Hepatitis E virus (HEV) infections are known to run a self-limiting course. Recently, chronic hepatitis E has been described in immunosuppressed patients after solid-organ transplantation. Besides the general recommendation to lower the immunosuppressive medication in these patients, there is currently no specific treatment. We here describe the successful use of pegylated <em>interferon</em> <em>alpha</em>-2b in the treatment of 2 liver transplant recipients who suffered a chronic HEV infection for 9 years (case A) or 9 months (case B). After 4 weeks of therapy, a 2-log decrease (case A) and a <em>3</em>-log decrease (case B) in the viral load were observed. In case A, who received treatment for 1 year, serum viral RNA became undetectable from week 20 onward, and serum liver enzymes normalized completely. In case B, <em>interferon</em> was discontinued at week 16 because of a lack of a further decline in the viral load. However, 4 weeks after the cessation of therapy, viral RNA was no longer detectable in the serum, and this was probably related to a further decline in the immunosuppressive medication. Liver tests normalized completely. In both cases, no relapse has been noted so far. We conclude that pegylated <em>interferon</em> <em>alpha</em>-2b may be useful in the treatment of chronic HEV infections in patients in whom the reduction of the immunosuppressive medication alone is not sufficient.

BACKGROUND

Optimization of the current dendritic cell (DC) culture protocol in order to promote the therapeutic efficacy of DC-based immunotherapy is warranted. Alternative differentiation of monocyte-derived DCs using granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-15 has been propagated as an attractive strategy in that regard. The applicability of these so-called IL-15 DCs has not yet been firmly established. We therefore developed a novel pre-clinical approach for the generation of IL-15 DCs with potent immunostimulatory properties.

METHODS

Human CD14+ monocytes were differentiated with GM-CSF and IL-15 into immature DCs. Monocyte-derived DCs, conventionally differentiated in the presence of GM-CSF and IL-4, served as control. Subsequent maturation of IL-15 DCs was induced using two clinical grade maturation protocols: (i) a classic combination of pro-inflammatory cytokines (tumor necrosis factor-<em>alpha</em>, IL-1beta, IL-6, prostaglandin E2) and (ii) a Toll-like receptor (TLR)7/8 agonist-based cocktail (R-848, <em>interferon</em>-gamma, TNF-<em>alpha</em> and prostaglandin E2). In addition, both short-term (2-<em>3</em> days) and long-term (6-7 days) DC culture protocols were compared. The different DC populations were characterized with respect to their phenotypic profile, migratory properties, cytokine production and T cell stimulation capacity.

RESULTS

The use of a TLR7/8 agonist-based cocktail resulted in a more optimal maturation of IL-15 DCs, as reflected by the higher phenotypic expression of CD8<em>3</em> and costimulatory molecules (CD70, CD80, CD86). The functional superiority of TLR7/8-activated IL-15 DCs over conventionally matured IL-15 DCs was evidenced by their (i) higher migratory potential, (ii) advantageous cytokine secretion profile (<em>interferon</em>-gamma, IL-12p70) and (iii) superior capacity to stimulate autologous, antigen-specific T cell responses after passive peptide pulsing. Aside from a less pronounced production of bioactive IL-12p70, short-term versus long-term culture of TLR7/8-activated IL-15 DCs resulted in a migratory profile and T cell stimulation capacity that was in favour of short-term DC culture. In addition, we demonstrate that mRNA electroporation serves as an efficient antigen loading strategy of IL-15 DCs.

CONCLUSIONS

Here we show that short-term cultured and TLR7/8-activated IL-15 DCs fulfill all pre-clinical prerequisites of immunostimulatory DCs. The results of the present study might pave the way for the implementation of IL-15 DCs in immunotherapy protocols.

<em>Interferon</em> <em>alpha</em> is the only treatment option for hepatitis delta virus (HDV). Trials investigating the efficacy of pegylated <em>interferon</em> <em>alpha</em> (PEG-IFNa) showed HDV RNA negativity rates of 25-<em>3</em>0% 24 weeks after therapy. However, the clinical and virological long-term outcome of HDV-infected patients treated with PEG-IFNa is unknown. We performed a retrospective-prospective follow-up of 77 patients treated for 48 weeks with either PEG-alfa-2a and adefovir (ADV) or either drug alone in the Hep-Net-International-Delta-Hepatitis-Intervention-Study 1 (HIDIT-1) trial. Long-term follow-up data were available for 58 out of 77 patients (75%) with a median time of follow-up of 4.5 (0.5-5.5) years and a median <em>3</em> visits per patient. Patients treated with ADV alone received retreatment with PEG-IFNa (48% versus 19%; P = 0.02) more often. Hepatitis B virus surface antigen (HBsAg) became negative in six PEG-IFNa-treated patients until the end of long-term follow-up (10%). Sixteen patients tested HDV RNA-negative 6 months after PEG-IFNa treatment who were entered in the long-term follow-up study. Out of these, nine individuals tested HDV RNA-positive at least once during further long-term follow-up, with seven patients being HDV RNA-positive at the most recent visit. Clinical endpoints (liver-related death, liver transplantation, hepatic decompensation, hepatocellular carcinoma) were observed in three PEG-IFNa-treated (8%) and three ADV-treated (14%) patients during posttreatment long-term follow-up with an overall annual event rate of 2.5% (4.9% in cirrhosis). Sequencing confirmed the reappearance of pretreatment virus strains in all cases.

CONCLUSIONS

Late HDV RNA relapses may occur after PEG-IFNa therapy of hepatitis delta and thus the term sustained virological response should be avoided in HDV infection. The annual posttreatment rate of clinical events in hepatitis delta patients eligible for PEG-IFNa therapy is about 2.5% and 4.9% in patients with cirrhosis.

Increasing evidence demonstrates that melatonin has an anti-inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll-like receptor 4 (TLR4)-mediated molecule myeloid differentiation factor 88 (MyD88)-dependent and TRIF-dependent signaling pathways in lipopolysaccharide (LPS)-stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 μg/mL) in the absence or presence of melatonin (10, 100, 1000 μm). As expected, melatonin inhibited TLR4-mediated tumor necrosis factor <em>alpha</em> (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10 in LPS-stimulated macrophages. In addition, melatonin significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS-stimulated macrophages. Although it had no effect on TLR4-mediated phosphorylation of c-Jun N-terminal kinase (JNK), p<em>3</em>8, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. In addition, melatonin inhibited TLR4-mediated Akt phosphorylation in LPS-stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of <em>interferon</em> (IFN)-regulated factor-<em>3</em> (IRF<em>3</em>), which was involved in TLR4-mediated TRIF-dependent signaling pathway, in LPS-stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS-induced IFN-β in macrophages. In conclusion, melatonin modulates TLR4-mediated inflammatory genes through MyD88-dependent and TRIF-dependent signaling pathways.

OBJECTIVE

Approximately half of hepatitis C virus (HCV)-infected patients do not respond to current interferon (IFN)-alpha combination therapy. To understand IFN-alpha resistance in vivo, we examined the dynamic responses to both type I and type II IFNs, human IFN (hIFN)-alpha, -gamma, and consensus IFN, in the chimpanzee model.

METHODS

Naive and HCV-infected chimpanzees were treated with 3 forms of hIFNs in vivo. Quantitative real-time polymerase chain reaction was performed to evaluate the expression of IFN-stimulated genes (ISGs) in both peripheral blood mononuclear cells and liver to compare the responses to hIFN between naive and infected chimpanzees. The hepatic expression of IFN signaling components and inhibitory regulators including suppressor of cytokine signaling 3 (SOCS3) were assessed. SOCS3 expression was also evaluated in the liver of HCV-infected patients undergoing IFN treatment.

RESULTS

The in vivo responses to all 3 hIFNs were much lower in the HCV-infected chimpanzees than those in the naive chimpanzees. This defect was particularly evident in the liver because induction of hepatic ISGs was barely detectable in the infected animals. Following IFN administration, the expression of SOCS3 was significantly up-regulated, possibly through induction of interleukin-6, in the liver of HCV-infected chimpanzees. HCV-infected humans also showed a differential pattern of hepatic SOCS3 expression in response to IFN that is associated with treatment response.

CONCLUSIONS

Our data indicate a predominantly defective hepatic response to IFN in HCV-infected chimpanzees, which is probably mediated through the activation of SOCS3 and may explain the nonresponse of many HCV patients to IFN-based therapy.

BACKGROUND

The present study evaluated the efficacy and safety of pegylated interferon (PegIFN)/ribavirin treatment in elderly patients with hepatitis C virus (HCV) infection.

METHODS

Seventy elderly patients with hepatitis C virus (HCV) infection (group A; age,>> or = 65 years) and 140 sex- and HCV genotype-matched controls (group B; age, 50-64 years) were allocated to receive a PegIFN-alpha-2a/ribavirin standard-of-care regimen.

RESULTS

Group A had a significantly higher rate of treatment discontinuation (21.4% vs 6.4%; P = .001) and grade 3 or 4 adverse events (34.3% vs 20%; P = .002) than group B. In intention-to-treat analysis, the sustained virologic response (SVR) rate was substantially lower in group A than in group B (67.1% vs 78.6%; P = .07). The inferiority of the SVR rate in group A was observed among patients with HCV genotype 1 (HCV-1) (51.9% vs 75.9%; P = .03) but not among patients with HCV genotype 2 or 3 (HCV-2/3) (76.7% vs 80.2%; P = .65). Among patients in group A who had a rapid virologic response, those infected with HCV-1 and those infected with HCV-2/3 had similar SVR rates (80% and 87.9%, respectively). For patients receiving treatment for >80% of its expected duration, SVR rates were similar between the 2 groups (80.4% vs 82.6%, respectively), regardless of viral genotype.

CONCLUSIONS

Older patients with HCV infection, especially those in the subgroup infected with HCV-1, had a greater frequency of adverse events and poorer adherence to the standard-of-care regimen, which may be the major reason for treatment inferiority.

BACKGROUND

Clinicaltrials.gov identifier NCT00629824 .

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells <em>3</em>:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of <em>interferon</em>-<em>alpha</em>/beta. SeV C proteins thus appear to be quite versatile.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the swine industry since the late 1980s. Accumulating evidences have revealed that PRRSV infection fails to induce type I <em>interferon</em> (IFN-<em>alpha</em>/beta), which are normally induced rapidly during virus replication in virus-infected cells. However, the potential mechanisms remain largely unclear. In this study, we showed that PRRSV infection activated the signal transduction components of NF-kappaB and AP-1, but not of <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>), an essential IFN-beta transcription factor. Furthermore, PRRSV infection significantly blocked synthetic dsRNA-induced IFN-beta production and IRF<em>3</em> nuclear translocation. To better understand the upstream signaling events that suppress IRF<em>3</em> activation, we further investigated the roles of individual components of the retinoic acid-inducible gene I (RIG-I)- and Toll-like receptor <em>3</em> (TLR<em>3</em>)-mediated signaling pathway for IFN-beta production during PRRSV infection. We observed that PRRSV infection significantly inhibited dsRNA-induced IRF<em>3</em> activation and IFN-beta generation by inactivating IFN-beta promoter stimulator 1 (IPS-1), an adaptor molecule of RIG-I. In contrast, PRRSV infection only partially reduced the activation of TIR domain-containing adaptor inducing IFN-beta (TRIF), an adaptor molecule of TLR<em>3</em>. Our results suggest that PRRSV infection suppresses production of IFN-beta primarily by interfering with the IPS-1 activation in the RIG-I signaling pathway.

Aldosterone infusion results in left ventricular hypertrophy (LVH) and hypertension and may involve profibrotic and proinflammatory mechanisms. In turn, hypertension is the major cause of diastolic heart failure (HF). Adiponectin, an adipose-derived plasma protein, exerts antiinflammatory and anti-hypertrophic effects and is implicated in the development of hypertension and systolic HF. We thus tested the hypothesis that hypoadiponectinemia in aldosterone-induced hypertension exacerbated cardiac remodeling and diastolic HF. Wild-type (WT) or adiponectin-deficient (APNKO) mice underwent saline or aldosterone infusion and uninephrectomy and were fed 1% salt water for 4 wk. Blood pressure was increased in aldosterone-infused WT (1<em>3</em>2 +/- 2 vs. 109 +/- <em>3</em> mm Hg; P < 0.01) and further augmented in APNKO mice (140 +/- <em>3</em> mm Hg; P < 0.05 vs. aldosterone-infused WT). LVH was increased in aldosterone-infused WT vs. WT mice (LV/body weight ratio, 4.8 +/- 0.2 vs. 4.1 +/- 0.2 mg/g) and further increased in aldosterone-infused APNKO mice (LV/body weight ratio, 6.0 +/- 0.4 mg/g). Left ventricular ejection fraction was not decreased in either aldosterone-infused WT or APNKO hearts. Pulmonary congestion however was worse in APNKO mice (P < 0.01). The ratio of early ventricular filling over late ventricular filling (E/A) and the ratio of mitral peak velocity of early filling to early diastolic mitral annular velocity (E/e'), measures of diastolic function, were increased in aldosterone-infused WT hearts and further increased in APNKO hearts (P < 0.05 for both). Renal function and cardiac fibrosis were no different between both aldosterone-infused groups. Aldosterone increased matrix metalloproteinase-2 expression in WT hearts (P < 0.05 vs. WT and P < 0.01 vs. APNKO). Myocardial atrial natriuretic peptide, <em>interferon</em>-gamma, and TNF-<em>alpha</em> expression were increased in aldosterone-infused WT hearts. Expression of these proteins was further increased in aldosterone-infused APNKO hearts. Therefore, hypoadiponectinemia in hypertension-induced diastolic HF exacerbates LVH, diastolic dysfunction, and diastolic HF. Whether or not adiponectin replacement prevents the progression to diastolic HF will warrant further study.

OBJECTIVE

Interleukin-10 knockout (IL-10(-/-)) mice spontaneously develop colitis characterized by T-helper cell type 1-polarized inflammation. We tested the possible therapeutic activity of the peroxisome proliferator-activated receptor alpha (PPARalpha) ligand fenofibrate, and the PPARdelta ligand GW0742, in IL-10(-/-) mice and investigated the cellular/molecular mechanisms for fenofibrate action.

METHODS

The effect of fenofibrate or GW0742 on the progression of colitis in C3H.IL-10(-/-) mice was evaluated. Effects of fenofibrate on cytokine and chemokine gene expression were studied in cultured splenocytes, pathogenic T cells isolated from C3H/HeJBir mice, and HT-29 colorectal cancer cells.

RESULTS

Treatment of C3H.IL-10(-/-) mice with fenofibrate delayed the onset of colitis, decreased the colonic histopathology score, and decreased colonic expression of genes encoding the inflammatory cytokines interferon-gamma and interleukin (IL)-17. The target for fenofibrate, PPARalpha, was expressed in lymphocytes, macrophages, and crypt and surface epithelial cells of the colon. The mean number of lymphocytes was decreased by more than 75% in colonic sections of fenofibrate-treated as compared with control IL-10(-/-) mice, and fenofibrate repressed interferon-gamma and IL-17 expression in isolated T cells. Fenofibrate also repressed the expression of the genes encoding 3 chemokines, CXCL10, CCL2, and CCL20, and repressed CXCL10 gene promoter activity in tumor necrosis factor-alpha-treated HT-29 cells. In contrast to the beneficial effect of fenofibrate, the PPARdelta ligand GW0742 accelerated the onset of colitis in IL-10(-/-) mice.

CONCLUSIONS

The immunopathology observed in IL-10(-/-) mice resembles that seen in Crohn's disease. The novel therapeutic activity of fenofibrate in this mouse model suggests that it may also have activity in Crohn's disease.

OBJECTIVE

The objective of this study was to investigate the efficacy of mesenchymal stem cell (MSC) in the treatment of arthritis.

METHODS

Mesenchymal stem cells were injected intravenously into mice with collagen-induced arthritis (CIA). Arthritic indexes were evaluated, and the levels of the pro- and anti-inflammatory cytokines interleukin-10 (IL-10), gamma <em>interferon</em> (IFN-gamma)-inducible protein 10 (IP-10), chemokine (C-X-C motif) receptor <em>3</em> (CXCR<em>3</em>), interleukin-17A (IL-17A), and tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) in serum or splenic cells were determined using real-time RT-PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA). The proliferation of dendritic cell line D2SC cells was determined using (<em>3</em>)H-TdR incorporation assay.

RESULTS

Upon injection of MSCs, overall arthritis symptoms were significantly improved in the CIA mouse models as indicated by the paw edema. Consistent with this observation, serum levels of pro-inflammatory cytokine TNF-<em>alpha</em> and inflammatory cell infiltration decreased significantly 12 days after MSC injection, while the expression of anti-inflammatory cytokines IL-10, IP-10, and CXCR<em>3</em> was increased in splenocytes. In addition, we provided evidence that MSCs may directly promote the proliferation of D2SC cells and the expression of IP-10 in D2SC cells in vitro.

CONCLUSIONS

Mesenchymal stem cells significantly enhance the efficacy of collagen-induced arthritis treatment, likely through the modulation of the expression of various cytokines.

Dendritic cells are the most potent antigen-presenting cells of the immune system. Although dendritic cells are likely to secrete selective cytokines that facilitate antigen presentation, the difficulty in isolating pure dendritic cells in sufficient numbers has made assessment of this function imprecise. In this study, pure populations of CD8<em>3</em>+ human blood dendritic cells were isolated by previously established enrichment procedures and subsequent cell sorting. Cytokine gene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) amplification of mRNA. Resting CD8<em>3</em>+ dendritic cells expressed interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), and transforming growth factor-beta 1 (TGF-beta 1) mRNA, while activation of cells with phorbol myristate acetate induced IL-1 <em>alpha</em> and beta, IL-9, TNF-beta, <em>interferon</em>-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, and G-CSF mRNA expression. Resting CD8<em>3</em>+ cells also expressed the Rantes, MCP-1, MIP-1 <em>alpha</em>, and MIP-1 beta chemokines, with 1-<em>3</em>09 expression induced upon activation. Resting and activated CD8<em>3</em>+ dendritic cells also expressed receptors for IL-2 (CD25), TGF-beta 1 and -beta <em>3</em>, and GM-CSF as determined by indirect immunofluorescence staining. These results indicate that dendritic cells have the ability to produce a variety of soluble factors which are likely to contribute substantially to the potent allostimulatory activity of these cells.

OBJECTIVE

In acute liver failure, hepatocytes are exposed to various cytokines that activate both cell survival and apoptotic pathways. NF-kappaB is a central transcription factor in these responses. Recent studies indicate that blocking NF-kappaB causes apoptosis, indicating the existence of NF-kappaB-regulated anti-apoptotic genes. In the present study the relationship between NF-kappaB activation and apoptosis has been investigated in hepatocytes.

METHODS

Primary rat hepatocytes were exposed to a cytokine mixture of tumor necrosis factor <em>alpha</em>, interleukin-1beta, <em>interferon</em>-gamma and lipopolysaccharide. Modulation of signalling pathways was performed by using dominant negative adenoviral constructs. Apoptosis and NF-kappaB activation were determined by caspase-<em>3</em> activity, Hoechst staining and electrophoretic mobility shift assay, respectively. Furthermore, expression and regulation of apoptosis-related genes were investigated.

RESULTS

(1) Inhibition of NF-kappaB activation results in apoptosis. (2) Inhibitor of apoptosis protein (IAP) family members, inhibitor of apoptosis protein1 (cIAP1), and X-chromosome-linked IAP, are expressed in rat hepatocytes. cIAP2 is induced by cytokines in an NF-kappaB-dependent manner and overexpression of cIAP2 inhibits apoptosis. (<em>3</em>) The anti-apoptotic Bcl-2 family member A1/Bfl-1 and the pro-apoptotic members Bak and Bid are induced by cytokines and NF-kappaB-dependent. (4) Nitric oxide inhibits caspase-<em>3</em> activity in hepatocytes.

CONCLUSIONS

In inflammatory conditions, hepatocyte survival is dependent on NF-kappaB activation and cIAP2 contributes significantly to this protection.

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 29<em>3</em>:99<em>3</em>-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-<em>3</em>, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-<em>3</em> was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S2<em>3</em>2I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by <em>alpha</em> <em>interferon</em>. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.

In the present study, we assessed the feasibility, toxicity, immunologic response, and clinical efficacy of vaccination with allogeneic dendritic cell (DC)/tumor fusions in patients with metastatic renal cell carcinoma (RCC). Patients with stage IV RCC with accessible tumor lesions or independent therapeutic indications for nephrectomy were eligible for enrollment. Tumors were processed into single cell suspensions and cryopreserved. DCs were generated from adherent peripheral blood mononuclear cells isolated from normal volunteers and cultured with granulocyte macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-<em>alpha</em>. DCs were fused to patient derived RCC with serial electrical pulses. Patients received up to <em>3</em> vaccinations at a fixed dose of 4x10(7) to 1x10(8) cells administered at 6-week intervals. Twenty-four patients underwent vaccination. Twenty-one and 20 patients were evaluable for immunologic and clinical response, respectively. DCs demonstrated a characteristic phenotype with prominent expression of HLA class II and costimulatory molecules. A mean fusion efficiency of 20% was observed, determined by the percent of cells coexpressing DC and tumor antigens. No evidence of significant treatment related toxicity or auto-immunity was observed. Vaccination resulted in antitumor immune responses in 10/21 evaluable patients as manifested by an increase in CD4 and/or CD8 T-cell expression of <em>interferon</em>-gamma after ex vivo exposure to tumor lysate. Two patients demonstrated a partial clinical response by Response Evaluation Criteria in Solid Tumors criteria and 8 patients had stabilization of their disease. Vaccination of patients with RCC with allogeneic DC/tumor fusions was feasible, well tolerated, and resulted in immunologic and clinical responses in a subset of patients.

Angiogenesis, the development of new capillaries, is involved in leukocyte ingress into the synovium during the development and progression of rheumatoid arthritis. Several soluble and cell surface-bound mediators including growth factors, cytokines, chemokines, proteolytic matrix-degrading enzymes, cell adhesion molecules and others may promote synovial neovascularization. On the other hand, endogenous angiostatic factors, such as angiostatin, endostatin, interleukin-4 (IL-4), IL-1<em>3</em>, <em>interferons</em> and some angiostatic chemokines are also produced within the rheumatoid synovium, however, their effects are insufficient to control synovial angiogenesis and inflammation. Several specific and non-specific strategies have been developed to block the action of angiogenic mediators. The first line of angiostatic agents include vascular endothelial growth factor (VEGF), angiopoietin, <em>alpha</em>(V)beta(<em>3</em>) integrin antagonist, as well as non-specific angiogenesis inhibitors including traditional disease-modifying agents (DMARDs), anti-tumor necrosis factor biologics, angiostatin, endostatin, fumagillin analogues or thalidomide. Potentially any angiostatic compound could be introduced to studies using animal models of arthritis or even to human rheumatoid arthritis trials.

Commercially available human acellular dermal matrix (HADM), AlloDerm((R)), was implanted as an interpositional graft in the abdominal wall of adult vervet monkeys. Host response to implanted HADM was evaluated and compared with a human cellular dermal matrix (HCDM) and a primate acellular dermal matrix (PADM). Clinical acceptance of the acellular grafts (HADM and PADM) and graft remodeling were evidenced by fibroblast repopulation and neoangiogenesis. A mild inflammatory response marked predominantly by macrophages and T-cells was present in both HADM and PADM during the first month but was absent by <em>3</em> months. Similarly, antibody and complement deposition into the grafts as well as in the serum was evident only at the early time points. Interleukin-6 (IL-6) or IL-10 was induced in some acellular graft-implanted monkeys at the early time points, but tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), <em>interferon</em>-gamma (IFN-gamma), or IL-2 was not detected over the study period. In contrast, significant inflammation was observed in HCDM-implanted animals, as evidenced by immune cell infiltration (p </= 0.0001), immunoglobulin G (IgG) binding (p < 0.001), complement (C5b) deposition (p < 0.05), TNF-<em>alpha</em> deposition (p < 0.001), and macrophage activation (p < 0.05). Abdominal wall repair in the vervet monkey is an immunologically relevant model to evaluate functional efficacy and host immune response to implanted biomaterials and may be predictive of clinical response and surgical outcomes in humans.

Human monocytes were isolated and their ability to harbour growth of virulent tubercle bacilli was assessed, in the presence or absence of various immunomodulators. Calcitriol (1,25(OH2), vitamin D<em>3</em>) alone, at doses of 10(-7)-10(-9) M endowed human monocytes with a significant ability to restrict intracellular growth of the tubercle bacilli. Crude immune lymphokines as well as recombinant <em>interferon</em>-gamma (IFN-gamma) endowed monocytes with no tuberculostatic activity. Similarly, other recombinant cytokines tested, notably colony-stimulating factor-1 (CSF-1), interleukin-1 (IL-1), interleukin-<em>3</em> (IL-<em>3</em>) and interleukin-6 (IL-6) all failed to stimulate anti-tuberculous properties, and even increased growth of the tubercle bacilli in monocytes, in the case of CSF-1. Conversely, incubation of crude lymphokines in combination with calcitriol led to total stasis of the growth of M. tuberculosis. Experiments with recombinant cytokines and immunologically active vitamins showed that a combination of IFN-gamma tumour necrosis factor-<em>alpha</em> and calcitriol induced a significant amount of intramonocyte killing of M. tuberculosis. Addition of this cocktail of factors to already infected monocytes led to substantial killing of tubercle bacilli. These sets of experiments establish clearly that combinations of recombinant cytokines and vitamins may induce substantial intramonocyte killing of M. tuberculosis. The mechanism involved in this killing activity was not clarified.

1. Increased levels of several pro-inflammatory cytokines including interleukin-1 beta (IL-1 beta) and tumour necrosis factor-<em>alpha</em> (TNF <em>alpha</em>) have been found in bronchoalveolar lavage fluid from symptomatic asthmatic patients. IL-1 beta, TNF <em>alpha</em> and <em>interferon</em>-gamma (IFN gamma) are known to stimulate a number of cells to produce inflammatory mediators such as prostaglandins. Although airway smooth muscle (ASM) is known to be a rich source of prostaglandins, the regulation of cyclo-oxygenase (COX) isoforms and prostanoid production by proinflammatory cytokines have not been studied in human airway smooth muscle. 2. We studied the effects of IL-1 beta, TNF <em>alpha</em> and IFN gamma on the induction of two isoforms of cyclo-oxygenase and its relation to prostaglandin E2 (PGE2) release and COX activity (reflected by PGE2 synthesis from exogenous arachidonic acid) in human cultured airway smooth muscle cells. <em>3</em>. IL-1 beta, but not TNF <em>alpha</em> or IFN gamma, caused a time- and concentration-dependent enhancement in PGE2 and other prostanoid (6-keto-PGF1 <em>alpha</em>, PGF2 <em>alpha</em>, thromboxane B2 (TXB2) and PGD2) production, with PGE2 and 6-keto-PGF1 <em>alpha</em> as the principal products. This stimulation was accompanied by a corresponding increase in COX activity. 4. COX-2 protein measured by Western blot analysis was not detectable in untreated cells, but was increased in a time- and concentration-dependent manner by IL-1 beta, but not TNF <em>alpha</em> or IFN gamma. In contrast, no variation in the expression of COX-1 protein was observed. 5. Pretreatment with the conventional non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and ibuprofen, and the selective COX-2 inhibitors, NS-<em>3</em>98 and nimesulide, completely blocked IL-1 beta-induced PGE2 release and COX activity. The glucocorticosteroid dexamethasone and protein synthesis inhibitors, cycloheximide and actinomycin D, not only markedly inhibited IL-1 beta-stimulated PGE2 release and COX activity but also suppressed IL-1 beta-induced COX-2 induction. 6. This study demonstrates that human cultured ASM cells release prostanoids in response to IL-1 beta stimulation and that the response is mostly mediated by the induction of COX-2 rather than COX-1 isoenzyme, implying that airway smooth muscle may be an important source of prostaglandins in human airways and that COX-2 may play an important role in the regulation of the inflammatory process in asthma.

CXC chemokines modulate host immunity, neovascularization, growth and invasive behaviour of tumours. Despite their relevance in tumour biology, chemokine expression in intestinal- and diffuse-type gastric carcinoma, which exhibit a completely different growth pattern, has not been investigated in detail. In this study, expression of the CXC chemokines CXCL8 [interleukin (IL)-8], CXCL1 [growth-related oncogene <em>alpha</em> (Gro <em>alpha</em>)], CXCL9 [monokine induced by <em>interferon</em> (IFN)-gamma] and CXCL10 [IFN-gamma-inducible protein-10 (IP-10)] and the corresponding chemokine receptors CXCR1-<em>3</em> was investigated by immunohistochemistry in intestinal- and diffuse-type gastric carcinoma. Tumour cells of all patients expressed CXCL8. CXCL8 expression was significantly stronger in tumour cells of diffuse- rather than intestinal-type gastric carcinoma (P < 0.01) as determined by a semiquantitative score. CXCL1 was expressed almost exclusively by diffuse- but not intestinal-type carcinoma cells. The corresponding chemokine receptors, CXCR1 and CXCR2, were found on carcinoma cells. Furthermore, CXCL8 expression correlated with number of tumour vessels (P < 0.01), suggesting an angiogenetic function in gastric carcinoma not only in vitro but also in vivo. CXCL10 and CXCL9, attractants for T cells, were expressed by peritumorous macrophages in close proximity to IFN-gamma-producing CXCR<em>3</em>-positive T cells in both tumour types. These chemokines may attract gastric carcinoma-infiltrating T cells via an IFN-gamma-mediated pathway and enhance host immunity against the tumour. In gastric carcinoma a complex interplay between CXC-chemokine signals derived from both tumour cells and tumour-infiltrating immune cells may exhibit pleiotropic effects in tumour biology that go far beyond their originally described functions as leucocyte chemoattractants. Because CXCL8 and CXCL1, which are known to increase growth and invasive behaviour of malignant tumours, are significantly stronger expressed in diffuse- than intestinal-type gastric carcinoma, one may speculate that these chemokines influence the different growth pattern of gastric carcinoma types.

OBJECTIVE

Interferon-alphaalpha) injections may be capable of triggering depression in some individuals. The first objective was to further characterize this depression and, secondly, to examine whether pre-treatment temperament was correlated with subsequent vulnerability to IFN-alpha.

METHODS

Twenty-three initially euthymic adults undergoing year-long PEG-IFN-alpha treatment for hepatitis C were evaluated at baseline and then prospectively monitored using both the Structured Clinical Interview for DSM-IV (SCID) and self-report questionnaires.

RESULTS

A major depressive episode developed within 3 months in 39%. Principal component analysis of the change in self-report scores after 1 month of treatment demonstrated three orthogonal factors: (i) a specific increase in depression as manifested in the Beck Depression Inventory (BDI) and Hospital Anxiety and Depression Scale (HADS), (ii) an increase in hostility and anxiety, (iii) and a generalized combination of worse symptoms including somatic symptoms on the Symptom Check List (SCL-90). BDI at 1 month was predicted by baseline BDI (r=0.76, P=.004). Hostility at 1 month was predicted by low baseline agreeableness (r=0.75, P=.01). Controlling for baseline BDI scores, categorical major depression was predicted by combined high baseline neuroticism and low agreeableness (combined r=0.66, P=.03).

CONCLUSIONS

These initial results (i) support the depressogenic nature of IFN-alpha treatment in a subset of vulnerable individuals, (ii) indicate that some individuals are also independently vulnerable to worsened hostility, and (iii) suggest that it may be possible to clinically predict these vulnerabilities in initially euthymic subjects.

Selective accumulation of eosinophils and activated CD4+ cells is now considered a central event in the pathogenesis of asthma, and this process is thought to be mediated by a number of cytokines including tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the Type 2 cytokines interleukin-4 (IL-4) and IL-5. To carry out a detailed time-course analysis of cellular changes in the bronchoalveolar lavage fluid (BAL), peripheral blood (PB), and bone marrow (BM), and of changes in the aforementioned cytokines in BAL and serum, Balb/c mice were sensitized by intraperitoneal injection with ovalbumin (OVA) adsorbed to aluminum hydroxide on two occasions 5 days apart, and were subjected to an OVA aerosol challenge 12 days after the second sensitization. This resulted in an airways inflammatory response characterized by early transient neutrophilia, marked eosinophilia, and, to a lesser extent, lymphocytosis in the BAL. Inflammatory events were first observed <em>3</em> h and 24 h after antigen challenge in the lung tissue and BAL, respectively, and lasted for 21 days. In the BM, we detected a 1.5- and 5-fold increase in the total number of cells and eosinophils, respectively, 4 days after the second sensitization. This was followed by a decrease, although BM eosinophilia remained clearly present at the time of antigen challenge. A second eosinopoietic event was observed in the BM shortly after challenge and reached a peak at day <em>3</em>. BM cellularity returned to normal at day 21 after challenge. Serum OVA-specific IgE was first detected <em>3</em> days following the second sensitization (150 ng/ml). IgE levels then decreased but remained at the 75 ng/ml range at the time of the aerosol challenge. During the sensitization period, TNF-<em>alpha</em> (approximately 25 pg/ml), IL-4 (approximately 40 pg/ml), and IL-5 (approximately 250 pg/ml) were detected in serum, but not in the BAL fluid (BALF) and returned to background levels at the time of the antigen challenge. After antigen challenge, TNF-<em>alpha</em>, IL-4, IL-5, and GM-CSF were detected in serum. Peak levels were observed at <em>3</em> h (approximately 40 pg/ml), <em>3</em> h (approximately 120 pg/ml), 12 h (approximately <em>3</em>50 pg/ml), and <em>3</em> h (approximately 10 pg/ml), respectively, and returned to background levels 24 h after challenge. In the BALF, we detected peak levels of TNF-<em>alpha</em>, IL-4, IL-5, and GM-CSF at 6 h (approximately 250 pg/ml), 24 h (approximately 140 pg/ml), 24 h (<em>3</em>50 pg/ml), and <em>3</em> h (approximately 10 pg/ml), respectively, with a return to background levels 5 days after challenge. No IL-10 could be detected at any time point during sensitization or after challenge in either serum or BAL. We also detected approximately 40 pg/ml of <em>interferon</em>-gamma (IFN-gamma) in the serum of normal untreated mice. Serum IFN-gamma levels fluctuated during sensitization and after challenge, but never exceeded those observed in untreated mice. Thus, the cytokine profile observed in this experimental model of allergic inflammation is characterized by IL-4 and IL-5 dominance, with an apparently minor TNF-<em>alpha</em> and GM-CSF contribution and relatively low or undetectable levels of IFN-gamma and IL-10.

BACKGROUND

Genital warts are a common sexually transmitted disease caused by human papillomavirus. Imiquimod is a novel immune-response modifier capable of inducing a variety of cytokines, including interferon alfa, tumor necrosis factor-alpha, as well as interleukins 1, 6, and 8. In animal models imiquimod has demonstrated antiviral, antitumor, and adjuvant activity. In vitro, imiquimod has no antiviral or antitumor activity.

OBJECTIVE

Our purpose was to determine the safety and efficacy of topical imiquimod for the treatment of external genital warts.

METHODS

This prospective double-blind, placebo-controlled, parallel design clinical trial was performed in three outpatient centers, a public health clinic, a university-based clinic, and a private practice. One hundred eight patients with external genital warts (predominantly white men) were entered into the trial. Fifty-one patients were randomly selected to receive 5% imiquimod cream; 57 patients were randomly chosen to receive placebo cream. Study medication was applied three times weekly for up to 8 weeks. Patients whose warts cleared completely were observed for up to 10 weeks to determine recurrence rates.

RESULTS

In the intent-to-treat analysis, the warts of 37% (19 of 51) of the imiquimod-treated patients and 0% (0 of 57) of the placebo group cleared completely (p < 0.001). In addition, many patients experienced a partial response. A reduction in baseline wart area of 80% or more was observed in 62% of imiquimod-treated patients (28 of 45) and 4% of the placebo group (2 of 50) (p < 0.001); a 50% reduction or more in wart area was noted in 76% of imiquimod-treated patients (34 of 45) and 8% of placebo recipients (4 of 50) (p < 0.001). Of imiquimod-treated patients whose warts cleared completely and who finished the 10-week follow-up period, 19% (3 of 16) experienced recurrences of warts. Imiquimod-treated patients experienced a significantly greater number of local inflammatory reactions than the placebo group. Symptoms and signs associated with the local inflammatory reactions included itching (54.2%), erythema (33.3%), burning (31.3%), irritation (16.7%), tenderness (12.5%), ulceration (10.4%), erosion (10.4%), and pain (8.3%). There were no differences in systemic reactions or laboratory abnormalities between treatment groups.

CONCLUSIONS

Topical 5% imiquimod cream appears to have a significant therapeutic effect in the treatment of external genital warts.

Hepatic lipocytes (perisinusoidal, Ito cells) are the primary matrix-producing cells in liver fibrosis. During liver injury they undergo activation, a process characterized by cell proliferation and increased fibrogenesis. We and others have established a culture model in which in vivo features of lipocyte activation can be mimicked by cells grown on plastic. Additionally, we recently showed that activation is associated with new expression of smooth muscle-specific <em>alpha</em>-actin both in vivo and in culture. Although <em>interferon</em>-gamma is known to inhibit collagen production in some systems, its action as a general modulator of lipocyte activation has not been examined; this issue forms the basis for our study. In culture-activated lipocytes, <em>interferon</em>-gamma (1,000 U/ml) significantly inhibited lipocyte proliferation as assessed by [<em>3</em>H]thymidine incorporation assay and nuclear autoradiography. In time-course studies of activation, it also markedly reduced expression of smooth muscle-specific <em>alpha</em>-actin and its messenger RNA. In dose-response experiments, maximal inhibitory effects on smooth muscle-specific <em>alpha</em>-actin mRNA gene expression were achieved with as little as 10 U <em>interferon</em>-gamma/ml. Inhibition of cellular activation was reversible; after <em>interferon</em>-gamma withdrawal, messenger RNA levels of smooth muscle-specific <em>alpha</em>-actin returned to untreated control levels. The effect of <em>interferon</em>-gamma extended to extracellular matrix gene expression, with reduction of type I collagen, type IV collagen and total fibronectin messenger RNAs to <em>3</em>%, 24% and 15% of untreated control levels, respectively. In contrast to the marked effects on smooth muscle-specific <em>alpha</em>-actin and extracellular matrix gene expression, <em>interferon</em>-gamma reduced total protein synthesis by only 17.7%.(ABSTRACT TRUNCATED AT 250 WORDS)