Endometrial glands are necessary for conceptus implantation and growth. In the ovine uterine gland knockout (UGKO) model, blastocysts hatch normally but fail to survive or elongate. This peri-implantation defect in UGKO ewes may be due to the absence of endometrial glands or, alternatively, to the lack of certain epithelial adhesion molecules or the inability of the endometrium to respond to signals from the conceptus. Two studies were performed to examine these hypotheses. In study one, normal (n = 8) and UGKO (n = 12) ewes were mated at oestrus (day 0) with intact rams and their uteri were flushed 14 days after oestrus. Normal ewes (n = 4) were also flushed on 14 days after oestrus. Uterine flushes from bred normal ewes contained filamentous conceptuses (n = 7 of 8), whereas those from UGKO ewes contained no conceptus (n = 5 of 12), a growth-retarded, tubular conceptus (n = 6 of 12), or a fragmented, filamentous conceptus (n = 1 of 12). In all groups, expression of mucin 1 and integrin <em>alpha</em>(v), <em>alpha</em>(5), beta(<em>3</em>) and beta(5) was localized at the apical surface of the endometrial luminal epithelium with no detectable differences between normal and UGKO ewes. Uterine flushes from pregnant ewes, but not cyclic or UGKO ewes, contained abundant immunoreactive <em>interferon</em> tau and the cell adhesion proteins, osteopontin and glycosylation-dependent cell adhesion molecule one. In study two, UGKO ewes were fitted with uterine catheters 5 days after oestrus, infused with recombinant ovine <em>interferon</em> tau or control proteins from 11 to 15 days after oestrus, and underwent hysterectomy 16 days after oestrus. Expression of several <em>interferon</em> tau-stimulated genes (ISG17, STAT1, STAT2 and IRF-1) was increased in the endometrium from <em>interferon</em> tau-infused UGKO ewes. These results support the hypothesis that the defects in conceptus elongation and survival in UGKO ewes are due to the absence of endometrial glands and their secretions rather than to alterations in expression of anti-adhesive or adhesive molecules on the endometrial luminal epithelium or to the responsiveness of the endometrium to the conceptus pregnancy recognition signal.

We report preliminary characterization of a gene designated beta-R1, which is selectively expressed in response to interferon beta (IFN-beta) compared with IFN-alpha. In human astrocytoma cells, beta-R1 was induced to an equivalent extent by 10 IU/mL IFN-beta or 2500 IU/mL IFN-alphaalphaalpha Con1, or a mixture of IFN-alpha subtypes. IFN-alphaA, U4A, and U6A, which lack, respectively, p48, STAT1, JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-alpha and -beta receptor, also failed to express beta-R1. U1A cells are partially responsive to IFN-beta and IFN-alpha plus an additional component, which is more efficiently formed on induction by IFN-beta compared with IFN-alpha.

Human peripheral blood mononuclear cells, monocytes-macrophages and T-cells were stimulated with human recombinant <em>interferon</em>-gamma, <em>interferon</em>-<em>alpha</em> and phytohemagglutinin. The culture supernatants were analyzed for tryptophan, kynurenine, <em>3</em>-hydroxyanthranilic acid, anthranilic acid and neopterin by high performance liquid chromatography. Tryptophan was decreased and the four other compounds were increased in supernatants of peripheral blood mononuclear cells activated by <em>interferon</em>-gamma (250 U/ml), <em>interferon</em>-<em>alpha</em> (10.000 U/ml) and phytohemagglutinin (1 microgram/ml). After splitting of peripheral blood mononuclear cells by adherence, the monocytes and macrophages but not the T-cells degraded tryptophan upon stimulation by <em>interferon</em>-gamma in a dose dependent manner. Supernatants of phytohemagglutinin stimulated but not of resting T-cells were found to induce tryptophan degradation by macrophages, the active principle being neutralized by an antiserum for <em>interferon</em>-gamma. Thus phytohemagglutinin acts by activating T-cells to release <em>interferon</em>-gamma which in turn induces macrophages to degrade tryptophan. In all experiments the appearance of neopterin in the culture media was correlated to the observed tryptophan degradation.

Cytokines are released in the central nervous system following brain injury and disease. Several of those conditions are thought to involve the accumulation of extracellular glutamate at excitotoxic concentrations, and may involve compromised glial glutamate uptake. Using primary cultures of postnatal rat hippocampus, we studied the effect of three cytokines on astrocytic high-affinity glutamate uptake. After 24 hours incubation with either tumor necrosis factors <em>alpha</em> (TNF-<em>alpha</em>), <em>interferon</em> gamma (IFN-gamma) or interleukin-1 beta (IL-1 beta), astrocytic glutamate uptake was markedly attenuated in a dose-dependent manner. Cytokine effects were reversed by inhibition of nitric oxide synthase (NOS) using N omega-nitro-L-arginine (LNA), NG-monomethyl-L-arginine acetate (L-NMMA) or N omega-nitro-L-arginine methyl ester (LNAME). Moreover, application of the NO donors <em>3</em>-morpholinosydnonimine (SIN-1) and s-nitroso-n-acetylpenicillamine (SNAP) mimicked cytokine inhibition of glutamate uptake. These data suggest that cytokine release can inhibit astrocytic glutamate uptake through a pathway that involves the liberation of nitric oxide. Astrocytic glutamate uptake may thus be compromised under conditions that are known to cause cytokine release such as nervous system injury, inflammation and ischemia.

OBJECTIVE

Rheumatoid arthritis (RA) is a T-cell-mediated systematic disease and is usually accompanied by articular cartilage damage. In the present study, we explored the effects of bone marrow-derived mesenchymal stem cells (MSCs) and MSC-differentiated chondrocytes (MSC-chondrocytes) on the responses of antigen-specific T cells in RA to type II collagen (CII) to evaluate the potential therapeutic value of MSCs in RA treatment.

METHODS

The effects of both MSCs and MSC-chondrocytes on the proliferation, activation-antigen expression (CD69 and CD25) and cytokine production [interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-10 and IL-4] of CII-reactive T cells in RA patients were investigated with the stimulation of CII or otherwise. CD3/annexin V staining was used to evaluate T-cell apoptosis in the inhibition. The role of transforming growth factor-beta1 (TGF-beta1) underlying the inhibition was also investigated.

RESULTS

MSCs failed to elicit positive responses of CII-reactive T cells, whereas they significantly suppressed CII-stimulated T-cell proliferation and activation-antigen expression in a dose-dependent fashion without inducing T-cell apoptosis. The inhibition was observed even after MSCs were added as late as 3 days after the initiation of stimulation. Moreover, MSCs inhibited both CD4+ and CD8+ T cells from producing IFN-gamma and TNF-alpha, while they up-regulated the levels of IL-10 and restored the secretion of IL-4. TGF-beta1 was confirmed to play a critical role in the inhibition. Throughout our study, MSC-chondrocytes shared similar properties with MSCs.

CONCLUSIONS

Both MSCs and MSC-chondrocytes suppressed CII-reactive T-cell responses to CII in RA, which suggested that MSCs could be a potential candidate for RA treatment in future if further confirmed in vivo.

OBJECTIVE

A phase II trial that uses liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) in patients with relapsed osteosarcoma is underway. To determine if in vivo cytokine induction plays a role in the mechanism of action of L-MTP-PE, we investigated the circulating cytokine levels of 16 patients who were undergoing therapy.

METHODS

Patients had histologically proven osteosarcoma and pulmonary metastases that developed either during adjuvant chemotherapy or that were present at diagnosis and persisted despite chemotherapy. Patients were rendered disease-free by surgery. The major goal of the study was to improve the disease-free interval in this high-risk group. L-MTP-PE 2 mg/m2 was infused during a 1-hour period twice a week for 12 weeks, then once a week for 12 weeks. Serial blood samples were collected after L-MTP-PE administration and were assayed for cytokine levels (tumor necrosis factor-alpha [TNF alpha] interleukin-1 alpha [IL-1 alpha], IL-1 beta, IL-6, interferon-gamma [IFN-gamma], neopterin, C-reactive protein).

RESULTS

After the infusion of L-MTP-PE, there was rapid induction of circulating TNF alpha and IL-6. TNF alpha levels peaked 1 to 2 hours after infusion in 10 of 16 patients, whereas peak IL-6 levels were detected at 2 to 3 hours in all patients. Induction of circulating TNF alpha and IL-6 was evident only after the first dose of L-MTP-PE. Neither IL-1 alpha nor IL-1 beta was detected in the plasma. Neopterin levels increased at 24 hours postinfusion, which indicated macrophage activation, and were not related to the induction of circulating IFN-gamma. C-reactive protein was elevated in all patients at 24 hours and decreased by 72 hours. Unlike circulating TNF alpha and IL-6, elevations in C-reactive protein and neopterin could be detected throughout the treatment course.

CONCLUSIONS

It is concluded that L-MTP-PE has specific biologic effects in patients with osteosarcoma that may be important to the drug's immunostimulatory capacity and its effectiveness as an antitumor agent.

The presence of mRNA transcripts for cytokines in normal and neoplastic human breast tissue has been investigated. Using reverse transcriptase-linked polymerase chain reaction (RT-PCR), we have specifically screened for the following cytokines: interleukin (IL)-1<em>alpha</em>, IL-1beta, IL-2, IL-<em>3</em>, IL-4, IL-5, IL-6, IL-7, IL-8, tumour necrosis factor (TNF)-<em>alpha</em>, TNF-beta and <em>interferon</em> (IFN)-gamma. No significant differences in expression of IL-1<em>alpha</em>, IL-1beta, IL-4, IL-6, TNF-<em>alpha</em> or TNF-beta were observed between the 2 groups of tissues. However, there was a significant difference in expression of IL-8 transcripts (p = 0.0017) which was higher in the neoplastic population. Transcripts for IL-2, IL-<em>3</em>, IL-5, IL-7 and IFN-gamma were not detected in either group. There was no evidence of associations between cytokine expression and tumour histological grade, patient age or lymph node metastases. Correlating tumour types with specific cytokine transcripts revealed high expression of IL-8, and to a lesser extent, IL-8 and TNF-beta irrespective of tumour origin. Analysis of primary epithelial and stromal cultures derived from both types of tissue showed that increased levels of IL-8, but not IL-6, were secreted by cells obtained from tumours. Thus, breast tissue of both normal and neoplastic origin expresses a wide range of cytokines. Increased or aberrant expression of cytokines, in particular IL-8, may be involved in the development/progression of breast cancer.

The studies reviewed here evaluated the role cellular immune system components play in control of brucellosis by conducting comparative studies with brucella-resistant C57BL/10 or C57BL/6 mice and susceptible BALB/c mice. We have shown by both in vitro and in vivo studies that activation of macrophages with <em>interferon</em>-gamma (IFN-gamma) is an important factor for control of infection with B. abortus in the mouse model and that the mechanism of anti-brucella activity largely involved reactive oxygen intermediates. Differences in control of the organism by resistant and susceptible mice was not related to inherent differences in the ability of their macrophages to control infection either with or without IFN-gamma activation nor was it attributable to NK cells since we found no role for them in control of brucellosis in either mouse strain. However, relative resistance to brucellosis did correlate with increased production of IFN-gamma by CD4 T cells during the first weeks after infection while IL-10 contributed to susceptibility in BALB/c mice. Moreover, by <em>3</em> weeks post-infection splenocytes from the susceptible BALB/c mice failed to produce IFN-gamma and relied on TNF-<em>alpha</em> as well as CD8 T cells to control infection until the end of the plateau phase around 6 weeks post-infection when IFN-gamma production resumed and clearance began. In contrast, IFN-gamma was crucial for control throughout the infection in the more resistant C57BL/6 mice and the mice died in its absence by 6 weeks post-infection compared to 12 weeks for the more susceptible mice that relied on additional mechanisms of control. In contrast to the IFN-gamma knock-out mice, both beta2 microglobulin knock-out C57BL/6 mice, which do not express conventional MHC class I molecules and thus cannot present antigen to CD8 T cells, or perforin knock-out C57BL/6 mice, which have no T cell cytotoxic activity, controlled and cleared the infection as well as normal C57BL/6 mice. The hiatus of IFN-gamma production in BALB/c mice correlated with very high levels of total IL-12 and it was postulated that the lack of IFN-gamma was a consequence of p40 homodimer blocking activity. However, reduction of p40 IL-12 in vivo through administration of indomethacin reduced the infection without a concomitant measurable increase in IFN-gamma. Current studies are aimed at elucidating the mechanism of the IFN-gamma hiatus.

<em>Interferon</em>-gamma (IFN-gamma) is a proinflammatory cytokine that plays a pivotal role in pathology of diseases in the central nervous system (CNS), such as multiple sclerosis. However, the direct effect of IFN-gamma on neuronal cells has yet to be elucidated. We show here that IFN-gamma directly induces neuronal dysfunction, which appears as dendritic bead formation in mouse cortical neurons and enhances glutamate neurotoxicity mediated via <em>alpha</em>-amino-<em>3</em>-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptors but not N-methyl-D-aspartate receptors. In the CNS, IFN-gamma receptor forms a unique, neuron-specific, calcium-permeable receptor complex with AMPA receptor subunit GluR1. Through this receptor complex, IFN-gamma phosphorylates GluR1 at serine 845 position by JAK1.2/STAT1 pathway, increases Ca(2+) influx and following nitric oxide production, and subsequently decreases ATP production, leading to the dendritic bead formation. These findings provide novel mechanisms of neuronal excitotoxicity, which may occur in both inflammatory and neurodegenerative diseases in the CNS.

The pathogenesis of Langerhans cell histiocytosis (LCH) remains poorly understood. To further elucidate LCH pathogenesis, we analyzed the expression of 10 cytokines relevant to cellular recruitment and activation at the protein level in 14 patients and identified the lesional cells responsible for cytokine production in situ by immunohistochemistry. The cytokines investigated included the hematopoietic growth factors interleukin-<em>3</em> (IL-<em>3</em>), IL-7, and granulocyte-macrophage colony-stimulating factor (GM-CSF); the lymphocyte regulatory cytokines IL-2, IL-4, and IL-10; the inflammatory regulators IL-1<em>alpha</em> and tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>); and the effector cell-activating cytokines IL-5 and <em>interferon</em>-gamma (IFN-gamma). In all specimens, CD1a(+) histiocytes (LCH cells) and CD<em>3</em>(+) T cells produced large amounts of cytokines, creating a true cytokine storm. IL-2, IL-4, IL-5, and TNF-<em>alpha</em> were produced exclusively by T cells, whereas only IL-1<em>alpha</em> was produced by LCH cells. Equal numbers of LCH cells, T cells, and macrophages produced GM-CSF and IFN-gamma. Equal numbers of LCH cells and macrophages produced IL-10, whereas IL-<em>3</em> was produced by T cells and macrophages. IL-7 was only produced by macrophages. Eosinophils, present in some specimens, were partially responsible for the production of IL-5, IFN-gamma, GM-CSF, IL-10, IL-<em>3</em>, and IL-7. Expression of all cytokines, abundant in most biopsies, was irrespective of age, gender, or site of biopsy. These findings emphasize the role of T cells in LCH. The juxtaposition of T cells and LCH cells suggests that both cells interact in a cytokine amplification cascade, resulting from stimulation of autocrine and paracrine stimulatory loops. This cascade can be linked directly to the development of LCH through recruitment, maturation, and proliferation of LCH cells. The cytokines studied are known to be involved in the development of other characteristic features of LCH, such as fibrosis, necrosis, and osteolysis.

BACKGROUND

Galectin-<em>3</em> is a beta-galactoside-binding protein which is implicated in diverse physiological and pathological processes including human liver cirrhosis and a mouse lung fibrosis model. The aim of this study is to determine whether galectin-<em>3</em> is involved in human lung fibrosis.

METHODS

We measured galectin-<em>3</em> concentration in bronchoalveolar lavage fluid (BALF) and examined its expression in alveolar macrophages from patients with interstitial lung disorders using ELISA and immunohistochemical staining, respectively. Using monocyte/macrophage cell lines in vitro, we examined the effect of cytokines on galectin-<em>3</em> expression, and the opposite similarly by RT-PCR and Western blotting. Finally, we performed Micro Boyden chamber assay and Sircoll assay to determine whether galectin-<em>3</em> induces migration and collagen synthesis, respectively, in fibroblasts.

RESULTS

Galectin-<em>3</em> was specifically increased in BALF from patients with idiopathic pulmonary fibrosis (IPF) and interstitial pneumonia associated with collagen vascular disease (CVD-IP). Galectin-<em>3</em> levels in BALF seemed to be lower in IPF and CVD-IP patients receiving corticosteroid therapy. Alveolar macrophages from IPF patients expressed more galectin-<em>3</em> compared with those from control. Galectin-<em>3</em> expression was induced by tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN)-gamma in a monocytic cell line U9<em>3</em>7. Galectin-<em>3</em> also induced mRNA expression and protein production of TNF-alpha and interleukin (IL)-8 in a macrophage cell line THP-1. This lectin stimulated NIH-<em>3</em>T<em>3</em> fibroblast to induce migration and collagen synthesis in vitro.

CONCLUSIONS

These results suggest that galectin-<em>3</em> is involved in the pathogenesis of human IPF and CVD-IP by activating macrophages and fibroblasts.

An immunoperoxidase technique was used to examine IP-10 (<em>interferon</em>-gamma inducible protein 10), RANTES (regulated on activation normal T cell expressed and secreted), MCP-1 (monocyte chemoattractant protein-1), and MIP-1 <em>alpha</em> (macrophage inflammatory protein-1 <em>alpha</em>) in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into <em>3</em> groups according to the size of infiltrate. MIP-1 <em>alpha</em>+ cells were more abundant than the other chemokines with few MCP-1+ cells. The mean percent MIP-1 <em>alpha</em>+ cells was higher than the percent MCP-1+ cells (P = 0.02) in group 2 (intermediate size infiltrates) lesions from periodontitis subjects, other differences not being significant due to the large variations between tissue samples. Analysis of positive cells in relation to CD4/CD8 ratios showed that with an increased proportion of CD8+ cells, the mean percent MIP-1 <em>alpha</em>+ cells was significantly higher in comparison with the mean percent RANTES+ and MCP-1+ cells (P < 0.015). Endothelial cells were MCP-1+ although positive capillaries were found on the periphery of infiltrates only. Keratinocyte expression of chemokines was weak and while the numbers of healthy/gingivitis and periodontitis tissue sections positive for IP-10, RANTES and MCP-1 reduced with increasing inflammation, those positive for MIP-1 <em>alpha</em> remained constant for all groups. In conclusion, fewer leucocytes expressed MCP-1 in gingival tissue sections, however, the percent MIP-1 <em>alpha</em>+ cells was increased particularly in tissues with increased proportions of CD8 cells and B cells with increasing inflammation and also in tissues with higher numbers of macrophages with little inflammation. Further studies are required to determine the significance of MIP-1 <em>alpha</em> in periodontal disease.

Currently available evidence suggests that in the steady state, the majority of hematopoietic stem cells are dormant in cell cycle and reside in the so-called G0 period. Studies in our laboratory indicated that once a stem cell leaves G0, its subsequent proliferation requires the presence of interleukin-<em>3</em> (IL-<em>3</em>). Recently it was reported that interleukin-1 (IL-1) may stimulate stem cells to become sensitive to IL-<em>3</em>. In a separate study, we observed that interleukin-6 (IL-6, also known as B cell stimulatory factor-2/<em>interferon</em> beta 2) possesses synergism with IL-<em>3</em>, shortening the G0 period of murine hematopoietic stem cells. We report here that human IL-6 and IL-<em>3</em> act synergistically in support of the proliferation of progenitors for human blast cell colonies and that IL-1 <em>alpha</em> reveals no synergism with IL-<em>3</em> when tested against purified human marrow progenitors. Panned My-10+ human marrow cells were plated in culture and on day 14 of incubation, either IL-<em>3</em>, IL-6, IL-1 <em>alpha</em> or a combination of these factors was added to the cultures. Blast cell colony formation was analyzed daily between days 18 and <em>3</em>2 of culture. IL-6 or IL-1 <em>alpha</em> alone failed to support blast cell colony formation. In the presence of IL-<em>3</em> alone, blast cell colonies continued to emerge between days 21 and 27. When a combination of IL-<em>3</em> and IL-6 was added, blast cell colonies developed earlier than in cultures with IL-<em>3</em> alone and twice as many blast cell colonies were identified. IL-1 <em>alpha</em> failed to augment IL-<em>3</em>-dependent blast cell colony formation. Replating studies of the individual blast cell colonies revealed various types of single as well as multilineage colonies. These observations suggest that IL-6 shortens the G0 period of human hematopoietic stem cells and that the reported synergistic activities of IL-1 on primitive hematopoietic cells may be indirect.

OBJECTIVE

To study the occurrence of relapse of herpes simplex encephalitis (HSE) and to find out whether soluble activity markers in cerebrospinal fluid (CSF) indicate direct viral or immune- mediated events.

METHODS

A consecutive series of 32 adult survivors of HSE were followed to determine the incidence of clinical relapse of HSE. Four patients had neurological deterioration interpreted as relapsing HSE. Four non-relapsing HSE cases were selected as matched controls. Fifty nine batched, paired CSF and serum samples from the eight HSE patients were analysed for soluble activity markers, predominantly cytokines and mediators (interferon-gamma, soluble CD8, tumour necrosis factor-alpha, and interleukin-10), amount of HSV-DNA and markers of glial and neuronal destruction (neurofilament protein, glial fibrillary acidic protein, S-100-beta, and neuron specific enolase).

RESULTS

Relapse of HSE was diagnosed in 3 of 26 (12 %) acyclovir-treated patients (5 episodes during 6.1 years of followup) and in 1 of 6 vidarabine-recipients. All relapses occurred from 1 to 4 months after acute HSE, except for a second relapse after 3.3 years in one patient. Computer tomography at relapses revealed few abnormalities apart from those found during the primary disease. Intravenous acyclovir and corticosteroids were given for 7-21 days in all the relapse patients. All relapse patients seemed to recover to the pre-relapse condition. HSV-DNA was demonstrated in CSF in all patients during the acute stage but not in any of 13 CSF samples taken during relapse phases. The HSV viral load during the acute stage of HSE was not higher or of longer duration in the relapsing patients than in the non-relapsing HSE controls. The levels of sCD8 were increased in nearly all CSF samples tested with peaks of sCD8 at one month of acute HSE. In all episodes of relapse, sCD8 peaks were detected during the first week at high levels. CSF levels of neuron-specific enolase, S-100 and glial fibrillary acidic protein were markedly lower at relapse than at the acute stage of HSV-1 encephalitis.

CONCLUSIONS

The lack of demonstrable HSV DNA in CSF, the lack of acute CSF signs and the lack of signs of neural and glia cells destruction indicate that a direct viral cytotoxicity is not the major pathogenic mechanism in relapse. Instead, the pronounced CSF proinflammatory immunological response and the relative lack of CSF anti-inflammatory cytokine IL-10 response suggest immunologically-mediated pathogenicity.

<em>Interferon</em>-<em>alpha</em> therapy is of proven efficacy in chronic hepatitis C, but it is not universally effective and may be associated with intolerable side effects. Ribavirin is a nucleoside analog with a broad spectrum of antiviral action. We conducted an uncontrolled pilot study of ribavirin therapy in 1<em>3</em> patients with chronic hepatitis C. Ribavirin was given for 6 mo, in a dose that was increased, at 2-mo intervals, from 600 mg to 1,000 mg to 1,200 mg/day. Serum ALT levels gradually decreased in all 1<em>3</em> treated patients; the mean percentage of decrease was 67% (from 210 U/L [range = 109 to 59<em>3</em>] to 6<em>3</em> U/L [range = 22 to 108 U/L]; p = 0.0006) after 6 mo of treatment. Serum aminotransferase levels fell to the normal range in four patients (<em>3</em>1%). In the <em>3</em> to 6 mo after cessation of ribavirin therapy, serum aminotransferase activities gradually rose to near pretreatment levels in all but one patient. Therapy was associated with a significant decrease in the geometric mean titer of hepatitis C virus RNA in serum (1:1,981 vs. 1:199; p less than 0.02) although no patients lost hepatitis C virus RNA from serum during therapy. No significant improvement was seen in liver histological appearance. Ribavirin therapy resulted in mild, reversible hemolysis; no patient exhibited symptomatic anemia. These findings suggest that ribavirin has a beneficial effect in patients with chronic hepatitis C, although further studies are needed to determine how ribavirin is best used.

<em>Interferon</em> (IFN) signal transduction involves <em>interferon</em> regulatory factors (IRF). Kaposi's sarcoma-associated herpesvirus (KSHV) encodes four IRF homologues: viral IRF 1 (vIRF-1) to vIRF-4. Previous functional studies revealed that the first exon of vIRF-2 inhibited <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) signaling. We now show that full-length vIRF-2 protein, translated from two spliced exons, inhibited both IFN-<em>alpha</em>- and IFN-lambda-driven transactivation of a reporter promoter containing the <em>interferon</em> stimulated response element (ISRE). Transactivation of the ISRE promoter by IRF-1 was negatively regulated by vIRF-2 protein as well. Transactivation of a full-length IFN-beta reporter promoter by either IRF-<em>3</em> or IRF-1, but not IRF-7, was also inhibited by vIRF-2 protein. Thus, vIRF-2 protein is an <em>interferon</em> induction antagonist that acts pleiotropically, presumably facilitating KSHV infection and dissemination in vivo.

To investigate the mechanism by which viruses are cleared from neurons in the central nervous system, we have utilized a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (OBLV60). After intranasal inoculation, OBLV60 grew preferentially in the olfactory bulbs of BALB/c mice. Using in situ hybridization, we found that viral RNA localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. Virus was cleared rapidly from the olfactory bulb between 5 and 11 days. Athymic nude mice failed to eliminate the virus, demonstrating a requirement for T lymphocytes. Immunosuppression of normal mice with cyclophosphamide also prevented clearance. Both CD4+ and CD8+ T-cell subsets were important, as depletion of either of these subsets delayed viral clearance. Gliosis and infiltrates of CD4+ and CD8+ cells were detected by immunohistochemical analysis at 6 days. The role of cytokines in clearance was investigated by using an RNase protection assay for interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>), IL-1 beta, IL-2, IL-<em>3</em>, IL-4, IL-5, IL-6, tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), TNF-beta, and gamma <em>interferon</em> (IFN-gamma). In immunocompetent mice there was upregulation of RNA for IL-1 <em>alpha</em>, IL-1 beta, IL-6, TNF-<em>alpha</em>, and IFN-gamma at the time of clearance. Nude mice had comparable increases in these cytokine messages, with the exception of IFN-gamma. Induction of major histocompatibility complex class I (MHC-I) molecules on cells in infected brains was demonstrated by immunohistochemical analyses in normal and nude mice, suggesting that IFN-gamma may not be necessary for induction of MHC-I on neural cells in vivo.

Recombinant human interleukin-1 (IL-1), injected into rabbits, induces the synthesis of endogenous IL-1. Also, IL-1 induces its own gene expression and synthesis in human peripheral blood mononuclear cells (PBMC). In this study, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) production by PBMC of 40 individuals stimulated with IL-1 <em>alpha</em> or IL-1 beta was determined by specific radioimmunoassay (RIA). After <em>3</em> h of PBMC incubation with IL-1, TNF-<em>alpha</em> mRNA was detected. IL-1 <em>alpha</em> stimulated both IL-1 beta and TNF-<em>alpha</em>, but there was no correlation in the amount of TNF-<em>alpha</em> or IL-1 beta synthesized in the PBMC of 29 individuals. IL-1-stimulated adherent cells produced approximately 50% more TNF-<em>alpha</em> than did unfractionated PBMC. Coincubation with <em>interferon</em>-gamma (IFN-gamma) did not change the amount of IL-1-induced TNF-<em>alpha</em>, whereas in the same culture IFN-gamma inhibited (greater than 70%) IL-1-induced IL-1 production. Endogenous pyrogen and TNF-like activity were detected in the sera of rabbits <em>3</em>.5 h after injection of either IL-1 <em>alpha</em> or -1 beta. These studies demonstrate that IL-1 induced TNF-<em>alpha</em> production in vivo and in vitro.

Transcript expression of 24 chemokines (CKs) was examined throughout 8 days in mouse lungs with type-1 (Th1) or type-2 (Th2) cytokine-mediated granulomas induced by bead-immobilized mycobacterial purified protein derivative or Schistosoma mansoni egg antigens. Where possible, CK protein levels were also measured. In addition, we examined effects of in vivo cytokine depletions. Findings were as follows: 1) bead challenge induced increases in 18 of 24 CK transcripts with type-1 and type-2 responses displaying different patterns. CKs fell into four categories: a) type-1-dominant (gamma-<em>interferon</em>-inducible protein (IP-10), monokine induced by INF-gamma (MIG), macrophage inflammatory protein-2 (MIP-2), lipopolysaccharide-induced chemokine (LIX), rodent growth-related oncogene homologue (KP), macrophage inflammatory protein-1<em>alpha</em> (MIP-1<em>alpha</em>) and -1beta (MIP-1beta), lymphotactin), b) type-2-dominant (eotaxin, monocyte chemotactic protein-2 (MCP-2) and -<em>3</em> (MCP-<em>3</em>), liver and activation-regulated chemokine (LARC), T cell activation protein-<em>3</em> (TCA-<em>3</em>), c) type-1 and type-2 co-dominant (MCP-1, MCP-5, monocyte-derived chemokine (MDC), thymus and activation-related chemokine (TARC), C10), and d) constitutive (lungkine, secondary lymphoid-tissue chemokine (SLC), EBI1-ligand chemokine (ELC), fractalkine, macrophage inflammatory protein-1gamma (MIP1-gamma), and stromal cell derived factor-1<em>alpha</em> (SDF1-<em>alpha</em>). 2) CKs displayed characteristic temporal patterns. CXC (IP-10, MIG, MIP-2, LIX, KC) and certain CC (MCP-1, MCP-5, MIP-1<em>alpha</em>, MIP-1beta) CKs were produced maximally within 1 to 2 days. Others (MCP-2, MCP-<em>3</em>, eotaxin, lymphotactin, LARC, TCA-<em>3</em>) displayed peak expression later. <em>3</em>) <em>Interferon</em>-gamma neutralization profoundly abrogated MIG, but had little effect on other CKs. Tumor necrosis factor-<em>alpha</em> neutralization caused up to 50% reduction in a range of CKs. These findings indicate that type-1 and type-2 granulomas display characteristic CK profiles with coordinated expression that is under cytokine-mediated regulation.

Porcine reproductive and respiratory syndrome is a major cause of economic loss for the swine industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) triggers weak and atypical innate immune responses, but key genes and mechanisms by which the virus interferes with the host innate immunity have not yet been elucidated. In this study, genes that control the response of the main target of PRRSV, porcine alveolar macrophages (PAMs), were profiled in vitro with a time-course experiment spanning the first round of virus replication. PAMs were obtained from six piglets and challenged with the Lelystad PRRSV strain, and gene expression was investigated using Affymetrix microarrays and real-time PCR. Of the 1409 differentially expressed transcripts identified by analysis of variance, two, five, 25, 16 and 100 differed from controls by a minimum of 1.5-fold at 1, <em>3</em>, 6, 9 and 12 h post-infection (p.i.), respectively. A PRRSV infection effect was detectable between <em>3</em> and 6 h p.i., and was characterized by a consistent downregulation of gene expression, followed by the start of the host innate immune response at 9 h p.i. The expression of beta <em>interferon</em> 1 (IFN-beta), but not of IFN-<em>alpha</em>, was strongly upregulated, whilst few genes commonly expressed in response to viral infections and/or induced by <em>interferons</em> were found to be differentially expressed. A predominance of anti-apoptotic transcripts (e.g. interleukin-10), a shift towards a T-helper cell type 2 response and a weak upregulation of tumour necrosis factor-<em>alpha</em> expression were observed within 12 h p.i., reinforcing the hypotheses that PRRSV has developed sophisticated mechanisms to escape the host defence.

We have recently reported that administration of recombinant tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) to hepatitis B virus (HBV) transgenic mice reduces the hepatic steady-state content of HBV-specific mRNA by up to 80% in the absence of liver cell injury. In the current study, we analyzed the regulatory effects of several other inflammatory cytokines in the same transgenic model system. Hepatic HBV mRNA content was reduced by up to 90% following administration of a single noncytopathic dose (100,000 U) of interleukin 2 (IL-2). Comparable effects were produced by administration of <em>alpha</em> and beta <em>interferons</em> (IFN-<em>alpha</em> and IFN-beta), but only after multiple injections of at least 500,000 U per mouse. Importantly, the regulatory effect of IL-2 was completely blocked by the prior administration of antibodies to tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), which did not block the effect of IFN-<em>alpha</em> or IFN-beta. In contrast to these observations, recombinant IFN-gamma, IL-1, IL-<em>3</em>, IL-6, TNF-beta, transforming growth factor beta, and granulocyte-monocyte colony-stimulating factor were inactive in this system. These results suggest that selected inflammatory cytokines can down-regulate HBV gene expression in vivo by at least two pathways, one that is dependent on TNF-<em>alpha</em> and another that is not. These results imply that antigen-nonspecific products of the intrahepatic HBV-specific inflammatory response may contribute to viral clearance or persistence during HBV infection.

BACKGROUND

The early evolution of acute kidney injury (AKI) in humans is difficult to study noninvasively. We hypothesized that urine proteomics could provide insight into the early pathophysiology of human AKI.

METHODS

A prospective nested case-control study (n = 250) compared serial urinary proteomes of 22 patients with AKI and 22 patients without AKI before, during, and after cardiopulmonary bypass surgery.

RESULTS

AKI was defined as a greater than 50% increase in serum creatinine level, and non-AKI, as less than 10% increase from baseline.

METHODS

Serum creatinine, urine protein-creatinine ratio, neutrophil gelatinase-associated lipocalin (NGAL), alphainterferon-inducible protein-10 (IP-10), monokine induced by interferon gamma (Mig), interferon-inducible T cell alpha chemoatractant (I-TAC), interleukin 6 (IL-6), IL-1beta, and IL-10. Urine protein profiling by means of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS).

RESULTS

SELDI-TOF-MS showed intraoperative tubular stress in both groups on arrival to the intensive care unit, evidenced by beta2-microglobulinuria. Non-AKI proteomes returned toward baseline postoperatively. In contrast, AKI proteomes showed a second phase of tubular injury/stress with the reappearance of beta2-microglobulin and multiple unidentified peaks (3 to 5 and 6 to 8 kDa) and the appearance of established tubular injury markers: urinary protein, <em>alpha</em>1-microglobulin, and NGAL. Furthermore, 2 novel peaks (2.43 and 2.78 kDa) were found to be dominant in postoperative non-AKI urine samples. The 2.78-kDa protein was identified as the active 25-amino acid form of hepcidin (hepcidin-25), a key regulator of iron homeostasis. Finally, an inflammatory component of reperfusion injury was evaluated by means of enzyme-linked immunosorbent assay analysis of candidate chemokines (IP-10, I-TAC, and Mig) and cytokines (IL-6, IL-1beta, and IL-10). Of these, IP-10 was upregulated in patients with versus without AKI postoperatively.

CONCLUSIONS

This is an observational study. SELDI-TOF-MS is a semiquantitative technique.

CONCLUSIONS

Evaluation of human AKI revealed early intraoperative tubular stress in all patients. A second phase of injury observed in patients with AKI may involve IP-10 recruitment of inflammatory cells. The enhancement of hepcidin-25 in patients without AKI may suggest a novel role for iron sequestration in modulating AKI.

Cytokines are a heterogenous group of polypeptide mediators that have been associated with activation of numerous functions, including the immune system and inflammatory responses. The cytokine families include, but are not limited to, interleukins (IL-I <em>alpha</em>, IL-I beta, ILIra and IL-2-IL-15), chemokines (IL-8/ NAP-I, NAP-2, MIP-I <em>alpha</em> and beta, MCAF/MCP-1, MGSA and RANTES), tumor necrosis factors (TNF-<em>alpha</em> and TNF-beta), <em>interferons</em> (INF-<em>alpha</em>, beta and gamma), colony stimulating factors (G-CSF, M-CSF, GM-CSF, IL-<em>3</em> and some of the other ILs), growth factors (EGF, FGF, PDGF, TGF <em>alpha</em>, TGF beta and ECGF), neuropoietins (LIF, CNTF, OM and IL-6), and neurotrophins (BDNF, NGF, NT-<em>3</em>-NT-6 and GDNF). The neurotrophins represent a family of survival and differentiation factors that exert profound effects in the central and peripheral nervous system (PNS). The neurotrophins are currently under investigation as therapeutic agents for the treatment of neurodegenerative disorders and nerve injury either individually or in combination with other trophic factors such as ciliary neurotrophic factor (CNTF) or fibroblast growth factor (FGF). Responsiveness of neurons to a given neurotrophin is governed by the expression of two classes of cell surface receptor. For nerve growth factor (NGF), these are p75NTR (p75) and p140trk (referred to as trk or trkA), which binds both BDNF and neurotrophin (NT)-4/5, and trkC receptor, which binds only NT-<em>3</em>. After binding ligand, the neurotrophin-receptor complex is internalized and retrogradely transported in the axon to the soma. Both receptors undergo ligand-induced dimerization, which activates multiple signal transduction pathways. These include the ras-dependent pathway utilized by trk to mediate neurotrophin effects such as survival and differentiation. Indeed, cellular diversity in the nervous system evolves from the concerted processes of cell proliferation, differentiation, migration, survival, and synapse formation. Neural adhesion and extracellular matrix molecules have been shown to play crucial roles in axonal migration, guidance, and growth cone targeting. Proinflammatory cytokines, released by activated macrophages and monocytes during infection, can act on neural targets that control thermogenesis, behavior, and mood. In addition to induction of fever, cytokines induce other biological functions associated with the acute phase response, including hypophagia and sleep. Cytokine production has been detected within the central nervous system as a result of brain injury, following stab wound to the brain, during viral and bacterial infections (AIDS and meningitis), and in neurodegenerative processes (multiple sclerosis and Alzheimer's disease). Novel cytokine therapies, such as anticytokine antibodies or specific receptor antagonists acting on the cytokine network may provide an optimistic feature for treatment of multiple sclerosis and other diseases in which cytokines have been implicated.

We report here the results of therapeutic trials in 200 patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) conducted in our department between 1986 and 199<em>3</em>. Motor disability grades were improved by more than one grade in 69.5% (91/1<em>3</em>1) of patients by oral administration of prednisolone, 50% (<em>3</em>/6) by eperisone hydrochloride only, 4<em>3</em>.8% (7/16) by blood purification (lymphocytapheresis and plasmapheresis), 40.0% (2/5) by intrathecal injection of hydrocortisone, <em>3</em>0.0% (<em>3</em>/10) by intravenous injection of high-dose methylprednisolone, 2<em>3</em>.<em>3</em>% (10/4<em>3</em>) by <em>interferon</em>-<em>alpha</em> (intramuscular injection and inhalation), 22.2% (2/9) by azathioprine, 20.0% (4/20) by high-dose vitamin C, 16.0% (4/25) by erythromycin, 12.5% (<em>3</em>/24) by salazosulfapyridine, 11.8% (2/17) by mizoribine, 7.1% (1/14) by fosfomycin, and 6.<em>3</em>% (1/16) by thyrotropin releasing hormone. No critical side effects of these therapies were seen with the exceptions of one patient with adult respiratory distress syndrome due to cytomegalovirus infection and one patient with drug-induced hepatitis/hepatic failure. Selection of these treatments for patients with HAM/ TSP must be considered on the basis of age, sex, disease severity and complications to reduce adverse events and to improve quality of life. Although the results were a synopsis of different treatments given to 200 patients with HAM/ TSP as an open trial, we consider this the first report of a large-scale therapeutic trial in patients with HAM/TSP. The results of this study indicate that immunomodulatory therapies have some beneficial effects in HAM/TSP, and the functions of these agents are related to the pathophysiology of this disease.