Dermatomyositis has been modeled as an autoimmune disease largely mediated by the adaptive immune system, including a local humorally mediated response with B and T helper cell muscle infiltration, antibody and complement-mediated injury of capillaries, and perifascicular atrophy of muscle fibers caused by ischemia. To further understand the pathophysiology of dermatomyositis, we used microarrays, computational methods, immunohistochemistry and electron microscopy to study muscle specimens from 67 patients, 54 with inflammatory myopathies, 14 with dermatomyositis. In dermatomyositis, genes induced by <em>interferon</em>-<em>alpha</em>/beta were highly overexpressed, and immunohistochemistry for the <em>interferon</em>-<em>alpha</em>/beta inducible protein MxA showed dense staining of perifascicular, and, sometimes all myofibers in 8/14 patients and on capillaries in 1<em>3</em>/14 patients. Of <em>3</em>6 patients with other inflammatory myopathies, 1 patient had faint MxA staining of myofibers and <em>3</em> of capillaries. Plasmacytoid dendritic cells, potent CD4+ cellular sources of <em>interferon</em>-<em>alpha</em>, are present in substantial numbers in dermatomyositis and may account for most of the cells previously identified as T helper cells. In addition to an adaptive immune response, an innate immune response characterized by plasmacytoid dendritic cell infiltration and <em>interferon</em>-<em>alpha</em>/beta inducible gene and protein expression may be an important part of the pathogenesis of dermatomyositis, as it appears to be in systemic lupus erythematosus.

The mosquito-borne Zika virus (ZIKV) is responsible for an explosive ongoing outbreak of febrile illness across the Americas. ZIKV was previously thought to cause only a mild, flu-like illness, but during the current outbreak, an association with Guillain-Barré syndrome and microcephaly in neonates has been detected. A previous study showed that ZIKV requires murine adaptation to generate reproducible murine disease. In our study, a low-passage Cambodian isolate caused disease and mortality in mice lacking the <em>interferon</em> (IFN) <em>alpha</em> receptor (A129 mice) in an age-dependent manner, but not in similarly aged immunocompetent mice. In A129 mice, viremia peaked at ∼10(7) plaque-forming units/mL by day 2 postinfection (PI) and reached high titers in the spleen by day 1. ZIKV was detected in the brain on day <em>3</em> PI and caused signs of neurologic disease, including tremors, by day 6. Robust replication was also noted in the testis. In this model, all mice infected at the youngest age (<em>3</em> weeks) succumbed to illness by day 7 PI. Older mice (11 weeks) showed signs of illness, viremia, and weight loss but recovered starting on day 8. In addition, AG129 mice, which lack both type I and II IFN responses, supported similar infection kinetics to A129 mice, but with exaggerated disease signs. This characterization of an Asian lineage ZIKV strain in a murine model, and one of the few studies reporting a model of Zika disease and demonstrating age-dependent morbidity and mortality, could provide a platform for testing the efficacy of antivirals and vaccines.

Mutants in complementation group U<em>3</em>, completely defective in the response of all genes tested to <em>interferons</em> (IFNs) <em>alpha</em> and gamma, do not express the 91 and 84 kDa polypeptide components of <em>interferon</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>), a transcription factor known to play a primary role in the IFN-<em>alpha</em> response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a <em>3</em>8 amino acid extension at the C-terminus of the 91 kDa polypeptide. Complementation of U<em>3</em> mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild-type cells completely restored the response to both IFN-<em>alpha</em> and -gamma and the ability to form ISGF<em>3</em>. Complementation with the 84 kDa component similarly restored the ability to form ISGF<em>3</em> and, albeit to a lower level, the IFN-<em>alpha</em> response of all genes tested so far. It failed, however, to restore the IFN-gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN-<em>alpha</em> and -gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN.

OBJECTIVE

We review the status of systemic therapy for patients with advanced renal cell carcinoma.

METHODS

A literature search was performed on MEDLINE and CANCERLIT to identify results of systemic therapy for patients with renal cell carcinoma published from January 1990 through December 1998. Treatment results of chemotherapy agents, immunotherapy, combination programs and adjuvant therapy were reviewed.

RESULTS

No chemotherapy agent has produced response rates that justify its use as a single agent. <em>Interferon</em>-<em>alpha</em> and interleukin (IL)-2 demonstrated low response rates ranging from 10% to 20%. The results of 2 randomized trials suggest that treatment with <em>interferon</em>-<em>alpha</em> compared to vinblastine or medroxyprogesterone achieves a small improvement in survival. Response rates in patients treated with low dose IL-2 are similar to those achieved with a high dose bolus schedule but whether the responses are as durable is being addressed in an ongoing randomized trial. A randomized trial of <em>interferon</em>-<em>alpha</em> plus IL-2 compared to monotherapy with either agent showed increased toxicity but no improvement in survival. In <em>3</em> randomized trials no survival benefit was associated with adjuvant <em>interferon</em>-<em>alpha</em> therapy following complete resection of locally advanced renal cell carcinoma.

CONCLUSIONS

Despite extensive evaluation of many different treatment modalities, metastatic renal cell carcinoma remains highly resistant to systemic therapy. A few patients exhibit complete or partial responses to interferon and/or IL-2 but most do not respond, and there are few long-term survivors. Preclinical research, and clinical evaluation of new agents and treatment programs to identify improved antitumor activity against metastases remain the highest priorities in this refractory disease.

ISGF-<em>3</em> is a multiprotein transcription factor that is very quickly activated in the cell cytoplasm only after attachment of <em>interferon</em> <em>alpha</em> to the cell surface. To understand the specific cytoplasmic activation of proteins that move to the nucleus and direct increased transcription of specific genes, we have purified and now report completion of the cloning of cDNA encoding the four proteins of ISGF-<em>3</em>. With all of the sequences available, it is clear that three of these proteins are encoded by members of a previously unrecognized gene family. We suggest that proteins encoded by this gene family serve the function of interpreting the fact that a cell surface receptor has bound its ligand so that specific signal transduction to the nucleus can occur.

To study the capacity of human neurons to mount innate immunity responses to viral infections, we infected cells of a human postmitotic neuron-derivative cell line, NT2-N, with rabies virus (RABV) and herpes simplex type 1 (HSV-1). Changes in neuronal gene expression were analyzed by use of Affymetrix microarrays. Applying a twofold cutoff, RABV increased the transcription of 228 genes, and HSV-1 increased the transcription of 26<em>3</em> genes. The most striking difference between the two infections concerns genes involved in immunity. These genes represent 24% of the RABV-upregulated genes and only 4.9% of the HSV-1-upregulated genes. Following RABV infection, the most upregulated genes belong to the immunity cluster and included almost exclusively genes for beta <em>interferon</em> (IFN-beta) primary and secondary responses as well as genes for chemokines (CCL-5, CXCL-10) and inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor <em>alpha</em>, interleukin 1 <em>alpha</em>). In contrast, HSV-1 infection did not increase IFN-beta gene transcripts and triggered the production of only IL-6 and <em>interferon</em> regulatory factor 1 mRNAs. The microarray results were confirmed by real-time PCR, immunocytochemistry, and enzyme-linked immunosorbent assay. Human neurons were found to express Toll-like receptor <em>3</em>. They produced IFN-beta after treatment with poly(I:C) but not with lipopolysaccharide. Thus, human neurons can mount an innate immunity response to double-stranded RNA. These observations firmly establish that human neurons, in absence of glia, have the intrinsic machinery to sense virus infection.

The discovery and initial description of the <em>interferon</em>-lambda (IFN-lambda) family in early 200<em>3</em> opened an exciting new chapter in the field of IFN research. There are <em>3</em> IFN-lambda genes that encode <em>3</em> distinct but highly related proteins denoted IFN-lambda1, -lambda2, and -lambda<em>3</em>. These proteins are also known as interleukin-29 (IL-29), IL-28A, and IL-28B, respectively. Collectively, these <em>3</em> cytokines comprise the type III subset of IFNs. They are distinct from both type I and type II IFNs for a number of reasons, including the fact that they signal through a heterodimeric receptor complex that is different from the receptors used by type I or type II IFNs. Although type I IFNs (IFN-<em>alpha</em>/beta) and type III IFNs (IFN-lambda) signal via distinct receptor complexes, they activate the same intracellular signaling pathway and many of the same biological activities, including antiviral activity, in a wide variety of target cells. Consistent with their antiviral activity, expression of the IFN-lambda genes and their corresponding proteins is inducible by infection with many types of viruses. Therefore, expression of the type III IFNs (IFN-lambdas) and their primary biological activity are very similar to the type I IFNs. However, unlike IFN-<em>alpha</em> receptors which are broadly expressed on most cell types, including leukocytes, IFN-lambda receptors are largely restricted to cells of epithelial origin. The potential clinical importance of IFN-lambda as a novel antiviral therapeutic agent is already apparent. In addition, preclinical studies by several groups indicate that IFN-lambda may also be useful as a potential therapeutic agent for other clinical indications, including certain types of cancer.

Ischemia reperfusion injury results from tissue damage during ischemia and ongoing inflammation and injury during reperfusion. Liver reperfusion injury is reduced by lymphocyte depletion or activation of adenosine A2A receptors (A2ARs) with the selective agonist 4-{<em>3</em>-[6-amino-9-(5-ethylcarbamoyl-<em>3</em>,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]- prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e). We show that NKT cells are stimulated to produce <em>interferon</em> (IFN)-gamma by 2 h after the initiation of reperfusion, and the use of antibodies to deplete NK1.1-positive cells (NK and NKT) or to block CD1d-mediated glycolipid presentation to NKT cells replicates, but is not additive to, the protection afforded by ATL146e, as assessed by serum alanine aminotransferase elevation, histological necrosis, neutrophil accumulation, and serum IFN-gamma elevation. Reduced reperfusion injury observed in RAG-1 knockout (KO) mice is restored to the wild-type (WT) level by adoptive transfer of NKT cells purified from WT or A2AR KO mice but not IFN-gamma KO mice. Additionally, animals with transferred A2AR-/- NKT cells are not protected from hepatic reperfusion injury by ATL146e. In vitro, ATL146e potently inhibits both anti-CD<em>3</em> and <em>alpha</em>-galactosylceramide-triggered production of IFN-gamma by NKT cells. These findings suggest that hepatic reperfusion injury is initiated by the CD1d-dependent activation of NKT cells, and the activation of these cells is inhibited by A2AR activation.

OBJECTIVE

Patients infected with the hepatitis C virus (HCV) often have liver steatosis, suggesting the possibility of a viral cytopathic effect. The aim of this study was to correlate the occurrence and severity of liver steatosis with HCV RNA type, level and sequence of the core-encoding region.

METHODS

We scored the liver steatosis in 101 HCV-infected individuals carefully selected to exclude other risk factors of a fatty liver. Results were compared with HCV RNA genotype and level in serum and liver. In selected patients, we assessed the effect of antiviral therapy on steatosis and the relationship between nucleocapsid sequence heterogeneity and fat infiltration.

RESULTS

Steatosis was found in 41 (40.6%) patients, irrespective of sex, age or route of infection. HCV genotype <em>3</em> was associated with higher steatosis scores than other genotypes. A significant correlation between steatosis score and titer of intrahepatic HCV RNA was found in patients infected with genotype <em>3</em>, but not in those infected with genotype 1. In selected patients, response to <em>alpha</em>-<em>interferon</em> was associated with the disappearance of steatosis. Analysis of the nucleocapsid of 14 HCV isolates failed to identify a sequence specifically associated with the development of steatosis.

CONCLUSIONS

We provide virological and clinical evidence that the steatosis of the liver is the morphological expression of a viral cytopathic effect in patients infected with HCV genotype <em>3</em>. At variance with published evidence from experimental models, the HCV nucleocapsid protein does not seem to fully explain the lipid accumulation in these patients.

Interleukin-6 (IL-6, also known as B-cell stimulatory factor 2/<em>interferon</em> beta 2) was previously shown to support the proliferation of granulocyte/macrophage progenitors and indirectly support the formation of multilineage and blast cell colonies in cultures of spleen cells from normal mice. We report here that IL-<em>3</em> and IL-6 act synergistically in support of the proliferation of murine multipotential progenitors in culture. The time course of total colony formation by spleen cells isolated from mice 4 days after injection of 5-fluorouracil (150 mg/kg) was significantly shortened in cultures containing both lymphokines relative to cultures supported by either of the two factors. Serial observations (mapping) of individual blast cell colonies in culture revealed that blast cell colonies emerged after random time intervals in the presence of IL-<em>3</em>. The average time of appearance in IL-6 alone was somewhat delayed, and in cultures containing both factors the appearance of multilineage blast cell colonies was significantly hastened relative to cultures grown in the presence of the individual lymphokines. In cultures of day-2 post-5-fluorouracil bone marrow cells, IL-6 failed to support colony formation; IL-<em>3</em> alone supported the formation of a few granulocyte/macrophage colonies, but the combination of factors acted synergistically to yield multilineage and a variety of other types of colonies. In this system, IL-1 <em>alpha</em> also acted synergistically with IL-<em>3</em>, but the effect was smaller, and no multilineage colonies were seen. Together these results indicate that IL-<em>3</em> and IL-6 act synergistically to support the proliferation of hemopoietic progenitors and that at least part of the effect results from a decrease in the G0 period of the individual stem cells.

It has been shown that all three classes of <em>interferons</em> enhance the expression of the major histocompatibility class I antigens (HLA-A,B,C;H-2) on a wide variety of cell types (1-10). However, their effect on the expression of the class II antigens (HLA-DR, Ia), which play a major part in cellular interactions that initiate an immune response, is more controversial. The predominate findings have been that the <em>interferons</em> specifically increase the synthesis and expression of only the class I antigens (<em>3</em>, 4, 6, 8, 10, 11). We report here that recombinant <em>interferon</em>-gamma (IFN-gamma) increases the synthesis and expression of the HLA-DR (la-like) antigens as well as beta 2-microglobulin (beta 2-m), a low m.w. subunit of HLA, on human melanoma cells. No increase in HLA-DR was detected on these melanoma cells with leukocyte <em>interferon</em> (IFN-<em>alpha</em>) at doses 400 times higher than the maximum dose of IFN-gamma. These findings were extended to show that pure IFN-gamma also increases the expression of the HLA-DR antigens on normal peripheral blood monocytes, whereas recombinant IFN-<em>alpha</em> at a similar dose had little effect on the expression of this surface antigen. These findings suggest a specialized role for IFN-gamma in immune regulation in comparison with IFN-<em>alpha</em>.

OBJECTIVE

Maintenance of the mucosal barrier is a critical function of intestinal epithelia. Myosin regulatory light chain (MLC) phosphorylation is a common intermediate in the pathophysiologic regulation of this barrier. The aim of this study was to determine whether a membrane permeant inhibitor of MLC kinase (PIK) could inhibit intracellular MLC kinase and regulate paracellular permeability.

METHODS

Recombinant MLC and Caco-2 MLC kinase were used for kinase assays. T84 and Caco-2 monolayers were treated with enteropathogenic Escherichia coli (EPEC) or tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma to induce barrier dysfunction.

RESULTS

PIK inhibited MLC kinase in vitro and was able to cross cell membranes and concentrate at the perijunctional actomyosin ring. Consistent with these properties, apical addition of PIK reduced intracellular MLC phosphorylation by 22% +/- 2%, increased transepithelial resistance (TER) by 50% +/- 1%, and decreased paracellular mannitol flux rates by 5.2 +/- 0.2-fold. EPEC infection induced TER decreases of 37% +/- 6% that were limited to 16% +/- 5% by PIK. TNF-alpha and IFN-gamma induced TER decreases of 22% +/- 3% that were associated with a 172% +/- 1% increase in MLC phosphorylation. Subsequent PIK addition caused MLC phosphorylation to decrease by 25% +/- 4% while TER increased to 97% +/- 6% of control.

CONCLUSIONS

PIK can prevent TER defects induced by EPEC and reverse MLC phosphorylation increases and TER decreases induced by TNF-alpha and IFN-gamma. The data also suggest that TNF-alpha and IFN-gamma regulate TER, at least in part, via the perijunctional cytoskeleton. Thus, PIK may be the prototype for a new class of targeted therapeutic agents that can restore barrier function in intestinal disease states.

RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of <em>interferon</em> (IFN) regulatory factor <em>3</em> target genes and IFN-stimulated genes, including several subtypes of <em>alpha</em> IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.

Although the immune system has long been implicated in the control of cancer, evidence for specific and efficacious immune responses in human cancer has been lacking. In the case of chronic myelogenous leukemia (CML), either allogeneic bone marrow transplant (BMT) or <em>interferon</em>-<em>alpha</em>2b (IFN-<em>alpha</em>2b) therapy can result in complete remission, but the mechanism for prolonged disease control is unknown and may involve immune anti-leukemic responses. We previously demonstrated that PR1, a peptide derived from proteinase <em>3</em>, is a potential target for CML-specific T cells. Here we studied <em>3</em>8 CML patients treated with allogeneic BMT, IFN- <em>alpha</em>2b or chemotherapy to look for PR1-specific T cells using PR1/HLA-A*0201 tetrameric complexes. There was a strong correlation between the presence of PR1-specific T cells and clinical responses after IFN-<em>alpha</em> and allogeneic BMT. This provides for the first time direct evidence of a role for T-cell immunity in clearing malignant cells.

<em>Interferons</em> (IFN) <em>alpha</em>/beta and gamma induce the formation of two transcriptional activators: gamma-activating factor (GAF) and <em>interferon</em>-stimulated gamma factor <em>3</em> (ISGF<em>3</em>). We report a natural heterozygous germline STAT1 mutation associated with susceptibility to mycobacterial but not viral disease. This mutation causes a loss of GAF and ISGF<em>3</em> activation but is dominant for one cellular phenotype and recessive for the other. It impairs the nuclear accumulation of GAF but not of ISGF<em>3</em> in heterozygous cells stimulated by IFNs. Thus, the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF.

The influenza A virus NS1 protein (NS1A protein) binds and inhibits the function of the <em>3</em>0-kDa subunit of CPSF, a cellular factor that is required for the <em>3</em>'-end processing of cellular pre-mRNAs. Here we generate a recombinant influenza A/Udorn/72 virus that encodes an NS1A protein containing a mutated binding site for the <em>3</em>0-kDa subunit of CPSF. This mutant virus is substantially attenuated, indicating that this binding site in the NS1A protein is required for efficient virus replication. Using this mutant virus, we show that NS1A binding to CPSF mediates the viral posttranscriptional countermeasure against the initial cellular antiviral response--the <em>interferon</em>-<em>alpha</em>/beta (IFN-<em>alpha</em>/beta)-independent activation of the transcription of cellular antiviral genes, which requires the <em>interferon</em> regulatory factor-<em>3</em> (IRF-<em>3</em>) transcription factor that is activated by virus infection. Whereas the posttranscriptional processing of these cellular antiviral pre-mRNAs is inhibited in cells infected by wild-type influenza A virus, functional antiviral mRNAs are produced in cells infected by the mutant virus. These results establish that the binding of <em>3</em>0-kDa CPSF to the NS1A protein is largely responsible for the posttranscriptional inhibition of the processing of these cellular antiviral pre-mRNAs. Mutation of this binding site in the NS1A protein also affects a second cellular antiviral response: in cells infected by the mutant virus, IFN-beta mRNA is produced earlier and in larger amounts.

<em>Interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) is a nonleukemogenic treatment of polycythemia vera (PV) able to induce cytogenetic remissions. Its use is limited by toxicity, leading to treatment discontinuation in approximately 20% of patients. We completed a phase 2 multicenter study of pegylated IFN-<em>alpha</em>-2a in 40 PV patients. Objectives included evaluation of efficacy, safety, and monitoring of residual disease using JAK2V617F quantification (%V617F). Median follow-up was <em>3</em>1.4 months. At 12 months, all <em>3</em>7 evaluable patients had hematologic response, including 94.6% complete responses (CRs). Only <em>3</em> patients (8%) had stopped treatment. After the first year, <em>3</em>5 patients remained in hematologic CR, including 5 who had stopped pegylated IFN-<em>alpha</em>-2a. Sequential samples for %V617F monitoring, available in 29 patients, showed %V617F decrease in 26 (89.6%). Median %V617F decreased from 45% before pegylated IFN-<em>alpha</em>-2a to 22.5%, 17.5%, 5%, and <em>3</em>% after 12, 18, 24, and <em>3</em>6 months, respectively. Molecular CR (JAK2V617F undetectable) was achieved in 7 patients, lasting from 6(+) to 18(+) months, and persisted after pegylated IFN-<em>alpha</em>-2a discontinuation in 5. No vascular event was recorded. These results show that pegylated IFN-<em>alpha</em>-2a yields high rates of hematologic and molecular response in PV with limited toxicity, and could even eliminate the JAK2 mutated clone in selected cases. Available at www.clinicaltrials.gov as #NCT00241241.

We tested the hypothesis that the inflammatory cytokines can regulate fibroblast extracellular matrix metabolism. Neonatal and adult rat cardiac fibroblasts cultures in vitro were exposed to interleukin (IL)-1beta (4 ng/mL), tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>; 100 ng/mL), IL-6 (10 ng/mL), or <em>interferon</em>-gamma (IFN-gamma; 500 U/mL) for 24 hours. IL-1beta, and to a lesser extent TNF-<em>alpha</em>, decreased collagen synthesis, which was measured as collagenase-sensitive [(<em>3</em>)H]proline incorporation, but had no effect on cell number or total protein synthesis. IL-1beta decreased the expression of procollagen <em>alpha</em>(1)(I), <em>alpha</em>(2)(I), and <em>alpha</em>1(III) mRNA, but increased the expression of procollagen <em>alpha</em>(1)(IV), <em>alpha</em>(2)(IV), and fibronectin mRNA, indicating a selective transcriptional downregulation of fibrillar collagen synthesis. IL-1beta and TNF-<em>alpha</em> each increased total matrix metalloproteinase (MMP) activity as measured by in-gel zymography, causing specific increases in the bands corresponding to MMP-1<em>3</em>, MMP-2, and MMP-9. IL-1beta increased the expression of proMMP-2 and proMMP-<em>3</em> mRNA, suggesting that increased metalloproteinase activity is due, at least in part, to increased transcription. The effects of IL-1beta were not dependent on NO production. Thus, IL-1beta and TNF-<em>alpha</em> decrease collagen synthesis and activate MMPs that degrade collagen. These observations suggest that IL-1beta and TNF-<em>alpha</em> may contribute to ventricular dilation and myocardial failure by promoting the remodeling of interstitial collagen.

OBJECTIVE

Acute hepatitis C virus infection accounts for approximately 20% of cases of acute hepatitis today. The aim of this study was to define the natural course of the disease and to contribute to the development of treatment strategies for acute hepatitis C virus.

METHODS

The diagnosis of acute hepatitis C virus in 60 patients was based on seroconversion to anti-hepatitis C virus antibodies or clinical and biochemical criteria and on the presence of hepatitis C virus RNA in the first serum sample.

RESULTS

Fifty-one of 60 (85%) patients presented with symptomatic acute hepatitis C virus. In the natural (untreated) course of acute symptomatic hepatitis C (n = 46), spontaneous clearance was observed in 24 patients (52%), usually within 12 weeks after the onset of symptoms, whereas all asymptomatic patients (n = 9) developed chronic hepatitis C. The start of antiviral therapy (<em>interferon</em>-<em>alpha</em> with or without ribavirin) beyond <em>3</em> months after the onset of acute hepatitis induced sustained viral clearance in 80% of treated patients.

CONCLUSIONS

The management of acute hepatitis C has to take into account the high rate of spontaneous viral clearance within 12 weeks after the onset of symptomatic disease. Treatment of only those patients who remain hepatitis C virus RNA positive for more than <em>3</em> months after the onset of disease led to an overall viral clearance (self-limited and treatment induced) in 91% of patients, and unnecessary treatment was avoided in those with spontaneous viral clearance. Patients with asymptomatic acute hepatitis C virus infection are unlikely to clear the infection spontaneously and should be treated as early as possible.

Human interleukin (IL) 1 receptor-associated kinase 4 (IRAK-4) deficiency is a recently discovered primary immunodeficiency that impairs Toll/IL-1R immunity, except for the Toll-like receptor (TLR) <em>3</em>- and TLR4-<em>interferon</em> (IFN)-<em>alpha</em>/beta pathways. The clinical and immunological phenotype remains largely unknown. We diagnosed up to 28 patients with IRAK-4 deficiency, tested blood TLR responses for individual leukocyte subsets, and TLR responses for multiple cytokines. The patients' peripheral blood mononuclear cells (PBMCs) did not induce the 11 non-IFN cytokines tested upon activation with TLR agonists other than the nonspecific TLR<em>3</em> agonist poly(I:C). The patients' individual cell subsets from both myeloid (granulocytes, monocytes, monocyte-derived dendritic cells [MDDCs], myeloid DCs [MDCs], and plasmacytoid DCs) and lymphoid (B, T, and NK cells) lineages did not respond to the TLR agonists that stimulated control cells, with the exception of residual responses to poly(I:C) and lipopolysaccharide in MDCs and MDDCs. Most patients (22 out of 28; 79%) suffered from invasive pneumococcal disease, which was often recurrent (1<em>3</em> out of 22; 59%). Other infections were rare, with the exception of severe staphylococcal disease (9 out of 28; <em>3</em>2%). Almost half of the patients died (12 out of 28; 4<em>3</em>%). No death and no invasive infection occurred in patients older than 8 and 14 yr, respectively. The IRAK-4-dependent TLRs and IL-1Rs are therefore vital for childhood immunity to pyogenic bacteria, particularly Streptococcus pneumoniae. Conversely, IRAK-4-dependent human TLRs appear to play a redundant role in protective immunity to most infections, at most limited to childhood immunity to some pyogenic bacteria.

Binding of <em>interferons</em> IFN-<em>alpha</em> and IFN-gamma to their cell surface receptors promptly induces tyrosine phosphorylation of latent cytoplasmic transcriptional activators (or Stat proteins, for signal transducers and activators of transcription). <em>Interferon</em>-<em>alpha</em> activates both Stat91 (M(r) 91,000; ref. 1) and Stat11<em>3</em> (M(r) 11<em>3</em>,000; ref. 2) whereas IFN-gamma activates only Stat91 (refs <em>3</em>, 4). The activated proteins then move into the nucleus and directly activate genes induced by IFN-<em>alpha</em> and IFN-gamma. Somatic cell genetics experiments have demonstrated a requirement for tyrosine kinase-2 (Tyk2) in the IFN-<em>alpha</em> response pathway and for Jak2 (ref. 6), a kinase with similar sequence, in the IFN-gamma response pathway. Here we investigate the tyrosine phosphorylation events on Stat and Jak proteins after treatment of cells with IFNs <em>alpha</em> and gamma and with epidermal growth factor (EGF). Stat91 is phosphorylated on Tyr701 after cells are treated with IFN-<em>alpha</em> and EGF, as it was after treatment with IFN-gamma (ref. 8). We find that Jak1 also becomes phosphorylated on tyrosine after cells are treated with these same three ligands, although each ligand is shown to activate at least one other different kinase. Jak1 may therefore be the enzyme that phosphorylates Tyr 701 in Stat91.

The cytokine transforming growth factor (TGF)-beta, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-beta receptor (TGF-betaR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-<em>3</em>08 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-beta-sensitive CCL64 cells mediated by recombinant TGF-beta1 or TGF-beta2 or of TGF-beta-containing glioma cell supernatant at an EC(50) of 0.1 mumol/L. SD-208 blocks autocrine and paracrine TGF-beta signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-beta reporter assays and strongly inhibits constitutive and TGF-beta-evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-beta-releasing LN-<em>3</em>08 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-<em>3</em>08 targets. The release of <em>interferon</em> gamma and tumor necrosis factor <em>alpha</em> by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-beta or of TGF-beta-containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-beta-induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated <em>3</em> days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-beta receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-beta activity.

OBJECTIVE

To study antibody-independent contributions of B cells to inflammatory disease activity, and the immune consequences of B-cell depletion with rituximab, in patients with multiple sclerosis (MS).

METHODS

B-Cell effector-cytokine responses were compared between MS patients and matched controls using a <em>3</em>-signal model of activation. The effects of B-cell depletion on Th1/Th17 CD4 and CD8 T-cell responses in MS patients were assessed both ex vivo and in vivo, together with pharmacokinetic/pharmacodynamic studies as part of 2 rituximab clinical trials in relapsing-remitting MS.

RESULTS

B Cells of MS patients exhibited aberrant proinflammatory cytokine responses, including increased lymphotoxin (LT):interleukin-10 ratios and exaggerated LT and tumor necrosis factor (TNF)-alpha secretion, when activated in the context of the pathogen-associated TLR9-ligand CpG-DNA, or the Th1 cytokine interferon-gamma, respectively. B-Cell depletion, both ex vivo and in vivo, resulted in significantly diminished proinflammatory (Th1 and Th17) responses of both CD4 and CD8 T cells. Soluble products from activated B cells of untreated MS patients reconstituted the diminished T-cell responses observed following in vivo B-cell depletion in the same patients, and this effect appeared to be largely mediated by B-cell LT and TNFalpha.

CONCLUSIONS

We propose that episodic triggering of abnormal B-cell cytokine responses mediates 'bystander activation' of disease-relevant proinflammatory T cells, resulting in new relapsing MS disease activity. Our findings point to a plausible mechanism for the long-recognized association between infections and new MS relapses, and provide novel insights into B-cell roles in both health and disease, and into mechanisms contributing to therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.

BACKGROUND

Wharton's jelly derived stem cells (WJMSCs) are gaining attention as a possible clinical alternative to bone marrow derived mesenchymal stem cells (BMMSCs) owing to better accessibility, higher expansion potential and low immunogenicity. Usage of allogenic mesenchymal stem cells (MSC) could be permissible in vivo only if they retain their immune properties in an inflammatory setting. Thus the focus of this study is to understand and compare the immune properties of BMMSCs and WJMSCs primed with key pro-inflammatory cytokines, Interferon-gamma (IFNgamma) and Tumor Necrosis Factor-alpha (TNFalpha).

RESULTS

Initially the effect of priming on MSC mediated suppression of alloantigen and mitogen induced lymphoproliferation was evaluated in vitro. Treatment with IFNgamma or TNFalpha, did not ablate the immune-suppression caused by both the MSCs. Extent of immune-suppression was more with WJMSCs than BMMSCs in both the cases. Surprisingly, priming BMMSCs enhanced suppression of mitogen driven lymphoproliferation only; whereas IFNgamma primed WJMSCs were better suppressors of MLRs. Further, kinetic analysis of cytokine profiles in co-cultures of primed/unprimed MSCs and Phytohematoagglutinin (PHA) activated lymphocytes was evaluated. Results indicated a decrease in levels of pro-inflammatory cytokines. Interestingly, a change in kinetics and thresholds of Interleukin-2 (IL-2) secretion was observed only with BMMSCs. Analysis of activation markers on PHA-stimulated lymphocytes indicated different expression patterns in co-cultures of primed/unprimed WJMSCs and BMMSCs. Strikingly, co-culture with WJMSCs resulted in an early activation of a negative co-stimulatory molecule, CTLA4, which was not evident with BMMSCs. A screen for immune suppressive factors in primed/unprimed WJMSCs and BMMSCs indicated inherent differences in IFNgamma inducible Indoleamine 2, 3-dioxygenase (IDO) activity, Hepatocyte growth factor (HGF) and Prostaglandin E-2 (PGE2) levels which could possibly influence the mechanism of immune-modulation.

CONCLUSIONS

This study demonstrates that inflammation affects the immune properties of MSCs distinctly. Importantly different tissue derived MSCs could utilize unique mechanisms of immune-modulation.