Interferon-induced protein 10 and interleukin 8. C-X-C chemokines present in proliferative diabetic retinopathy

From May through November 1997, 1,011 samples of raw milk from bulk storage tanks were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. The samples originated from 1,011 different dairy herds located throughout the Netherlands. O157 VTEC was not isolated from any of the milk samples examined. Additionally, survival of O157 VTEC in raw and UHT-sterilized cow's milk at 7 and 15 degrees C was studied, both in the absence and presence of an activated lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS). Results indicated that the O157 VTEC strain tested was able to grow in raw milk at 7 degrees C as well as at 15 degrees C. Naturally occurring amounts of thiocyanate and hydrogen peroxide in the raw milk tested were not sufficient to activate the LPS. Although the LPS exhibited an antimicrobial activity against O157 VTEC in LPS-activated sterilized milk, O157 VTEC populations were not (or not as obviously) reduced in LPS-activated raw milk. Possibly background microflora were more sensitive to the LPS than the O157 VTEC test strain. It was concluded that raw milk contaminated with O157 VTEC will remain a hazard if kept at 7 or 15 degrees C. Effective pasteurization and avoiding postpasteurization contamination are necessary to ensure the safety of milk.     

Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colony-stimulating factor, and interferon-gamma release from alveolar macrophages in asthma

In the present study, we examined denervation-induced changes in the sensitivity of hypothalamic postsynaptic serotonin1A (5-HT1A) receptor function with respect to changes in the dose-dependent elevation in plasma hormones [adrenocorticotropic hormone (ACTH), corticosterone, prolactin, oxytocin, prolactin, renin and vasopressin] by the 5-HT1A agonist 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT). Rats received intracerebroventricular (i.c.v.) injections of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) or vehicle (0.1% ascorbate in saline) 3 weeks before challenge with increasing doses of 8-OH-DPAT (0, 10, 50 or 200 micrograms/kg s.c.). The effectiveness of 5,7-DHT-induced destruction of serotonergic neurons was confirmed by a 93% reduction in [3H]paroxetine-labeled 5-HT uptake sites in the hypothalamus. No changes in basal levels of ACTH, corticosterone, oxytocin, prolactin, renin and vasopressin were observed in rats that received i.c.v. 5,7-DHT injections. The dose-response curves for 8-OH-DPAT-induced elevations of plasma corticosterone and prolactin levels were shifted to the left in rats treated with 5,7-DHT, whereas no significant difference in the ACTH dose-response curve was observed between rats treated with vehicle and rats treated with 5,7-DHT. In contrast, the maximal oxytocin response to 8-OH-DPAT was attenuated in rats treated with 5,7-DHT. A 5,7-DHT-induced decline in the synthesis of oxytocin could explain this phenomenon. Although 8-OH-DPAT did not increase plasma levels of renin or vasopressin in rats treated with vehicle, 8-OH-DPAT produced an elevation (75%) in plasma renin concentration but not in vasopressin levels in rats that received i.c.v. injections of 5,7-DHT. No change was observed in [3H]8-OH-DPAT labeled 5-HT1A receptors in the hypothalamus. In summary, denervation of hypothalamic serotonergic nerve terminals produces supersensitivity of some neuroendocrine responses to 8-OH-DPAT independent of changes in the density of hypothalamic 5-HT1A receptors.     

Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-gamma inducible protein-10 and monocyte chemotactic protein-1

In viral meningitis the inflammatory response involves activated T cells and monocytes which are recruited into the subarachnoid space. To identify the chemotactic signals attracting the cells to the site of infection in the meninges, we measured the levels of two CXC chemokines, interferon-gamma (IFN-gamma) inducible protein (IP)-10 and monokine induced by IFN-gamma, four CC chemokines, monocyte chemotactic protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, as well as the cytokines interleukin (IL)-15 and IL-16 in the cerebrospinal fluid (CSF) of patients suffering from viral meningitis. The results point to an involvement of two chemokines, MCP-1 and IP-10, since (1) unlike the other cytokines, MCP-1 and IP-10 were present in 97% and 79% of the CSF, respectively, at concentrations sufficient to induce chemotaxis of mononuclear cells; (2) more than 90% of the CSF of viral meningitis induced chemotaxis of peripheral blood mononuclear cells (PBMC) and all of them induced chemotaxis of activated T cells, and (3) the CSF-mediated chemotaxis of PBMC was inhibited by anti-MCP-1 antibodies and chemotaxis of activated T cells was abolished by the combination of anti-MCP-1 and anti-IP-10 antibodies. Our data provide evidence that MCP-1 and IP-10 lead to accumulation of activated T cells and monocytes in the CSF compartment in viral meningitis.     

Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF-alpha

In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo. Injection of TNF-alpha into s.c. air pouches led to a rapid, transient accumulation of leukocytes. Maximal accumulation of leukocytes in the air pouch was observed at between 2 and 4 h after injection of TNF-alpha. The cellular exudate comprised predominantly neutrophils, with smaller numbers of eosinophils and mononuclear phagocytes also being recruited. However, lymphocyte recruitment was not observed. TNF-alpha injection induced a time-dependent increase in the levels of immunoreactive macrophage inflammatory protein (MIP)-2, MIP-1alpha, and JE in the pouch exudate as well as increased steady-state mRNA levels of KC, MIP-2, MIP-1alpha, and JE in the tissue lining the s.c. pouch and of MIP-2, MIP-1alpha, and JE in the exudate cell population. Passive immunization with specific Abs directed against each of these chemokines significantly inhibited the accumulation of neutrophils, mononuclear phagocytes, and eosinophils in response to TNF-alpha. Taken together, these data demonstrate the existence of a chemokine network in vivo involving at least four individual chemokines that regulates recruitment of the major peripheral blood granulocytes and mononuclear phagocytes to s.c. sites during acute inflammation. To our knowledge, these data are also the first demonstration that the C-C chemokine JE is involved in neutrophil recruitment in a physiologic system in vivo.     

Interleukin-17 and interferon-gamma synergize in the enhancement of proinflammatory cytokine production by human keratinocytes

OBJECTIVES: To investigate the correlation of epidermal growth factor receptor (EGFR) expression and its ligands EGF and transforming growth factor-alpha (TGF-alpha) with disease outcome in a cohort of patients with superficial bladder cancer. METHODS: Tumor samples of 21 patients with transitional cell carcinoma of the bladder were analyzed by immunohistochemistry for expression of EGFR, EGF, and TGF-alpha. Disease-related events were recorded during a routine clinical follow-up and analyzed for possible correlation with the expression status of the above-mentioned proteins. RESULTS: All Stage pT1 transitional cell carcinomas expressed EGFR, and 10 of 21 (48%) tumors showed focal areas of strong EGF and/or TGF-alpha expression. Of these, 80% with EGF positivity (8 of 10) had recurrences, whereas only 9% of patients without EGF staining (1 of 11) did so. The same pattern was observed with TGF-alpha. A strong association was confirmed between EGF/TGF-alpha positivity and tumor recurrence (P <0.005). We also found that EGF and TGF-alpha were expressed in stroma and/or around the vessels of tumor tissue in 48% and 38% of the tumors, respectively. No association was found between the recurrence rate/vascular invasion and the stromal/vascular wall expression of the growth factors. CONCLUSIONS: Expression of EGF and TGF-alpha is correlated with tumor recurrence. Also, there is the ability of vessel walls to express EGF and TGF-alpha in superficial bladder cancer. Further clarification of the impact of this expression on angioinvasion of tumor cells may be helpful in understanding the nature of local invasion and metastasis.     

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.     

Contribution of the CXC chemokines IP-10 and Mig to the antitumor effects of IL-12

DMP 728 showed a dose-dependent inhibition of platelet aggregation at doses of 0.05 to 0.9 mg per subject, with a maximal inhibition (> 90%) of platelet aggregation at doses of 0.9 mg per subject and higher. Minimal changes in bleeding time from baseline were observed at doses up to 0.6 mg per subject. At the 0.9 mg/subject dose level, bleeding time was prolonged by approximately twofold to threefold above the baseline. At higher doses (1.5 mg/subject to 3.9 mg/subject), bleeding time prolongation was > 30 minutes during the infusion. In all dose groups, bleeding times returned to the control value within 8 hours after cessation of the infusion. Maximum plasma concentration and area under the curve of DMP 728 increased linearly and proportionally to the dose. No clinical changes in vital signs, 12-lead electrocardiograms, physical examinations, coagulation tests, or stool hemoccult tests were observed at any of the doses. In conclusion, DMP 728 is a potent antiplatelet agent and well tolerated at doses ranging from 0.05 to 3.0 mg/subject.     

High-dose interferon-gamma alters the distribution of B lymphocytes and macrophages in rat spleen and lymph nodes

Continuous intravenous infusion of rat interferon-gamma (IFN-gamma) for 3 days provokes profound alterations of splenic architecture in LEW rats. The marginal zone of the white pulp is almost totally depleted of B lymphocytes and the follicles are reduced to small remnants. IgM kappa + plasmablasts and plasma cells increase substantially in the outer periarteriolar lymphatic sheath (PALS) and in the splenic red pulp. In addition, marginal metallophilic and marginal zone macrophages are augmented, partially by proliferation. It is discussed whether the activation and proliferation of these macrophages prevent replenishment of the marginal zone and follicles with recirculating B cells. Changes in B lymphocyte and medullary macrophage distribution are also present in submandibular and mesenteric lymph nodes.     

Immunity to Toxoplasma gondii induced in vitro in non-immune mouse macrophages with specifically immune lymphocytes

Male and female CBA mice were used to study in vitro the mechanisms involved in the development and expression of cellular immunity to toxoplasma infection. The lag phase preceding toxoplasma division was delayed in nonimmune macrophages obtained from peritoneal cavities stimulated with thioglycollate. Specific anti-toxoplasma activity was conferred on nonimmune macrophages incubated with toxoplasma-immune spleen lymphocytes and soluble toxoplasma antigen. Treatment of immune spleen cell populations with anti-theta serum plus complement abolished completely their activity of conferring anti-toxoplasma activity on nonimmune macrophages, demonstrating that the essential cells were T lymphocytes. The mediator(s) responsible for the acquisition of immunity to toxoplasma in the nonimmune macrophages were soluble. Heat-inactivated, toxoplasm-immune macrophages of fibroblasts. The findings are related to previous investigations of induced immunity in animals and man.     

Serum levels of sCD23, interleukin-10 and interferon-gamma in patients with coeliac disease

The role of cellular and humoral immunity coeliac disease was investigated by the measurement of serum levels of interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and soluble CD23 (sCD23). Coeliac disease was diagnosed by duodenal biopsy and response to a gluten-free diet (GFD). The results were compared with age and sex-matched patients with non-specific upper gastrointestinal symptoms and normal duodenal histology. While the levels of serum IL-10 were significantly elevated (P < 0.01) in patients with coeliac disease taken as a whole, the levels of serum IFN-gamma were normal and sCD23 significantly decreased (P < 0.002). The median serum sCD23 was significantly lower in the coeliac disease patients not on a GFD compared with those asymptomatic on a GFD (P < 0.03) and the control group (P < 0.0004). The coeliac disease patients on a GFD also had significantly lower serum sCD23 and higher IL-10 compared with the control group (P < 0.01 and P < 0.015). There was no significant difference in the serum IL-10 between the coeliac disease patients on a GFD and those not on a GFD and between the latter and the control group. The low levels of serum sCD23 in coeliac disease suggest diminished humoral immunity and, conversely, exaggerated cellular immunity. The aetiology of the raised levels of IL-10 in coeliac disease is unclear and similar to that observed in patients with inflammatory bowel disease. However, this may represent a regulatory response to the elevated levels of proinflammatory cytokines described in coeliac disease. A combination of diminished sCD23 and raised IL-10 is clearly unusual as both are associated with Th2-type functions. The possible causes of this finding are discussed.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Failure of 2- and 10-meter radio waves to induce genetic damage in Drosophila melanogaster

Over the past 20 years, more than 300 patients have been anesthetized in the lateral sitting position during neurosurgical procedures in the posterior fossa and the cervical and upper thoracic spine. Since the patient can be placed quickly and easily in the horizontal position, the lateral sitting position has a number of advantages over the conventional sitting position, particularly in the treatment of arterial hypotension and venous air embolism. Furthermore, with the patient in the lateral horizontal position, the surgical procedure can be completed satisfactorily.     

Evaluation of NR-LU-10 with the Neoprobe gamma detector in a mouse model

1. The effects of diethyl maleate (DEM) on the cytotoxicity of phenyl-hydroquinone (PHQ) and other hydroquinones were studied in freshly isolated rat hepatocytes. 2. Addition of PHQ (0.5 or 0.75 mM) to hepatocytes resulted in dose-dependent cell death accompanied by the abrupt depletion of both GSH and protein thiols and the accumulation of phenyl-benzoquinone (PBQ). 3. Pretreatment with DEM (1.25 mM), which causes an abrupt depletion of cellular GSH in hepatocytes, delayed the onset of PHQ-induced cytotoxicity. The delay correlated with inhibition of PBQ formation. 4. Although the pH of the cell suspension was increased slightly (mean pH 0.18) by incubation under carbogen flow, the addition of DEM to the cell suspension inhibited both the increase in pH and the formation of PBQ from PHQ. 5. In hepatocyte suspensions without DEM, PHQ cytotoxicity was dependent on pH, and toxicity was associated with oxidation of PHQ and accumulation of PBQ. 6. Among other hydroquinones (0.5 mM), tert-butyl-hydroquinone-induced cytotoxicity was decreased by DEM (1.25 mM), but DEM did not affect the cytotoxicity of 2,5-di(tert-butyl)-1,4-benzohydroquinone. 7. PHQ-induced cytotoxicity correlated with the accumulation of PBQ in the cell, and the inhibition of PHQ-induced cytotoxicity by DEM correlated with pH-dependent changes in PBQ formation.     

Release of interleukin-12 in experimental Escherichia coli septic shock in baboons: relation to plasma levels of interleukin-10 and interferon-gamma

Interleukin (IL)-12 is thought to be a key factor for the induction of interferon gamma (IFN-gamma), a cytokine essential for the lethal effects of endotoxin. We report here on the release of the nonfunctional subunit of IL-12, p40, as well as biologically active heterodimeric IL-12, p70, after administration of a lethal (n = 5) or sublethal (n = 8) dose of live Escherichia coli to baboons. Remarkably, on lethal challenge, peak levels of p40 were observed at 3 hours that were about twofold lower than those elicited after sublethal challenge (2,813 +/- 515 pg/mL v 4,972 +/- 732 pg/mL, P < .05). This disparity was also observed, although to a lesser extent, for IL-12 p70 antigen, of which maximum levels of 91 +/- 47 pg/mL and 151 +/- 41 pg/mL were measured 6 hours after a lethal or sublethal dose of E coli, respectively. Circulating p70 antigen correlated with IL-12 biologic activity (r = 0.869; P < .001). When comparing lethal to sublethal conditions, lower peak levels of IL-12 on lethal E coli sharply contrasted with higher levels of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 observed in these animals. Lower IL-12 concentrations in the lethal group may have resulted in part from the enhanced production of IL-10, a known inhibitor of IL-12 synthesis in vitro, as peak levels of this cytokine 3 hours postchallenge inversely correlated with peak levels of IL-12, in particular p40 (r = -0.802; P < .01). Contrary to what might be expected if IFN-gamma were solely induced by IL-12, lethally challenged baboons generated threefold more IFN-gamma at 6 hours than those receiving a sublethal dose (P < .05). Moreover, higher levels of IFN-gamma were associated with lower p40/p70 ratios, suggesting that, in agreement with observations in vitro, IFN-gamma may have preferentially upregulated the release of p70 over p40. These data show that IL-12 is released in experimental septic shock in nonhuman primates and suggest that IL-10 and IFN-gamma are involved in the regulation of this release. Furthermore, this study indicates that the systemic release of IL-12 might be essential, but is not likely sufficient, to promote lethal production of IFN-gamma in sepsis.     

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta1 (TGF-beta1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-beta1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-beta1 on the melanogenic activity of B16/F10 cells.