Biomedical subjects

R A Shapiro

Publications and source records attributed to R A Shapiro.

At least 37 records · Page 2Linked to original sources

PubMed1997

Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFNgamma, and LPS.

There is evidence that nitric oxide (NO) may mediate some of the functional myocardial changes caused by bacterial LPS and inflammatory cytokines. The expression of the inflammatory or inducible NO synthase (iNOS) in human cardiac myocytes, however, has not been well characterized. Therefore, we treated cultured, dedifferentiated human ventricular cardiac myocytes with the combination of TNF-alpha (500 U/ml), IL-1beta (30U/ml), IFNgamma (100 U/ml), and LPS (E.coli 0111:B4, 10 microg/ml). Northern blot analysis revealed a approximately 4.5 kb transcript for inducible NOS (iNOS) in the stimulated human heart cells but not in untreated cells. RT-PCR confirmed that iNOS mRNA was only present in stimulated cells. However, treatment of the myocytes for up to 96 h with cytokines and LPS did not result in NO synthesis as measured by nitrite + nitrate accumulation in the culture medium, and no iNOS enzymatic activity could be detected in the cell lysates. Western blot analysis failed to detect iNOS protein. Thus, despite high and persistent levels of iNOS mRNA in cytokine-treated cells, iNOS protein was absent in this experimental model. GTP-cyclohydrolase I was induced both at the mRNA and protein levels and resulted in increased biopterin levels, indicating sufficient amounts of the cofactor tetrahydrobiopterin (BH4) were present, and that the failure to express an inducible protein was specific to iNOS. To determine if the absence of iNOS protein was due to a novel cardiac iNOS gene or modified iNOS transcript in human myocytes, we cloned an iNOS cDNA from cytokine-treated myocytes. Sequencing and expression of the clone revealed a functional iNOS cDNA with >99% identity to other human iNOS cDNA clones. When human cardiac cells were transduced with a retroviral vector carrying only the coding region of the human hepatocyte iNOS cDNA, both iNOS mRNA and protein could be detected. In conclusion, these cells derived from cultured human cardiac myocytes lacked the capacity to express an endogenous iNOS protein, the basis of which appears to be a cell-specific suppression or failure of iNOS translation.

Argininosuccinate Synthase↗

PubMed1997

Identification of CD14 residues involved in specific lipopolysaccharide recognition.

CD14 is a key molecule responsible for the innate host inflammatory response to microbial infection. It is able to bind a wide variety of microbial ligands and facilitate the activation of both myeloid and nonmyeloid cells. However, its specific contribution to the innate recognition of bacteria is not known. Presently there is no information on the contribution of individual CD14 residues to Escherichia coli lipopolysaccharide (LPS) binding or on the molecular basis of the interaction between CD14 and LPS from other bacteria. LPS obtained from Porphyromonas gingivalis, a bacterium associated with chronic inflammatory disease, binds CD14 and activates myeloid cells but does not facilitate the activation of nonmyeloid cells. The transfer and binding of these two LPS species to soluble CD14 recombinant globulin proteins with single point mutations was examined. Functional activity of the mutant proteins was monitored by E-selectin expression on human umbilical cord endothelial cells. The analysis identified a charge reversal mutation in a single residue, E47, that demonstrated selective binding to E. coli LPS but not to P. gingivalis LPS. E-selectin activation assays indicated that proteins with mutations at position E47 maintained their structural integrity. Other mutations, including a charge reversal mutation of residue E58, did not significantly reduce the binding of either LPS ligand or the ability of the molecule to facilitate E-selectin activation. These data demonstrate that CD14 can selectively recognize different LPS ligands.

Amino Acid Sequence↗

PubMed1997

Dexamethasone suppresses iNOS gene expression by upregulating I-kappa B alpha and inhibiting NF-kappa B.

Cytokine-stimulated inducible nitric oxide synthase (iNOS) gene expression is dependent on nuclear factor-kappa B (NF-kappa B) activation and is suppressed by glucocorticoids (GC). In this study we examined the molecular mechanisms of GC inhibition of iNOS expression in rat hepatocytes. Combinations of tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma (cytokine mixture CM) induced high levels of iNOS mRNA and NO synthesis. The synthetic GC dexamethasone markedly repressed iNOS mRNA and protein expression, and nuclear run-on assays showed that this inhibition was occurring at the level of transcription. In addition, transfection studies showed that CM-stimulated activity of a 1.6-kb murine iNOS promoter fragment linked upstream of luciferase was suppressed by dexamethasone. Electromobility shift assays demonstrated that CM induced the appearance of an NF-kappa B complex composed of p50 and p65 subunits; the addition of dexamethasone markedly decreased this band shift. I-kappa B alpha expression was decreased by CM and upregulated in the presence of dexamethasone. Subsequently, nuclear p65 levels were decreased by dexamethasone compared with CM-treated cells. Thus GC repress NF-kappa B DNA-binding activity in rat hepatocytes in part through the upregulation of its inhibitor I-kappa B alpha. These data indicate that one mechanism by which GC block iNOS expression is through the inhibition of NF-kappa B activation resulting in decreased iNOS transcription.

Animals↗

PubMed1996

Identification of a region required for subtype-specific agonist-induced sequestration of the m2 muscarinic acetylcholine receptor.

When the m1 and m2 muscarinic acetylcholine receptors are transiently expressed in JEG-3 cells, the m2, but not the m1, receptor undergoes agonist-induced sequestration. Both receptors exhibit internalization when expressed in Y1 cells. These results suggest that the m1 and m2 receptors use distinct cellular mechanisms or pathways for agonist-induced internalization and that JEG-3 cells are deficient in the mechanism or pathway used by the m1 receptor. Transfection experiments with chimeric receptors indicate that the specificity for agonist-induced internalization for the m2 receptor lies in the carboxyl-terminal fifth of the receptor. The intracellular carboxyl-terminal tail of the m2 receptor is neither sufficient nor required for the m2-specific sequestration. Site-directed mutagenesis demonstrates that two amino acids in the carboxyl-terminal end of the third cytoplasmic loop of the m2 receptor are required for sequestration in JEG-3 cells. In addition, the sixth transmembrane domain, which is adjacent to this cytoplasmic domain, is also required. Thus, m2-specific agonist-induced sequestration requires sequences both in the carboxyl-terminal end of the third cytoplasmic loop and the adjacent transmembrane domain.

Amino Acid Sequence↗

PubMed1996

Early signal transduction by the receptor to the chemokine monocyte chemotactic protein-1 in a murine T cell hybrid.

Members of the C-C family of chemotactic cytokines promote chemotaxis and adhesion of leukocytes. In this study, we have identified a murine T cell hybrid that expresses receptors to the chemotactic cytokine monocyte chemotactic protein-1 (MCP-1). This cell line was used to examine MCP-1 receptor-mediated signal transduction events in a homologous system in the absence of interference with other receptors. Our results show that in the 3B4 M1.9 T cell hybrid, MCP-1 receptors mediate intracellular calcium mobilization and extracellular calcium import without detectable increases in total water-soluble inositol phosphates. In addition, MCP-1 regulates the tyrosine phosphorylation of specific substrates at 42 and 44 kDa and induces mobility shift of p42/44 mitogen-activated protein kinases. MCP-1-mediated calcium responses, tyrosine phosphorylation, and the electrophoretic mobility shift of p42/44 mitogen-activated protein kinases can be inhibited by pretreatment of cells with pertussis toxin, indicating a role for Gi-like G proteins in coupling the MCP-1R to signal transduction.

Animals↗

PubMed1996

Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

Blotting, Northern↗

PubMed1996

PGE2 and LTB4 inhibit cytokine-stimulated nitric oxide synthase type 2 expression in isolated rat hepatocytes.

Prostaglandins have been shown to have a wide range of effects on nitric oxide synthesis when studied in different cell populations. The proximity of hepatocytes to eicosanoid-producing endothelial cells and Kupffer cells prompted us to determine the effects of PGE2 and LTB4 on hepatocyte NO production by the inducible nitric oxide synthase (iNOS, NOS-2) in vitro. PGE2 decreased hepatocyte NO synthesis in a concentration-dependent manner when the cells were stimulated with a combination of cytokines or IL-1 alone. LTB4 had a similar effect. PGE2 had to be present at the time of cytokine exposure to produce maximal inhibition of NO synthesis. Reduced synthesis of NO2- was associated with reduced NOS-2 mRNA levels suggesting that the induction of NOS-2 was inhibited. These findings demonstrate that eicosanoids can regulate hepatocyte NO synthesis in vitro.

Animals↗

PubMed1996

Two amino acid differences in the sixth transmembrane domain are partially responsible for the pharmacological differences between the 5-HT1D beta and 5-HT1E 5-hydroxytryptamine receptors.

5-Hydroxytryptamine elicits its physiological effects by interacting with a diverse group of receptors. Two of these receptors, the 5-HT1D beta and the 5-HT1E receptors, are approximately 60% identical in the transmembrane domains that presumably form the ligand binding site yet have very different pharmacological properties. Analysis of the pharmacological properties of a series of chimeric 5-HT1D beta/5-HT1E receptors indicates that sequences in the sixth and seventh transmembrane domains are responsible for the differential affinity of 5-carboxamidotryptamine for these two receptors. More detailed analysis shows that two amino acid differences in the sixth transmembrane domain (Ile333 and Ser334 in the 5-HT1D beta receptor, corresponding to Lys310 and Glu311 in the 5-HT1E receptor) are largely responsible for the differential affinities of some, but not all, ligands for the 5-HT1D beta and 5-HT1E receptors. It is likely that these two amino acids subtly determine the overall three-dimensional structure of the receptor rather than interact directly with individual ligands.

Amino Acid Sequence↗

PubMed1996

The management of pigmented lesions of the nail bed.

Pigmented lesions of the nail bed, especially without a history of trauma, represent a diagnostic challenge to the clinician. These lesions are often categorized as melanonychia striata (MS), which refers to any linear tan-brown-black pigmentation of the nail bed. The differential diagnosis of MS includes subungual hematomas, onchomycosis nigricans, junctional nevi, melanoma in situ (MIS), and malignant melanoma (MM). Our algorithm at the New York University (NYU) Medical Center for the treatment of pigmented lesions of the nail bed is presented. A histopathologic diagnosis with any evidence of melanocytic atypia, however subtle, requires absolute confirmation by complete excision. The absence of a clear margin or recurrence requires total nail bed excision and reconstruction using a full-thickness graft. The diagnosis of MIS is similarly treated. The surgical management of subungual MM is discussed. All cases of MM of the hand treated at NYU were reviewed. In all, 30 patients were treated from 1982 to 1995. Follow-up ranged from 6 months to 13 years. In our series, there were 8 cutaneous and 22 subungual melanomas. There was a marked delay in treatment of both groups, with subungual melanomas more often erroneously treated as other pathology prior to correct diagnosis. The 5-year survival rate was 100% for patients with cutaneous lesions, but only 80% for those with the subungual variety. There was a statistical difference in the depths of the lesions (subungual, 3.68 mm; cutaneous, 1.36 mm) with a p-value of 0.008. The role of elective lymph node dissection in the absence of clinical metastases as well as intraoperative sentinel lymphatic mapping remains controversial and is discussed.

Adolescent↗

PubMed1995

A central role for IL-1 beta in the in vitro and in vivo regulation of hepatic inducible nitric oxide synthase. IL-1 beta induces hepatic nitric oxide synthesis.

We have previously demonstrated that high levels of inducible nitric oxide synthase (iNOS) expression in hepatocytes required a combination of LPS and TNF-alpha, IL-1 beta, and IFN-gamma. The need for such a complex regulatory system seemed unwarranted based on the importance of NO in the liver. Therefore, we investigated whether individual cytokines could induce NO synthesis in hepatocytes and characterized some of the mechanisms involved. Rat hepatocytes were stimulated in vitro with escalating doses of TNF-alpha, IL-1 beta, or IFN-gamma. Only IL-1 beta induced high levels of iNOS mRNA and corresponding NO2- + NO3- production, and dexamethasone and cycloheximide blocked a majority of this response. Nuclear run-on experiments revealed that IL-1 beta upregulated iNOS gene transcription. IL-1 receptor antagonist protein (IL-1ra) competitively inhibited IL-1 beta-stimulated NO synthesis, implying activation through a cell-specific receptor. Rats injected with both LPS and IL-1ra showed decreased hepatic iNOS mRNA and plasma NO2- + NO3- compared with rats given LPS alone, indicating that IL-1 beta plays a role in regulating iNOS expression within the liver in vivo during endotoxemia. The soluble TNF receptor antagonist, PEG-(rsTNF-RI)2, also suppressed hepatic iNOS mRNA levels and plasma NO2- + NO3- increases, supporting a role for this cytokine in LPS-induced iNOS expression. Finally, IL-1 beta at high doses also induced iNOS mRNA and significant NO2- + NO3- production in cultures of primary human hepatocytes. These data indicate an important role for IL-1 beta in the regulation of hepatic NO synthesis.

Animals↗

PubMed1995

Autocrine/paracrine induction of tissue inhibitor of metalloproteinase-1 in Chinese hamster ovary cells by oncostatin M.

Chinese hamster ovary (CHO) cells expressing recombinant human oncostatin M (rOM) were found to secrete high levels of a 28-kDa protein. Sequence analysis of the protein suggested that it was hamster tissue inhibitor of metalloproteinase-1 (TIMP-1). In this study, we show that induction of TIMP-1 mRNA and protein by CHO cells is due to rOM action in an autocrine/paracrine mode. TIMP-1 expression in rOM-producing CHO cells increased concomitantly with methotrexate-induced rOM amplification. TIMP-1 upregulation was not caused by either transfection of nonspecific DNA nor was it a direct effect of treatment of the cells with methotrexate. These results suggest that oncostatin M is a potent inducer of TIMP-1 and that its receptor-mediated expression is conserved across species.

Animals↗

PubMed1995

Porphyromonas gingivalis lipopolysaccharide is poorly recognized by molecular components of innate host defense in a mouse model of early inflammation.

Porphyromonas gingivalis is a gram-negative bacterium that is associated with periodontitis. It has been hypothesized that destruction of bone and periodontal connective tissue is associated with colonization of the subgingival crevicular space by P. gingivalis, although how these bacteria overcome innate host defenses is largely unknown. To examine the early cellular and molecular events of P. gingivalis interaction with host tissues, we compared lipopolysaccharide (LPS) isolated from this bacterium with Escherichia coli LPS, a potent inflammatory mediator, in a mouse model of acute inflammation. In these studies, mice were given intramuscular injections of either P. gingivalis LPS or E. coli LPS and then sacrificed after 4 h. Reverse transcriptase-PCR analysis showed that expression of mRNAs for E- and P-selectins was higher in E. coli LPS-injected muscles than in P. gingivalis LPS-injected or control phosphate-buffered-saline-injected muscles. Similarly, monocyte chemotactic protein 1 and fibroblast-induced cytokine mRNAs were expressed in E. coli LPS-injected muscles whereas their expression was reduced or absent in P. gingivalis LPS-injected samples. These results were confirmed by in situ hybridization whereby stronger hybridization for selectin mRNAs was observed in the endothelium of capillaries from E. coli LPS-injected samples than in that from P. gingivalis LPS-injected muscles. In addition, many monocytes expressing monocyte chemotactic protein 1 mRNA and polymorphonuclear leukocytes expressing fibroblast-induced cytokine mRNA were observed in E. coli LPS-injected muscles whereas only a few cells were identified in P. gingivalis LPS-injected muscles. These results demonstrate that compared with E. coli, P. gingivalis has a low biologically reactive LPS as measured by its weak activation of inflammation. This may allow P. gingivalis to evade innate host defense mechanisms, resulting in colonization and chronic disease.

Animals↗

PubMed1995

The prevalence of sexually transmitted diseases in children and adolescents evaluated for sexual abuse in Cincinnati: rationale for limited STD testing in prepubertal girls.

OBJECTIVE: To determine the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, syphilis, and human immunodeficiency virus (HIV) infection in sexually abused children and to develop selective criteria for sexually transmitted disease (STD) testing in these children in our community. DESIGN: Prospective. SETTING: University-affiliated children's hospital in Ohio. PARTICIPANTS: All children evaluated at our hospital for sexual abuse were eligible. Eight hundred fifty-five children were evaluated over a 1-year period. The study included 704 girls and 151 boys. Children ranged in age from 3 weeks to 18 years old. METHODS AND RESULTS: Standard STD testing (American Academy of Pediatrics recommendations) was defined as serum rapid plasma reagin test, examination for Trichomonas, N gonorrhoeae culture of the throat, rectum, and genitalia and C trachomatis culture of the rectum and genitalia. STD testing in this study was recommended in children with 1) a history of genital discharge or contact with the perpetrator's genitalia, 2) examination findings of genital discharge or trauma, and 3) all adolescents. HIV testing was obtained in children with risk factors for HIV infection, those with contact with a perpetrator with HIV risk factors, or if the family was concerned about HIV acquisition. A total of 423 children were tested for N gonorrhoeae, 415 for C trachomatis, 275 for syphilis, 208 for Trichomonas, and 140 for HIV. Twelve children were determined to have N gonorrhoeae infection, 11 had C trachomatis infection, and four had Trichomonas infection. Overall, the prevalence of STDs in prepubertal girls was 3.2% and 14.6% in pubertal girls. The prevalence of N gonorrhoeae in prepubertal girls with vaginal discharge was 11.1% and 0% in prepubertal girls without discharge (P < .001). C trachomatis infection was diagnosed in 0.8% of prepubertal girls compared with 7.0% of pubertal girls (P < .001). None of the children tested positive for syphilis or HIV and no males had a STD. CONCLUSIONS: In our community, N gonorrhoeae testing in prepubertal girls can be limited to those with a vaginal discharge on examination unless other risk factors are present. The prevalence C trachomatis and Trichomonas in prepubertal girls is low and may be omitted from routine evaluations. All pubertal girls evaluated for sexual abuse should be tested for STDs because of the high prevalence of asymptomatic infection in this patient population.

Adolescent↗

PubMed1995

Cloning and expression of an endothelin receptor subtype B from human prostate that mediates contraction.

Recent evidence suggests a role for endothelin (ET) in contraction of human prostate [J. Urol. 149:495-499 (1993)]. Although both ETA and ETB receptors have been shown to mediate contraction of smooth muscle, the molecular identity of the contractile ETB receptor is controversial. The aim of this study was to examine the receptor subtype that mediates ET-induced contraction in prostate from patients with benign prostatic hyperplasia. Saturation binding with 125I-ET-1 and 125I-ET-3 in prostate stromal cells (PSC) indicated the presence of receptors with subnanomolar affinity for these radioligands, with equivalent receptor densities. Inhibition of specific 125I-ET-1 or 125I-ET-3 binding in PSC revealed a rank order of potency of ET-1 - ET-3 = sarafotoxin S6c >> BQ-123. These data are consistent with a predominance of ETB receptors in PSC. The functional effects of ET stimulation of PSC were examined in a collagen gel contraction assay. ET-1 and ET-3 caused contraction of underlying collagen gel matrices with EC50 values of 0.4 +/- 0.04 and 0.7 +/- 0.2 nM, respectively. To determine the molecular nature of the contractile ETB receptor in PSC, reverse transcription-polymerase chain reactions were conducted with oligonucleotide primers to the 5' and 3' ends of the coding sequence of the full length human ETB receptor. DNA sequence analysis of the 1.3-kilobase DNA product showed 99% homology to other human ETB receptor cDNAs. The encoded protein has a deduced amino acid sequence identical to that of other human ETB receptors, with the exception of two conservative substitutions. Expression of the PSC ETB cDNA in COS-7 cells resulted in a binding profile similar to that observed in parent cells. Polymerase chain reaction analysis revealed the presence of prepro-ET-1 mRNA in PSC. Collectively, these data indicate that PSC from patients with benign prostatic hyperplasia express ETB receptors that mediate ET-induced contraction.

Amino Acid Sequence↗

PubMed1995

Humeral fractures without obvious etiologies in children less than 3 years of age: when is it abuse?

OBJECTIVE: To determine the occurrence and frequency of abuse in children with humeral fractures without immediately obvious etiologies who are less than 3 years old and present with arm injuries. METHODS: A retrospective chart review was conducted of all children less than 3 years old treated for a humeral fracture at Children's Hospital Medical Center between July 1, 1990, and September 10, 1993. One hundred twenty-four charts of children with humeral fractures were reviewed for possible abuse using previously developed criteria. Charts were evaluated independently by the investigators. Consensus was reached on classification of each chart into the following categories: abuse, indeterminate, or not abuse. RESULTS: Abuse was diagnosed in 9 of 25 (36%) children less than 15 months of age, but in only 1 of 99 (1%) children older than 15 months (P < .05). Abuse was excluded in 91 of 124 (73%) children. No determination of abuse (indeterminate) could be made in 23 of 124 (18.5%) children. In children less than 15 months of age, abuse was diagnosed in 2 of 10 (20%) with supracondylar fractures and in 7 of 12 (58%) with spiral/oblique fractures. CONCLUSION: The prevalence of abuse in our children presenting with humeral fractures was much lower than in other published reports, especially in the children over the age of 15 months. However, we found a higher prevalence of supracondylar fractures associated with abuse than those same reports. Given these findings, abuse should be considered in all children less than 15 months of age with humeral fractures, including those with supracondylar fractures. The majority of humeral fractures in children are accidental, especially beyond the age of 15 months.

Child Abuse↗

PubMed1994

Molecular cloning, characterization, and tissue distribution of rat lipopolysaccharide binding protein. Evidence for extrahepatic expression.

LPS binding protein (LBP) is a glycoprotein present in normal serum that becomes markedly elevated during acute phase responses. LBP has been reported to greatly potentiate host responses to endotoxin or LPS. Therefore, LBP may play a critical role in the body's response to injury and infection. Little is known about the factors regulating production of LBP. To investigate the regulation of LBP expression, we have cloned the full-length cDNA for rat LBP. The deduced amino acid sequence of rat LBP was highly homologous with that reported for rabbit and human LBP. The sequence of rat LBP further refines the conserved regions found within the family of proteins that bind LPS; this family is comprised of bactericidal permeability-increasing protein and LBP from multiple species. Use of the rat LBP cDNA clone for Northern blot analysis reveals that LBP mRNA levels are markedly up-regulated in liver during acute phase responses. However, in contrast to previous reports, we also find evidence of extrahepatic expression of LBP under these induced conditions. The presence of LBP mRNA in activated tissues other than liver suggests that LBP may play a larger role in local tissue responses to LPS than previously appreciated.

Acute-Phase Proteins↗

PubMed1994

Differential induction of nitric oxide synthase in hepatocytes during endotoxemia and the acute-phase response.

OBJECTIVE: Nitric oxide (NO) is a potent biologic mediator produced by hepatocytes following exposure to cytokines and lipopolysaccharide (LPS). These cytokines are also known to regulate induction of the hepatic acute-phase response. The objective of this study was to determine whether inducible nitric oxide synthase (iNOS), the enzyme that produces NO, is expressed as part of the hepatic acute-phase response. DESIGN: The gene expression for inducible NOS (iNOS) as well as alpha 1-acid glycoprotein (AGP), an established acute-phase reactant, was measured by Northern blot analysis in rat hepatocytes in vivo during endotoxemia (LPS injection) and during the acute-phase response produced by hindlimb turpentine injection. Hepatocyte iNOS messenger RNA (mRNA) levels were correlated with iNOS activity and circulating plasma nitrite and nitrate levels. In vitro, iNOS and AGP mRNA levels were determined in cultured hepatocytes stimulated with interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), or dexamethasone. RESULTS: The AGP mRNA levels were increased in vivo following both LPS and turpentine injection, while iNOS expression was induced only by LPS injection. Hepatocyte iNOS activity and plasma nitrite and nitrate levels also increased after LPS treatment. In vitro, the cytokine combination IL-6, IL-1 beta, and TNF-alpha induced hepatocyte iNOS expression but had minimal effects on AGP in the absence of dexamethasone. Addition of dexamethasone alone markedly increased AGP mRNA levels, with further increases seen with TNF-alpha or IL-1 beta addition. In contrast, dexamethasone decreased iNOS expression. CONCLUSION: The results show that hepatocyte iNOS expression is not part of the acute-phase response induced by remote inflammation and indicates that iNOS is differentially regulated from the acute-phase reactant, AGP.

Acute-Phase Reaction↗

PubMed1994

T cell long-term hyporesponsiveness follows antigen receptor engagement and results from defective signal transduction.

T cell receptor (TCR)-mediated stimulation of T hybridomas leads to cell activation and lymphokine production that is followed by a long-term hyporesponsiveness. To investigate the biochemical events involved in the induction and maintenance of this antigen receptor hyporesponsiveness or anergy, we have expressed a G protein/PLC beta 1-coupled muscarinic subtype 1 acetylcholine receptor in a murine T cell hybrid. Transfected cells were capable of responding to both muscarinic agonists and TCR ligands by inducing interleukin-2 secretion that was sensitive to cyclosporin A and dexamethasone. Both receptors induced tyrosine kinase (TK) activity, but muscarinic stimulation did not affect tyrosine phosphorylation of PLC gamma 1, nor did the TK inhibitor, herbimycin, block muscarinic receptor-mediated calcium mobilization. These data indicate that in T cells, the muscarinic receptor mediates T cell effector functions by regulating a TK-independent proximal pathway which later converges with the TCR pathway. Using these cells, we have explored the long-term consequences of T cell stimulation via antigen or muscarinic receptors. Our results show that hyporesponsiveness specifically follows TCR engagement and appears to result from a defect in the early signal transduction initiated by TCR cross-linking. A study of TCR-mediated signaling supports this model by showing that tyrosine phosphorylation and calcium mobilization are deficient in hyporesponsive T cells.

Animals↗