Biomedical subjects

A V Wisnewski

Publications and source records attributed to A V Wisnewski.

15 recordsLinked to original sources

PubMed2005

Glutathione protects human airway proteins and epithelial cells from isocyanates.

BACKGROUND: Glutathione (GSH), one of the major anti-oxidants of the lung, has been linked to the human response to isocyanate exposure. However, the ability of GSH to modulate key chemical reactions, thought to be central to the development of human isocyanate allergy, has not been directly analyzed under biologic exposure conditions. OBJECTIVE: To better understand the potential role of GSH in the response to occupational isocyanate exposure, we evaluated its effects on two processes thought to be involved in the development of isocyanate allergy, isocyanate-protein conjugation and epithelial cell toxicity. METHODS: The effects of GSH on (1) isocyanate conjugation with albumin, its major target in the airway fluid and (2) isocyanate-induced toxicity to human airway epithelial cell lines, A549 and NCI-H292, were tested using two different in vitro models. For protein conjugation studies, a newly described vapour exposure system was used to model the air/liquid interface at the surface of the epithelial fluid in the airways. Epithelial cell exposures were performed in fluid phase to mimic the in vivo exposure of airway cells covered by epithelial lining fluid. RESULTS: Reduced GSH prevented hexamethylene diisocyanate (HDI) conjugation to albumin in a dose-dependent manner, while oxidized GSH (GSSG) conversely increased conjugation rates. GSH levels equivalent to those found in normal human airway fluid (100 microm) provided >90% protection against HDI-protein conjugation when albumin was exposed to HDI vapour levels 10-fold above permissible occupational limits. Physiologic levels of GSH, but not GSSG, also reduced HDI toxicity to human airway epithelial cells in a dose-dependent manner, when present extracellularly, however, drugs that modulate intra-cellular GSH levels did not significantly alter isocyanate toxicity. CONCLUSIONS: Together with previously reported genetic and toxicity studies, the data suggest that airway GSH plays an important role in protection against HDI exposure and may help prevent the development of allergic sensitization and asthma.

Air Pollutants, Occupational↗

PubMed2002

Diisocyanate-exposed auto body shop workers: a one-year follow-up.

BACKGROUND: Diisocyanates currently are the most commonly identified cause of occupational asthma in industrialized countries. Auto body shops, a common hexamethylene diisocyanate (HDI) exposure setting, are difficult to study due to their small size and episodic exposures. OBJECTIVES: A 1-year follow-up was undertaken as an adjunct to the cross-sectional SPRAY study (Survey of Painters & Repairers of Auto bodies by Yale) to investigate the effects of HDI on auto body shop workers over time and whether or not the healthy worker effect may exist in this industry. METHODS AND RESULTS: Forty-eight workers from seven shops were re-contacted. Thirty-four subjects who stayed at the same shop and 11 who left their original shop participated. No statistically significant changes in physiology, symptoms, and immunologic responses from baseline to follow-up were noted. However, significant differences between those who left the shops and those who stayed were noted. Those who left were younger, less experienced in the industry, and more likely to have a history of asthma (23 vs. 3%; P < 0.05), bronchial hyper-responsiveness (23 vs. 9%), HDI-specific IgG (64 vs. 29%; P < 0.05), and HDI-specific proliferation (S.I. 2.0 vs. 1.3; P < 0.05). CONCLUSIONS: The differences in workers who stayed at their shop compared to those who left, combined with the low asthma prevalence and high job turnover rate, all suggest that a healthy worker effect may exist in the auto body industry, and may in part account for the low prevalence of asthma noted in SPRAY and other cross-sectional studies of diisocyante workers.

Adult↗

PubMed2001

Subclinical immunologic and physiologic responses in hexamethylene diisocyanate-exposed auto body shop workers.

BACKGROUND: Diisocyanates are potent sensitizing agents and currently the most commonly identified cause of occupational asthma in industrialized countries. However, diisocyanate asthma is difficult to diagnose and exposure and host risk factors are unclear. Auto body shops, one of the most common hexamethylene diisocyanate (HDI) exposure settings, are particularly difficult to study due to their small size and episodic exposures. Surveillance studies of such workers are limited. OBJECTIVES: We have initiated a cross-sectional field epidemiologic study, Survey of Painters and Repairers of Auto bodies by Yale (SPRAY), to characterize the effects of diisocyanate exposures on actively employed auto body shop workers. Methods and Results We present here questionnaire, physiologic, immunologic, and exposure data on 75 subjects enrolled in the study. No overt cases of clinically apparent diisocyanate asthma were identified based on spirometry, methacholine challenge, peak flows, and symptoms. HDI-specific lymphocyte proliferation was present in 30% of HDI-exposed workers and HDI-specific IgG in 34% of HDI-exposed workers, but they were not associated. HDI-specific IgE was detected in two workers. HDI-specific lymphocyte proliferation, increased methacholine responsiveness, and symptoms of chest tightness and shortness of breath were more common in the most heavily HDI-exposed workers, the painters. More long-term follow-up of this cohort should clarify the significance of these HDI-specific immunologic responses, physiologic changes, and symptoms. CONCLUSIONS: These findings demonstrate the presence of HDI-specific immune responses in a large proportion of healthy HDI-exposed workers.

Adolescent↗

PubMed2001

Recent developments in diisocyanate asthma.

Despite recent advances in our understanding of diisocyanate-induced asthma, this disease remains a perplexing phenomenon. Studies reported in the past year have focused on: (i) diisocyanate antigens; (ii) the role of airway and skin epithelium; (iii) human immune responses to exposure; (iv) neurogenic pathways; and (v) genetic factors that may confer susceptibility. These studies support the hypothesis that diisocyanate asthma results from the host immune response to these chemicals, and may represent a mixed T helper type 1/2 response. A better understanding of the pathogenesis of diisocyanate asthma should facilitate the development of better diagnostic tests and strategies for disease surveillance and intervention.

Asthma↗

PubMed2001

Human gamma/delta T-cell lines derived from airway biopsies.

gamma/delta T cells have been postulated to play an important role in the immune response at epithelial boundaries, but have not been well described in human lung tissue. We have identified and characterized gamma/delta T-cell lines from human airway biopsies and compared them with T-cell lines from paired peripheral blood samples. Airway-derived T-cell lines stimulated with tetanus toxoid (TT) contained a greater proportion of gamma/delta T cells compared with T-cell lines stimulated with mitogens, other antigens, or without antigen. TT-stimulated airway T cells expressed different T-cell receptors (TCRs) than did blood- derived T cells, and used predominantly variable region (V)gamma I family genes rather than V gamma II family genes. Airway-derived gamma/delta T cells produced high levels of interferon-gamma and were associated with T helper 1--like cytokine profiles. This study describes the presence and antigen-dependent proliferation of gamma/delta T cells from human airway tissue, and demonstrates differences in lung-derived gamma/delta TCRs compared with gamma/delta T cells derived from peripheral blood. The data suggest that gamma/delta T cells may be functionally enriched in human airways relative to peripheral blood.

Biopsy↗

PubMed2001

Development of immunoassays for biomonitoring of hexamethylene diisocyanate exposure.

Hexamethylene diisocyanate (HDI) is used widely to manufacture polyurethanes for paints and coatings. It is an irritant and a chemical asthmagen. The U.S. Occupational Safety and Health Administration time-weighted average permissible exposure limit is 5 ppb and the ceiling limit is 20 ppb. We sought to develop a sensitive and specific immuno-bioassay to supplement workplace air monitoring and detect recent HDI exposure. For this, we produced rabbit antiserum to HDI-adducted keyhole limpet hemocyanin (HDI-KLH). The specificity of the antiserum was demonstrated by its reaction with a variety of HDI-conjugated proteins and the absence of reactions with conjugates of other diisocyanates, namely toluene diisocyanate and diphenyl methylene diisocyanate. Four immunoassays were developed and compared for their ability to detect decreasing quantities of HDI-adducted human serum albumin (HSA) containing 2 mol HDI adduct per mol HSA (HDI(2)-HSA) as determined by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. The sensitivities of some of the assays are within the range (0.82-45 nM) of current analytic methods. A Western analysis procedure has a sensitivity of 600 nM HDI adduct on HSA. ELISA inhibition assay, in which microtiter plates are coated with the HDI(2)-HSA antigen, has a sensitivity of 300 nM HDI adduct. An immunoblot assay has a sensitivity of 9 nM HDI adduct. The most sensitive bioassay (1.8 nM HDI adduct) is a three-antibody sandwich ELISA in which wells of microtiter plates are coated with the IgG fraction of the anti-HDI-KLH antisera. Compared with analytic methods for HDI biomonitoring, the immunoassays are faster and less costly and accommodate numerous samples simultaneously. The assays have the potential to affect industrial biomonitoring programs significantly.

Air Pollutants, Occupational↗

PubMed2000

Identification of human lung and skin proteins conjugated with hexamethylene diisocyanate in vitro and in vivo.

Diisocyanates are asthma-causing chemicals used in the commercial production of polyurethane. We have previously shown that human lung epithelial cell proteins can become conjugated with hexamethylene diisocyanate (HDI) and may be biologically important in diisocyanate-induced asthma. The objective of this study was to identify specific human lung and skin proteins that become conjugated with diisocyanate after in vitro and in vivo exposure. Following in vitro exposure of human airway epithelial cells (A549), keratin 18, the 78-kD glucose-regulated protein, trans-1, 2-dihyrobenzene-1,2-diol dehydrogenase, and actin were identified as prominent diisocyanate-conjugated proteins through use of a combination of immunocytochemical and mass spectrometric techniques. Following in vivo inhalation of an HDI aerosol, keratin 18 was also identified as the predominant diisocyanate-conjugated protein in human endobronchial biopsy samples, whereas albumin was the predominant diisocyanate-conjugated protein in bronchoalveolar lavage fluid. Keratin was also identified as a predominant diisocyanate-conjugated protein in human skin biopsy samples after epicutaneous exposure to liquid-phase HDI, although the major skin diisocyanate-conjugated protein (56-kD) differed from the predominant lung diisocyanate-conjugated keratin (47-kD). The data from this study identify keratin and other proteins as potential "carriers" for diisocyanates in vivo, and suggest that HDI conjugation of these proteins may play a role in the pathogenesis of diisocyanate-induced asthma.

Adult↗

PubMed1999

Isocyanate-conjugated human lung epithelial cell proteins: A link between exposure and asthma?

BACKGROUND: Isocyanates are a group of highly reactive cross-linking chemicals that cause airway inflammation and asthma in exposed individuals. Isocyanates have been detected along the airway epithelia of exposed workers and animals, prompting the hypothesis that isocyanates can directly bind to epithelial cell proteins. OBJECTIVE: We tested the hypothesis that hexamethylene diisocyanate (HDI) binds directly to lung epithelial cell proteins and initiated studies to evaluate the immunostimulatory potential of HDI-conjugated lung epithelial cell proteins. METHODS: Human lung epithelial cell lines were exposed to vapor- and liquid-phase HDI, and the cellular proteins were analyzed for HDI conjugation by Western blotting and tested for the ability to induce lymphocyte proliferation in vitro. RESULTS: A number of epithelial cell polypeptides, ranging from 25 to 110 kd in apparent molecular weight, were conjugated with HDI after exposure of the human lung epithelial cell lines (A549 and NCI-H292) to HDI concentrations greater than 0.005% (vol/vol) in the liquid phase. Vapor-phase HDI exposure resulted in a more restricted HDI conjugation pattern, with major HDI-conjugated polypeptides migrating at 47, 71, and 91 kd. HDI-conjugated epithelial cell proteins specifically stimulated proliferation of PBMCs from subjects with isocyanate-induced asthma but not HDI-exposed nonasthmatic individuals or atopic subjects with nonisocyanate-related asthma. CONCLUSIONS: The data demonstrate that epithelial cell proteins readily react with HDI and that HDI-conjugated epithelial cell proteins can stimulate lymphocyte proliferation. Further characterization and evaluation of HDI-conjugated epithelial cell proteins will elucidate their potential role in the pathogenesis of isocyanate-induced asthma.

Air Pollutants↗

PubMed1998

Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41.

The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype.

Amino Acid Sequence↗

PubMed1997

In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection.

Animals↗

PubMed1996

Function and expression of a human idiotypic network in Schistosomiasis japonica.

The cross-reactive idiotype (Hu-SJ-CRIM) is defined by polyclonal human anti-idiotypic antibodies derived from chronically S. japonicum infected patients. The present study shows that serum levels of Hu-SJ-CRIM expressed by antibodies to S. japonicum soluble egg antigen (SEA) are associated with acute infection and hepatosplenic disease. Xenogeneic anti-idiotypic antisera (anti-Hu-SJ-CRIM) suppressed human lymphocyte blastogenesis to SEA in vitro by 47-82% (P < 0.05). These anti-idiotypic antibodies also suppressed in vitro granuloma formation induced by SEA coated heads in a dose dependent manner. This immunosuppression was antigen specific in that mitogen (PHA) or non-related antigen (PPD) induced blastogenic responses were not suppressed. Surprisingly, anti-idiotypic antibodies (anti-SJ-CRIM), which describe the mouse correlate CRIM were not suppressive in the human blastogenesis or in vitro granuloma formation assays. These data indicate a dichotomy in the function and specificity of the idiotype/anti-idiotype human and murine immune networks in S. japonicum infection. Thus, only the patient derived molecules and serology form the basis for an immunoregulatory network in Schistosomiasis japonica.

Adult↗

PubMed1995

Induction of protective immunity to schistosomiasis with immunologically cross-reactive Lumbricus molecules.

Immunologically cross-reactive molecules of Schistosoma japonicum and Lumbricus terrestris were identified by antibodies derived from human and rodent sera. Pooled IgG from schistosomiasis patients but not uninfected individuals bound multiple antigens of identical molecular weight in both soluble S. japonicum worm antigen preparations (SWAP) and soluble earthworm preparations (SEWP). These antigens had molecular weights corresponding to 18, 40, 62, 64, 74, 97, and > 110 kDa. Three of these antigens of 74, 97 and > 110 kDa were immuno-affinity purified using antibodies derived from schistosomiasis patients' sera. Vaccination of mice with SEWP produced murine antibodies which bound parasite molecules of 40, 74, 97, and > 110 kDa and induced 36% protection from S. japonicum infection (P < 0.05). Antibody production to S. japonicum paramyosin, a molecule previously shown to induce protection from schistosome infection, was prominently expressed in the protected murine immune sera. The study shows that Lumbricus sp. represent a potential source for paramyosin and other candidate vaccine molecules for schistosomiasis.

Animals↗

PubMed1994

Human monoclonal antibodies heterogeneously express a human cross-reactive idiotype associated with immune function in Schistosoma japonicum infection.

Hybridomas secreting human monoclonal antibodies (hMAb) were derived from Epstein Barr Virus (EBV) transformed lymphocytes of a patient with acute Schistosoma japonicum infection. Three IgG1 hMAb SJ-D, SJ-E, and SJ-F bind soluble egg antigens (SEA) as determined by ELISA. These hMAb exhibit identical western blot profiles, recognizing an epitope(s) of multiple antigens with apparent molecular weights between 42 and 75 kDa. Serological analysis of these hMAb revealed a heterogeneity in their expression of a specific human S. japonicum anti-SEA associated cross reactive idiotype designated Hu SJ-CRIM. The differential expression of idiotypy by these hMAb correlates with immunosuppression of blastogenesis of lymphocytes from schistosomiasis patients. The level of suppression mediated by hMAb expressing high levels of Hu SJ-CRIM ranged from 41% to 52% (p < 0.05) for antigen and 36% to 43% for mitogen. In contrast, hMAb SJ-D which expressed over two fold lower levels Hu SR-CRIM, on a per weight basis showed no suppressive immune function. The data show the heterogeneous expression of human idiotype associated with S. japonicum infection and the correlation of idiotype expression with immune function.

Animals↗

PubMed1992

Human hybridoma generation by hypo-osmolar electrofusion: characterization of human monoclonal antibodies to Schistosoma mansoni parasite antigens.

Human monoclonal antibodies which bind Schistosoma mansoni worm and egg antigens were identified and characterized from hybridomas generated using the hypo-osmolar electrofusion technique of somatic cell fusion. Splenocytes from S. mansoni infected individuals were mitogen-activated in vitro and subsequently fused by electrofusion. The greatest number of HAT resistant hybridomas per helical fusion chamber was obtained with unfrozen splenocytes cultured for 4-6 days after introduction of mitogen. Hybridomas secreting IgG antibodies recognizing parasite antigens were identified by ELISA. Twenty-one cloned cell lines secreting IgG antibody were maintained for at least 6 months. Characterization of antigen reactivity by Western blot analysis of nien cloned cell lines revealed antibodies which bound stage specific parasitic antigens. The data show that the technique of hypo-osmolar electrofusion produces stable, antibody producing hybridomas. The human monoclonal antibodies screened represent candidate molecules useful in the investigations of the human pathogen S. mansoni.

Animals↗

PubMed1991

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay.

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.

Animals↗