 cells which were lysed by HSV-specific CTLs in the previous experiments remained sensitive to anti-HSV CTLs but two other clones derived from the same transfection were not lysed. Further, L cells transfected with the gC or gD gene coupled to the mouse mammary tumour virus promoter and capable of expressing high levels of the glycoproteins following dexamethasone induction were not lysed by H-2k-restricted anti-HSV CTLs. These results suggest that HSV-specific CTLs do not recognize gC, at least when it is expressed using an Ad vector and in most transfected cell lines, whereas a significant proportion of anti-viral CTLs recognize gB presented in some but not all murine haplotypes.

Adenoviridae↗
PubMed1990

Identification of C3b-binding regions on herpes simplex virus type 2 glycoprotein C.

Glycoprotein C from herpes simplex viruses types 1 and 2 (gC-1 and gC-2) acts as a receptor for the C3b fragment of the third component of complement. Our goal is to identify domains on gC involved in C3b receptor activity. Here, we used in-frame linker-insertion mutagenesis of the cloned gene for gC-2 to identify regions of the protein involved in C3b binding. We constructed 41 mutants of gC-2, each having a single, double, or triple insertion of four amino acids at sites spread across the protein. A transient transfection assay was used to characterize the expressed mutant proteins. All of the proteins were expressed on the transfected cell surface, exhibited processing of N-linked oligosaccharides, and bound one or more monoclonal antibodies recognizing distinct antigenic sites on native gC-2. This suggested that each of the mutant proteins was folded into a native structure and that a loss of C3b binding by any of the mutants could be attributed to the disruption of a specific functional domain. When the panel of insertion mutants was assayed for C3b receptor activity, we identified three distinct regions that are important for C3b binding, since an insertion within those regions abolished C3b receptor activity. Region I was located between amino acids 102 and 107, region II was located between residues 222 and 279, and region III was located between residues 307 and 379. In addition, region III has some structural features similar to a conserved motif found in complement receptor 1, the human C3b receptor. Finally, blocking experiments indicated that gC-1 and gC-2 bind to similar locations on the C3b molecule.

Amino Acid Sequence↗
PubMed1990

Herpes simplex virus type 1 encodes two Fc receptors which have different binding characteristics for monomeric immunoglobulin G (IgG) and IgG complexes.

Two herpes simplex virus type 1 glycoproteins, gE and gI, have been shown to form a complex that binds the Fc domain of immunoglobulin G (IgG). We demonstrate that this complex is required for the binding of monomeric nonimmune IgG but that gE alone is sufficient for binding polymeric IgG in the form of IgG complexes. Evidence that gE but not gI is required for binding IgG complexes is as follows. IgG complexes bound equally well to cells infected with gI-negative mutants or with wild-type virus, whereas cells infected with gE-negative mutants did not bind IgG complexes. Furthermore, L cells transiently transfected to express gE bound IgG complexes. Additional evidence that gI fails to augment binding of IgG complexes comes from experiments in which the gI gene was inducibly expressed in cells after infection. Inducible gI expression failed to increase binding of IgG complexes to infected cells in comparison with cells not capable of inducible gI expression. In contrast, expression of both gE and gI was necessary for binding of monomeric IgG, as demonstrated by flow cytometry using cells infected with gE-negative and gI-negative mutants. These observations demonstrate that herpes simplex virus type 1 Fc receptors (FcRs) have different binding characteristics for monomeric IgG and IgG complexes. Furthermore, it appears that gE is the FcR for IgG complexes and that gE and gI form the FcR for monomeric IgG.

Animals↗
PubMed1990

Conjugal temporal arteritis.

We report the simultaneous occurrence of biopsy-proven temporal arteritis in husband and wife. Serologic and viral studies were negative, including viral culture of the wife's temporal artery. The concurrent incidence of giant cell arteritis in a married couple would suggest a common exogenous exposure.

Aged↗
PubMed1989

Discrepant rotavirus results in two laboratories using the same enzyme immunoassay.

Rotavirus testing was performed on 66 stool samples in two separate laboratories using the same enzyme immunoassay. Sixteen of 46 of the positive results reported by the reference laboratory were negative in the hospital laboratory. In addition, RNA gel electrophoresis had negative results in the 16 discrepant samples. This indicates the need to repeat or confirm positive results.

Child↗
PubMed1989

Protective effects of Towne cytomegalovirus vaccine against low-passage cytomegalovirus administered as a challenge.

To test the protective effect of Towne live attenuated human cytomegalovirus (HCMV) vaccine in normal individuals, we developed a parenteral challenge consisting of a low-passage isolate (Toledo stain) inoculated subcutaneously in graded doses. This challenge virus caused a mild mononucleosis syndrome in seronegative individuals at doses of 10 or 100 pfu. The illness was accompanied by atypical lymphocytosis, raised hepatic enzymes, excretion of HCMV and HCMV-specific immune responses. Naturally seropositive volunteers also developed clinical and laboratory evidence of infection after challenge with 1,000 pfu of Toledo but resisted 10 or 100 pfu. Volunteers who had been vaccinated 1 y earlier also were resistant to disease caused by 10 or 100 pfu of Toledo, although some were asymptomatically infected by the 100 pfu dose. Vaccine-induced immunity to HCMV was as complete as naturally induced immunity when the challenge dose of Toledo was 10 pfu.

Antibodies, Viral↗
PubMed1989

A novel function of the herpes simplex virus type 1 Fc receptor: participation in bipolar bridging of antiviral immunoglobulin G.

We describe a novel function of the Fc receptor of herpes simplex virus type 1 (HSV-1), its ability to participate in antibody bipolar bridging. This refers to the binding of a single immunoglobulin G (IgG) molecule by its Fab end to its antigenic target and by its Fc end to an Fc receptor (FcR). We demonstrate that various immune IgG antibodies, including polyclonal rabbit antibodies to HSV-1 glycoproteins gC1 and gD1 and monoclonal human antibody to gD1 blocked rosetting of IgG-coated erythrocytes at IgG concentrations 100- to 2,000-fold lower than required for rosette inhibition with nonimmune IgG. Steric hindrance did not account for the observed differences between immune and nonimmune IgG since rabbit anti-gC1 F(ab')2 fragments did not block rosetting. Murine anti-gC1 or anti-gD1 IgG, a species of IgG incapable of binding by its Fc end to the HSV-1 FcR, also did not block rosetting. When cells were infected with a gC1-deficient mutant, anti-gC1 IgG inhibited rosetting to the same extent as nonimmune IgG. This indicates that binding by the Fab end of the IgG molecule was required for maximum inhibition of rosetting. Bipolar bridging was shown to occur even when small concentrations of immune IgG were present in physiologic concentrations of nonimmune IgG. The biologic relevance of antibody bipolar bridging was evaluated by comparing antibody- and complement-dependent virus neutralization of an FcR-negative mutant and its parent HSV-1 strain. By engaging the Fc end of antiviral IgG, the parent strain resisted neutralization mediated by the classical complement pathway. These observations provide insight into the role of the HSV-1 FcR in pathogenesis and may help explain the function of FcR detected on other microorganisms.

Animals↗
PubMed1989

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

Animals↗
PubMed1988

Increased adherence of human granulocytes to herpes simplex virus type 1 infected endothelial cells.

We studied the interaction of human polymorphonuclear leukocytes (PMNs) with umbilical vein endothelial cells infected with herpes simplex virus (HSV) type 1. PMNs labeled with 51Cr were added to endothelial monolayers at varying times after infection and their adherence assessed 1 h later. Granulocyte adherence (GA) to uninfected cells averaged 26.5 +/- 1.9%. Increased adherence began 6 h postinfection and rose to a maximum at 20 to 24 h. HSV-1 glycoproteins seemed to mediate the increase in GA: tunicamycin treatment of infected monolayers for 18 h abolished the increased GA as did incubation of infected cells with F(ab')2 fragments prepared from human antiserum containing HSV-1 antibody.

Cell Adhesion↗
PubMed1988

C3b receptor activity on transfected cells expressing glycoprotein C of herpes simplex virus types 1 and 2.

Glycoprotein C from herpes simplex virus type 1 (gC-1 from HSV-1) acts as a receptor for the C3b fragment of the third component of complement on HSV-1-infected cell surfaces. Direct binding assays with purified gC-1 and C3b demonstrate that other viral and cellular proteins are not required for this interaction. Although C3b receptor activity is not expressed on HSV-2-infected cell surfaces, purified gC-2 specifically binds C3b in direct binding assays, suggesting that gC-1 and gC-2 are functionally similar. Here, we used a transient transfection system to further characterize the role of gC-1 and gC-2 as C3b receptors and to localize the site(s) on gC involved in C3b binding. The genes for gC-1 and gC-2 were each cloned into a eucaryotic expression vector containing the Rous sarcoma virus long terminal repeat as the promoter and transfected into NIH 3T3 cells. The expressed proteins were similar in molecular size, extent of carbohydrate processing, and antigenic properties to gC-1 and gC-2 purified from infected cells. Using a double-label immunofluorescence assay, we found that both gC-1 and gC-2 were expressed on the surfaces of transfected cells and bound C3b. These results suggest that other proteins expressed during HSV-2 infection prevent receptor activity. We constructed three in-frame deletion mutants of gC-2 to identify domains on the protein important for C3b receptor activity. These mutants lacked amino acids 26 to 73, 219 to 244, or 318 to 346. The mutant protein lacking residues 26 to 73 was reactive with two monoclonal antibodies recognizing distinct epitopes, showed a wild-type pattern of carbohydrate processing, and bound C3b on the transfected cell surface. These results suggest that residues 26 to 73 are not involved in C3b binding. The other two mutant proteins were present on the cell surface, but did not bind C3b. In addition, these mutant proteins showed altered patterns of carbohydrate processing, formed aggregates, and were no longer recognized by the monoclonal antibodies. These properties indicate that removal of residues 219 to 244 or 318 to 346 disrupted the native conformation of gC-2, possibly owing to an alteration in the spacing between critical cysteine residues.

Antibodies, Monoclonal↗
PubMed1987

Hepatitis B surface antigen in corneal donors.

Using an enzyme immunoassay, we detected hepatitis B surface antigen in washings of ocular tissue from two of three corneal donors who had positive serum assays for hepatitis B surface antigen. Hepatitis B surface antigen was identified in emulsified corneal tissue from one of two donors from whom corneal tissue was available for study.

Adult↗
PubMed1987

Human leukemia-derived cell lines and clones as models for mechanistic analysis of natural killer cell-mediated cytotoxicity.

Tumor target cells (TC) are lysed by natural killer (NK) cells provided that they (1) form conjugates with the effector cells, (2) activate effector cells to release cytotoxic factors, and (3) they are susceptible to the lytic effect of these factors. While this cascade of events that leads to TC killing has been defined, the signal molecules responsible for each of the steps remain largely undetermined. A variety of human leukemia-derived TC lines and clones were analyzed for their sensitivity to NK cell-mediated lysis and for their ability to bind and activate NK cells. These characteristics have been correlated with TC surface expression of differentiation antigens and carbohydrate residues. Of the cell lines and clones tested, K562, SPI-802, MOLT-4, MOLT-4/C8-1, ZS, KG-1/A-3, and HL-60S were sensitive to NK cell-mediated lysis, while KG-1, THP-1-0, HL-60R, and LFM were resistant. KG-1, THP-1-0, HL-60R, and LFM cells were further studied to determine mechanisms responsible for their resistance to NK cells. It was found that HL-60R and LFM cells were unable to bind NK cells. In contrast, KG-1 and THP-1-0 cells were able to bind to and activate NK cells. Therefore, it is likely that the NK-resistance of KG-1 and THP-1-0 cells may be related to their lack of sensitivity to cytotoxic factors released by bound NK cells. All of the TC cell lines and clones capable of binding NK cells expressed the 3-fucosyl-N-acetyl-lactosamine hapten (Lex or SSEA-1 antigen) recognized by the monoclonal antibody Leu M1. These TC consistently lacked surface L-fucose residues, as shown by lack of Ulex europaeus agglutinin binding. In contrast, HL-60R and LFM which did not form conjugates with NK cells, did not express surface Lex determinants and avidly bound the Ulex agglutinin. Distinct subpopulations of NK-resistant KG-1 cells expressed Lex antigens or bound Ulex. We compared KG-1/A-3, a NK-sensitive cell clone, with the parental NK-resistant KG-1 cell line. KG-1/A-3 lost the ability to bind the Ulex lectin displayed by the parental cell line and showed increased expression of Lex determinants. Results from these phenotypic analyses suggest that expression of Lex determinants and Ulex binding sites on the TC membrane are mutually exclusive and their expression or absence may correlate with mechanisms which regulate TC-NK cell interactions.

Cell Line↗
PubMed1987

Suppression of matrix protein synthesis by herpes simplex virus in bovine smooth muscle cells.

We investigated the effect of herpes simplex virus type 1 infection on the synthesis of extracellular matrix proteins by bovine smooth muscle cells. The cells supported viral replication, which reached maximum at 24 hrs post-infection. Uninfected and infected (multiplicity of infection of 5 or 20) cultures of smooth muscle cells were labeled with [14C]proline at five hrs post-infection. Incorporation of radioactivity into non-dializable protein was determined following electrophoresis of the cell-matrix and medium fractions. The synthesis of fibronectin and collagen types I and III was almost completely suppressed. The data suggest that HSV-1 infection of smooth muscle cells in vitro alters extracellular matrix synthesis.

Animals↗
PubMed1987

Diagnosis of pulmonary nodules in the cancer patient: the role for routine culture and an unusual application of the human tumor colony-forming assay.

Many solid tumors that occur in humans tend to metastasize to the lungs. The etiology of pulmonary nodules in cancer patients, therefore, presents a diagnostic problem that can be resolved only by histologic evaluation or resection. We present two cases of cancer patients with preoperative and intraoperative diagnoses of metastatic lung tumors. Tissue culture using the Human Tumor Colony-Forming Assay (HTCFA), a procedure that predicts chemosensitivity patterns of tumors, was employed to successfully diagnose those nodules as fungal infections. In one case, results using the HTCFA were crucial in the identification of the opportunistic organism. These cases stress the importance of routine culture of all pulmonary nodules and present an unusual but noteworthy application of the HTCFA system.

Adolescent↗