Costimulation with dexamethasone and prostaglandin E2: a novel paradigm for the induction of T-cell anergy.

In this report, data are presented which indicate that anti-CD3 mAb-stimulated human peripheral blood T-cells treated with both dexamethasone (DEX) and prostaglandin E2 (PGE2) become anergic. This anergy can be reversed by the addition of IL-2. Further, experiments were performed to investigate this T-cell anergy. The results show that addition of DEX and PGE2 to anti-CD3 mAb-stimulated T-cells inhibits the induction of p56lck but not p59fyn kinase activity nor is the tyrosine phosphorylation of PLC gamma altered appreciably. Additionally, this treatment of anti-CD3 mAb-stimulated T-cells also results in decreased tyrosine phosphorylation of ERK1, suggesting that the Ras activation pathway may be inhibited. Interestingly, the induction of T-cell anergy is reproduced when an agonist for the cAMP-independent EP3 subtype of the PGE2 receptor is substituted for PGE2. Thus, while the mechanisms responsible for the dual action of DEX and PGE2 on the induction of T-cell anergy is unknown, these data suggest that a cAMP-independent mechanism may be involved. These data indicate that a state of anergy can be induced in normal human T-cells by the activation of these cells in the presence of physiologic concentrations of DEX and PGE2.

T cell receptor-mediated signaling is defective in T cells obtained from patients with primary intracranial tumors.

It has been well established that patients with malignant glioblastomas exhibit T cell anergy. In this report, we further investigate the nature of this T cell anergy. The results demonstrate that tumor size but not location correlates with decreased mitogen or anti-CD3 mAb responsiveness of T cells obtained from patients. Stimulation of the TCR/CD3 complex on these patients' T cells revealed defects in early transmembrane signaling. Both PHA and anti-CD3 mAb activated PBL and T cells obtained from patients exhibited a marked decrease in the tyrosine phosphorylation of a number of proteins. In particular, decreased phosphorylation of pp100 and phospholipase Cgamma1 (PLCgamma1) was observed. In addition, PLCgamma1 and p56(lck) protein levels were dramatically reduced in T cells obtained from patients harboring a glioma. In contrast, the protein levels of p59(fyn) were normal or only slightly reduced in T cells obtained from patients with gliomas. Quantitation of free intracellular calcium concentrations ([Ca2+]i) after mitogen (PHA) stimulation or ionomycin treatment of T cells obtained from patients revealed that they mobilize less calcium than do T cells obtained from normal subjects. Stimulation of T cells obtained from patients with PMA and ionomycin, which should bypass the requirement for PLCgamma1 activation as well as directly activate the p21(ras) signaling pathway, did not restore the proliferative capacity of these T cells to normal levels. These results indicate that the anergy observed in T cells obtained from these patients is a consequence of one or more defects in the early transmembrane signaling events associated with TCR/CD3 stimulation.

Prevalence of antibodies to arenaviruses in rodents from the southern and western United States: evidence for an arenavirus associated with the genus Neotoma.

The objectives of this study were to extend our knowledge of the geographic distribution and rodent host range of arenaviruses in North America. Sera from wild rodents collected from the southern and western United States were tested for antibody against Tamiami, Pichinde, Junin, and lymphocytic choriomeningitis viruses, using an indirect fluorescent antibody test. Antibody to at least one arenavirus was found in 220 (3.1%) of 7,106 rodents tested. The antibody-positive animals included Mus musculus from Florida and Texas; Neotoma albigula from Arizona, Colorado, and New Mexico; N. fuscipes and N. lepida from California: N. mexicana from Arizona, New Mexico, and Utah; N. stephensi from Arizona and New Mexico; and Oryzomys palustris and Sigmodon hispidus from Florida. Sigmodon hispidus seropositive for Tamiami virus were found only in Florida (156 [27.0%] of 578 tested), although 463 hispid cotton rats from outside that state were examined. High-titered antibodies to Tamiami virus were present in sera from S. hispidus, (geometric mean antibody titer [GMAT] of 1:792), whereas sera from Neotoma spp. reacted at high titer to both Tamiami (GMAT = 1:905) and Pichinde (GMAT = 1:433) viruses. The results suggest that arenaviruses are widely distributed in the southern United States and that one or more indigenous arenaviruses are associated with Neotoma spp. in North America.

Dexamethasone and prostaglandin E2 modulate T-cell receptor signaling through a cAMP-independent mechanism.

One possible explanation for the link between stress and increased incidence of infection can be attributed to concomitant increases in levels of glucocorticoids (GS) and prostaglandin E2 (PGE2), both of which possess potent immunoregulatory activities. We have previously demonstrated that concentrations of PGE2 and the synthetic glucocorticoid, dexamethasone (DEX), which individually do not inhibit human T-cell responsiveness to anti-CD3 monoclonal antibody (mAb), act synergistically to inhibit IL-2 secretion and subsequent T-cell proliferation. In the present paper, we demonstrate that treatment of anti-CD3 mAb-stimulated T-cells with low (10(-8) and 10(-9) M) concentrations of DEX and PGE2 results in the inhibition of steady-state levels of IL-2 mRNA. Initial studies to elucidate the biochemical mechanisms involved indicate that the inhibitory effects of DEX and PGE2 cannot be correlated with increased levels of intracellular cAMP or the induction of apoptosis. However, the data indicate that DEX and PGE2 when added together interrupt anti-CD3 mAb-induced tyrosine phosphorylation of substrate proteins. Furthermore, the synergistic effect of DEX and PGE2 is mimicked by agonists for the cAMP-independent EP3 subtype of the PGE2 receptor. These data suggest that DEX and PGE2 elicit cAMP-independent signaling pathways which interact to inhibit the T-cell receptor-linked signal transduction cascade in anti-CD3 mAb-stimulated T-cells.

Isolation of black creek canal virus, a new hantavirus from Sigmodon hispidus in Florida.

Numerous rodents were trapped for serologic and virologic studies following the identification of a hantavirus pulmonary syndrome (HPS) case in Dade County, Florida. Cotton rats (Sigmodon hispidus) were the most frequently capture rodent and displayed the highest seroprevalence to a variety of hantavirus antigens. Hantavirus genome RNA was detected in all the seropositive cotton rats tested, using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A virus was isolated from tissues of two seropositive cotton rats by cultivation of lung and spleen homogenates on Vero E6 cells. Nucleotide sequence information obtained by direct RT-PCR and the serologic relationships of this virus with the other hantaviruses indicate that this virus, Black Creek Canal virus, represents a new hantavirus distinct from the previously known serotypes.

Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells.

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind 125I-IGF-I with an affinity of 1.77 nM (Kd) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the beta chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.

Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome.

A previously unrecognized disease, hantavirus pulmonary syndrome, was described following an outbreak of severe, often lethal, pulmonary illness in the southwestern United States in May-June, 1993. We have now studied the morphologic features of the causative agent, Sin Nomber virus (SNV), by thin section electron microscopy and immunoelectron microscopy of infected Vero E6 cells. SNV virions were roughly spherical and had a mean diameter of 112 nm. They had a rather dense envelope and closely apposed fine surface projections, 7 nm in length. Filamentous nucleocapsids were present within virions. Viral inclusion bodies were present in the cytoplasm of infected cells; these appeared granular or filamentous, depending on the plane of section. All of these characteristics were similar to published descriptions of other hantaviruses; however, unlike all other hantaviruses and virtually all other member viruses of the family Bunyaviridae which bud upon smooth intracytoplasmic membranes, SNV budding occurred almost entirely upon the plasma membrane of infected cells. Virus budding was associated with the formation of long 28 nm diameter tubular projections. Occasional elongated 47 nm diameter virus-like particles were seen to bud upon intracytoplasmic membranes. As shown by immunoelectron microscopy, viral antigens were localized over virions, inclusions, and tubular projections associated with virion morphogenesis.

Isolation of the causative agent of hantavirus pulmonary syndrome.

Investigation of a recent outbreak of acute respiratory illness in the southwestern United States resulted in the recognition of a new disease, hantavirus pulmonary syndrome (HPS) with high mortality. Different animals and cell lines were used in attempts to isolate the causative agent. A previously unknown hantavirus was passaged in laboratory-bred deer mice, recovered from lung tissues of a deer mouse, Peromyscus maniculatus, and propagated in the E6 clone of Vero cells. Virus antigen was readily detected in the infected cells by an indirect immunofluorescence assay, using convalescent-phase sera from HPS patients. By electron microscopy, the virus was shown to have the typical morphologic features of members of the genus Hantavirus, family Bunyaviridae. Virus sequences corresponded to those previously detected by a nested reverse transcriptase-polymerase chain reaction assay of hantavirus-infected specimens from rodents and humans. This newly recognized virus, the etiologic agent of HPS, has been tentatively named Muerto Canyon virus.

Ro 32-0432, a selective and orally active inhibitor of protein kinase C prevents T-cell activation.

Several lines of circumstantial evidence support the assumption that protein kinase C (PKC) activation together with elevated levels of cytosolic Ca++ are necessary for T-cell activation and proliferation in response to a physiological stimulus, i.e., MHC class II restricted antigen presentation. By using a potent, cell-permeable and selective inhibitor of PKC, Ro 32-0432, we have tested this hypothesis. Ro 32-0432 inhibits interleukin-2 (IL-2) secretion, IL-2 receptor expression in, and proliferation of, peripheral human T-cells stimulated with phorbol ester together with phytohemagglutin or anti-CD3, but does not inhibit IL-2 induced proliferation in cells already stimulated to express IL-2 receptors. Proliferation of the influenza peptide antigen HA 307-319-specific human T-cell clone (HA27) after exposure to antigen-pulsed autologous presenting cells was also inhibited by Ro 32-0432. Oral administration of Ro 32-0432 inhibited subsequent phorbol ester-induced edema in rats demonstrating the systemic efficacy of the compound to inhibit PKC-driven responses. Induction of more physiologically T-cell driven responses such as host vs. graft responses and the secondary paw swelling in adjuvant-induced arthritis were also inhibited by Ro 32-0432. These data demonstrate the crucial role for PKC in T-cell activation and that selective p.o. bioavailable PKC inhibitors are efficacious in preventing T-cell driven chronic inflammatory responses in vivo. Inhibition of PKC represents an important mechanistic approach to prevent T-cell activation and compounds of this class may have important therapeutic applicability to chronic inflammatory and autoimmune diseases.

Improved specificity of testing methods for filovirus antibodies.

An epizootic among monkeys imported into the United States created an immediate need for detection of antibodies to filoviruses. Thousands of samples were submitted to the Centers for Disease Control and Prevention for testing. Problems of sensitivity and specificity existed in the methods available for these assays. The experiments described in this report resulted in improved methods for the detection of antibodies to filoviruses, both for indirect fluorescent antibody assays (IFA) by standardizing methods and the Western blot (WB) by minimizing antigen load and by incorporating skim milk in diluents.

Ebola protein analyses for the determination of genetic organization.

Amino-acid sequencing of the purified major nucleoprotein (NP), VP35 and VP40 from purified Ebola virus proved that they are the protein products of the first three genes, and that the open reading frame (ORF) of the NP begins at nucleotide 470. Because of the many unusual features of the ORFs of Ebola virus, we thought that our conclusions should be substantiated. Comparisons of in vitro-translation products to purified viral proteins were used to demonstrate conclusively that the NP, VP35 and VP40 were the protein products of genes one, two, and three, respectively. Studies using antibodies to synthetic peptides matching the N- and C-termini of the deduced sequences from these genes confirmed these conclusions and that the ORF for the NP begins at nucleotide 470. Subsequent studies confirmed that VP30 is encoded by the fifth gene.

Inhibitors of protein kinase C. 3. Potent and highly selective bisindolylmaleimides by conformational restriction.

The protein kinase inhibitor staurosporine has been used to design a series of selective bisindolylmaleimide inhibitors of protein kinase C (PKC). Guided by molecular graphics, conformational restriction of the cationic side chain has led to ATP competitive inhibitors of improved potency and selectivity. Two compounds have been further evaluated and were shown to inhibit PKC of human origin and prevent T-cell activation in a human allogeneic mixed lymphocyte reaction. One of these compounds was orally absorbed in mice and antagonized a phorbol ester induced paw edema in a dose-dependent manner. This compound also selectively inhibited the secondary T-cell mediated response in a developing adjuvant arthritis model in rats and provides evidence for the potential use of PKC inhibitors as therapeutic immunomodulators.

Suppression of high affinity IL-2 receptors on mitogen activated lymphocytes by glioma-derived suppressor factor.

Previously we have reported that human glial tumor cells secrete a factor(s) which suppresses the mitogen responsiveness of normal human peripheral blood lymphocytes (PBL) in a dose dependent manner. In this study we extend these observations and explore the possible mechanisms by which glioma-derived suppressor factor(s) (GSF) modulates lymphocyte reactivity. Preincubation of lymphocytes with GSF for 2 hrs induces suppression of lymphocyte mitogen responsiveness. GSF also inhibits production of interleukin-2 (IL-2) by mitogen activated human T-cells. Addition of delectinated or recombinant IL-2 to mitogen activated human T-cells in the presence of GSF does not restore the normal proliferative response of these cells. These findings suggest that GSF induces a defect in the expression of the receptor for IL-2 (IL-2R) on activated T-cells. Binding studies with radiolabeled IL-2 demonstrated that GSF suppresses and in some cases completely inhibits the expression of functional high affinity IL-2R on activated T-cells, thereby, preventing association of IL-2R with its receptor and the subsequent progression of the cell into the proliferative stage of the cell cycle. These cellular defects induced by GSF closely parallel the observed defects noted in T-cells obtained from patients with gliomas, indicating that the factors elicited from glial tumors may be responsible for the immunological deficits observed in patients with primary malignant intracranial tumors.

Inhibitors of protein kinase C. 2. Substituted bisindolylmaleimides with improved potency and selectivity.

A hypothetical mode of inhibition of protein kinase C (PKC) by the natural product staurosporine has been used as a basis for the design of substituted bisindolylmaleimides with improved potency over the parent compound. Structure-activity relationships were consistent with the interaction of a cationic group in the inhibitor with a carboxylate group in the enzyme, and the most potent compound had a Ki of 3 nM. The inhibitors were competitive with ATP but inhibited cAMP-dependent protein kinase (PKA) only at much higher concentrations despite the extensive sequence homology between the ATP-binding regions of PKA and PKC. Three compounds were evaluated further and found to inhibit a human allogeneic mixed lymphocyte reaction pointing to the potential utility of PKC inhibitors in immunosuppressive therapy. One of these compounds was orally absorbed in the rat and represents an attractive lead in the development of PKC inhibitors as drugs.

A novel conformationally restricted protein kinase C inhibitor, Ro 31-8425, inhibits human neutrophil superoxide generation by soluble, particulate and post-receptor stimuli.

A novel, bis-indolylmaleimide, Ro 31-8425, bearing a conformationally restricted side chain, inhibits protein kinase C isolated from rat brain and human neutrophils with a high degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. It also inhibits phorbol ester-induced intracellular events known to be mediated by protein kinase C (p47 phosphorylation in intact platelets, CD3 and CD4 down-regulation in T-cells). Ro 31-8425 inhibited superoxide generation in human neutrophils activated by both receptor stimuli (formyl-methionyl-leucylphenylalanine, opsonized zymosan, IgG and heat aggregated IgG) and post-receptor stimuli (1,2-dioctanoylglycerol and fluoride). The compound also blocked antigen driven, but not IL-2 induced, T-cell proliferation. These results support a central role for protein kinase C in the activation of the respiratory burst and antigen-driven T-cell proliferation.

Monoclonal antibodies to three strains of hantaviruses: Hantaan, R22, and Puumala.

Thirty hybrid cell lines that produce monoclonal antibodies to three strains of hantaviruses have been generated and characterized. One clone specific to Hantaan 76-118 strain, four clones specific to Rattus strains and one clone specific to Puumala virus have been identified. Most of the monoclones produced antibodies specific to nucleoproteins. Only two monoclones were found to produce glycoprotein specific, neutralizing antibodies. The immunofluorescent (IFA) staining patterns of the monoclonal antibodies show consistent correlation with viral protein specificities as described for other hemorrhagic fever viruses. Cross-reactivity studies with hantaviruses tested demonstrate conserved antigenic sites on nucleoproteins among these hantaviruses tested. Puumala specific monoclones, produced for the first time, reveal both conserved and strain specific sites on the viral nucleoproteins of the Scandinavian virus.

Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity.

Clarification of the precise role of protein kinase C (PKC) in cellular functional responses has been hampered by a lack of potent, selective inhibitors. The structural lead provided by staurosporine, a potent but non-selective protein kinase (PK) inhibitor, was used to derive a series of bis(indolyl)maleimides of which the most potent, Ro 31-8425 (I50: PKC = 8 nM) showed 350-fold selectivity for PKC over cAMP-dependent protein kinase. Ro 31-8425 antagonised cellular processes triggered by phorbol esters (potent, specific PKC activators) and inhibited the allogeneic mixed lymphocyte reaction, suggesting a role for PKC in T-cell activation. Methylation of the primary amine in Ro 31-8425 produced an analogue. Ro 31-8830 which, when administered orally, produced a dose-dependent inhibition of a phorbol ester-induced paw oedema in mice (minimum effective dose = 15 mg/kg). Ro 31-8830 also selectively inhibited the secondary inflammation in a developing adjuvant arthritis model in the rat. The results presented here suggest that these selective inhibitors of PKC may have therapeutic value in the treatment of T-cell-mediated autoimmune diseases.

K252a is a potent and selective inhibitor of phosphorylase kinase.

The inhibition of phosphorylase kinase by a number of protein kinase inhibitors was examined. Both K252a and staurosporine are potent inhibitors of phosphorylase kinase with IC50 values of 1.7 nM and 0.5 nM respectively. K252a shows a 300-fold selectivity for this enzyme over protein kinase C whereas staurosporine shows only a 20-fold selectivity for phosphorylase kinase. In contrast, the Roche bis-indolyl maleimides inhibit phosphorylase kinase with IC50 values of approximately 1 microM and are highly selective for protein kinase C.