In response to viral infection, host cells elicit a number of responses, including the expression of <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta). In these cells, IFN regulatory factor-<em>3</em> (IRF-<em>3</em>) undergoes a sequence of posttranslational modifications that allow it to act as a potent transcriptional coactivator of specific IFN genes, including IFN-beta. We investigated the mechanisms by which herpes simplex virus 1 (HSV-1) inhibits the production of IFN-beta mediated by the IRF-<em>3</em> signaling pathway. Here, we show that HSV-1 infection can block the accumulation of IFN-beta triggered by Sendai virus (SeV) infection. Our results indicate that HSV-1 infection blocks the nuclear accumulation of activated IRF-<em>3</em> but does not block the initial virus-induced phosphorylation of IRF-<em>3</em>. The former effect was at least partly mediated by increased turnover of IRF-<em>3</em> in HSV-1-infected cells. Using mutant viruses, we determined that the immediate-early protein ICP0 was necessary for the inhibition of IRF-<em>3</em> nuclear accumulation. Expression of ICP0 also had the ability to reduce IFN-beta production induced by SeV infection. ICP0 has been shown previously to play a role in HSV-1 sensitivity to IFN and in the inhibition of antiviral gene production. However, we observed that an ICP0 mutant virus still retained the ability to inhibit the production of IFN-beta. These results argue that HSV-1 has multiple mechanisms to inhibit the production of IFN-beta, providing additional ways in which HSV-1 can block the IFN-mediated host response.

Mantle-cell lymphoma (MCL) is characterized by poor prognosis with a median survival of only <em>3</em> to 4 years. To improve clinical outcome, the European MCL Network initiated a randomized trial comparing consolidation with myeloablative radiochemotherapy followed by autologous stem cell transplantation (ASCT) to <em>alpha</em>-<em>interferon</em> maintenance (IFN <em>alpha</em>) in first remission. Patients 65 years of age or younger with advanced-stage MCL were assigned to ASCT or IFN <em>alpha</em> after achievement of complete or partial remission by a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-like induction therapy. According to the International Prognostic Index (IPI), 4<em>3</em>% of patients had a low-risk, 41% a low-intermediate, 11% a high-intermediate, and 6% a high-risk profile. Sixty-two of 122 patients proceeded to ASCT and 60 received IFN <em>alpha</em>. Patients in the ASCT arm experienced a significantly longer progression-free survival (PFS) with a median of <em>3</em>9 months compared with 17 months for patients in the IFN <em>alpha</em> arm (P = .0108). The <em>3</em>-year overall survival (OS) was 8<em>3</em>% after ASCT versus 77% in the IFN group (P = .18). Early consolidation by myeloablative radiochemotherapy followed by ASCT is feasible and results in a significant prolongation of PFS in advanced-stage MCL. Longer follow-up is needed to determine the effect on OS.

To investigate the relationship between genotypes of hepatitis C virus and response to <em>interferon</em>-<em>alpha</em> therapy, hepatitis C virus RNA was assayed by polymerase chain reaction with three sets of primers and probes in 70 patients with non-A, non-B chronic hepatitis who received <em>interferon</em>-<em>alpha</em>. Twenty-four patients sustained long-term remissions (complete responders). Polymerase chain reaction for 5'-terminal noncoding region detected hepatitis C virus RNA in 94.<em>3</em>% (66 of 70) of the patients. Polymerase chain reaction for nonstructural region <em>3</em>, in which primers and a probe were synthesized to be identical to hepatitis C virus-J, detected hepatitis C virus RNA in 40 patients. Polymerase chain reaction for nonstructural region 5-in which sequences of primers and a probe were derived from hepatitis C virus-K2, a genotype different from hepatitis C virus-J--detected hepatitis C virus RNA in 17 patients. Only one patient was positive on both nonstructural region <em>3</em> and nonstructural region 5 polymerase chain reaction. Nucleotide sequence of clones obtained from 5' terminal noncoding region polymerase chain reaction products of two patients positive on polymerase chain reaction for nonstructural region <em>3</em> and negative on polymerase chain reaction for nonstructural region 5 (group 1) corresponded to that of the hepatitis C virus-J group, and those of clones from two patients negative on polymerase chain reaction for nonstructural region <em>3</em> and positive on polymerase chain reaction for nonstructural region 5 (group 2) corresponded to that of hepatitis C virus-K2.(ABSTRACT TRUNCATED AT 250 WORDS)

The type I <em>interferon</em> (IFN) <em>alpha</em> and beta promoters have been a leading paradigm of virus-activated transcriptional regulation for more than two decades, and have contributed substantially to our understanding of virus-inducible gene regulation, the coordinated activities of NF-kappaB and IRF transcription factors, the temporal and spatial recruitment of co-activators to the enhanceosome, and signaling pathways that trigger the innate antiviral response. In 200<em>3</em>, the ISICR Milstein Award was presented to John Hiscott of McGill University and Tom Maniatis of Harvard University for their ongoing research describing the mechanisms of regulation of type 1 <em>interferon</em> genes and specifically for the identification of key signaling kinases involved in phosphorylation of the transcription factors IRF-<em>3</em> and IRF-7. The specific roles played by IRFs and the IKK-related kinases TBK1 and IKKvarepsilon are now recognized within the broader framework of TLR and RIG-I signaling pathways. This review summarizes the unique features of the IKK-related kinases and offers a summary of recent advances in the regulation of the early host response to virus infection.

OBJECTIVE

To conduct a systematic review and meta-analysis of studies that measured cytokine and chemokine levels in individuals with major depressive disorder (MDD) compared to healthy controls (HCs).

METHODS

The PubMed/MEDLINE, EMBASE, and PsycINFO databases were searched up until May <em>3</em>0, 2016. Effect sizes were estimated with random-effects models.

RESULTS

Eighty-two studies comprising <em>3</em>212 participants with MDD and 2798 HCs met inclusion criteria. Peripheral levels of interleukin-6 (IL-6), tumor necrosis factor (TNF)-<em>alpha</em>, IL-10, the soluble IL-2 receptor, C-C chemokine ligand 2, IL-1<em>3</em>, IL-18, IL-12, the IL-1 receptor antagonist, and the soluble TNF receptor 2 were elevated in patients with MDD compared to HCs, whereas <em>interferon</em>-gamma levels were lower in MDD (Hedge's g = -0.477, P = 0.04<em>3</em>). Levels of IL-1β, IL-2, IL-4, IL-8, the soluble IL-6 receptor (sIL-6R), IL-5, CCL-<em>3</em>, IL-17, and transforming growth factor-beta 1 were not significantly altered in individuals with MDD compared to HCs. Heterogeneity was large (I2 : 51.6-97.7%), and sources of heterogeneity were explored (e.g., age, smoking status, and body mass index).

CONCLUSIONS

Our results further characterize a cytokine/chemokine profile associated with MDD. Future studies are warranted to further elucidate sources of heterogeneity, as well as biosignature cytokines secreted by other immune cells.

Infantile haemangiomas (IH) are the most common benign tumours of infancy. Although most IH are innocuous and 85-90% regress spontaneously, some may become life- or function-threatening and require immediate treatment. Previous standard therapeutic options include physical measures (laser surgery, cryosurgery) and systemic corticosteroids, in severe cases also vincristine, <em>alpha</em>-<em>interferon</em> or cyclophosphamide, all bearing the risk of serious side-effects. Oral propranolol is a very recent therapeutic option for complicated IH with impressive efficacy and generally good tolerance. The effects of propranolol on IH were discovered by chance, and very little is known about its mechanisms of action in IH. Here we present a summary of current knowledge of how propranolol interferes with endothelial cells, vascular tone, angiogenesis and apoptosis. Early, intermediate and long-term effects of propranolol on IH can be attributed to three different pharmacological targets. Early effects (brightening of the haemangioma surface within 1-<em>3</em> days after start of therapy) are attributable to vasoconstriction due to decreased release of nitric oxide. Intermediate effects are due to the blocking of proangiogenic signals (vascular endothelial growth factor, basic fibroblast growth factor, matrix metalloproteinase 2/9) and result in growth arrest. Long-term effects of propranolol are characterized by induction of apoptosis in proliferating endothelial cells, and result in tumour regression.

The role(s) of gamma <em>interferon</em> (IFN-gamma), tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), and interleukin-4 (IL-4) in establishment and maintenance of protective immunity to Francisella tularensis LVS in mice (C<em>3</em>H/HeN) was examined by selective removal of these cytokines in vivo with neutralizing antibodies. The 50% lethal dose (LD50) for mice infected intradermally with F. tularensis alone was 1<em>3</em>6,000 CFU; treatment of mice with anti-IFN-gamma or anti-TNF-<em>alpha</em> at the time of infection significantly reduced (P much less than 0.05) the LD50 to 2 and 5 CFU, respectively. Abrogation of protective immunity, however, was effective only when anti-IFN-gamma or anti-TNF-<em>alpha</em> was administered prior to day <em>3</em> postinfection. In contrast, the LD50 for mice treated with anti-IL-4 was repeatedly higher (555,000 CFU) than for controls; this difference, however, was not significant (P greater than 0.05). Thus, IL-4 may be detrimental, while IFN-gamma and TNF-<em>alpha</em> were clearly crucial to the establishment of protective immunity to F. tularensis during a primary infection. The importance of IFN-gamma and TNF-<em>alpha</em> during a secondary immune response to F. tularensis was also investigated. Spleen cells from immune mice passively transfer protective immunity to recipient mice in the absence of confounding antibody-mediated immunity. This passive transfer of immunity, however, was abrogated by treatment of recipient mice with anti-IFN-gamma or anti-TNF-<em>alpha</em> at the time of challenge infection. That anticytokines effectively abrogate protective immunity very early in the course of infection with F. tularensis suggests that T-cell-dependent activation of macrophages for microbicidal activity is unlikely. These T-cell-independent events early in the course of infection may suppress bacterial replication until a T-cell-dependent response ultimately clears the bacteria.

Toll-like receptor (TLR) signal transduction is mediated by an adaptor protein termed MyD88. In the case of TLR2 and TLR4, another adaptor related to MyD88 called Mal also participates in signalling. Two recent papers have added a third adaptor to the family, called Toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing <em>interferon</em>-beta (IFN-beta) (TRIF) or TIR-containing adaptor molecule-1 (TICAM-1), which is particularly important for IFN regulatory factor-<em>3</em> (IRF-<em>3</em>) activation by antiviral TLR<em>3</em>. Two additional adaptors are present in humans, termed Trif-related adaptor molecule (TRAM) and sterile <em>alpha</em> and HEAT-Armadillo motifs (SARM). It is probable that differential use of adaptors will help explain the distinct pathways activated by TLRs during host defence.

We treated seven patients who had progressive hairy-cell leukemia with daily doses of <em>3</em> million units of partially pure <em>alpha</em> (leukocyte) <em>interferon</em> by the intramuscular route. Three patients had a complete remission, and four had a partial remission, according to strict criteria for a response. After treatment, bone-marrow aspirates showed an absence of leukemia cells in three patients and 5 per cent or fewer in three others. Normalization of subnormal peripheral-blood values occurred in six of six patients with anemia, in seven of seven with granulocytopenia, and in four of four with thrombocytopenia. Remissions have been maintained for over 6 to over 10 months. <em>Alpha</em> <em>interferon</em> appears to be highly effective in patients with hairy-cell leukemia.

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I <em>interferon</em> in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary <em>interferon</em>-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-<em>alpha</em> and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-<em>alpha</em> and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-<em>3</em> and IFN-<em>alpha</em>; resiquimod more effectively matures pDC than either IL-<em>3</em> or IFN-<em>alpha</em> alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.

We recently developed a genome-length hepatitis C virus (HCV) RNA replication system (OR6) with luciferase as a reporter. The OR6 assay system has enabled prompt and precise quantification of HCV RNA replication. Pegylated <em>interferon</em> (IFN) and ribavirin combination therapy is the world standard for chronic hepatitis C, but its effectiveness is limited to about 55% of patients. Newer therapeutic approaches are needed. In the present study, we used the OR6 assay system to evaluate the anti-HCV activity of <em>3</em>-hydroxy-<em>3</em>-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, called statins, and their effects in combination with IFN-<em>alpha</em>. Five types of statins (atorvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin) were examined for their anti-HCV activities. Fluvastatin exhibited the strongest anti-HCV activity (IC50: 0.9 micromol/L), whereas atorvastatin and simvastatin showed moderate inhibitory effects. However, lovastatin, reported recently as an inhibitor of HCV replication, was shown to exhibit the weakest anti-HCV activity. The anti-HCV activities of statins were reversed by the addition of mevalonate or geranylgeraniol. Surprisingly, however, pravastatin exhibited no anti-HCV activity, although it worked as an inhibitor for HMG-CoA reductase. The combination of IFN and the statins (except for pravastatin) exhibited strong inhibitory effects on HCV RNA replication. In combination with IFN, fluvastatin also exhibited a synergistic inhibitory effect. In conclusion, statins, especially fluvastatin, could be potentially useful as new anti-HCV reagents in combination with IFN.

A 96-well microtiter infection assay for the human immunodeficiency virus (HIV) is described. The assay utilizes human T-cell lymphotropic virus type I-immortalized MT-2 cells as targets for infection and requires only 4 to 5 days for completion. Cytolysis was quantitated by vital dye uptake of poly-L-lysine-adhered cells as an endpoint for infection. The assay's efficacy was proven by the sensitive and accurate assessment of several known anti-HIV agents including two inhibitors of reverse transcription (<em>3</em>'-azido-<em>3</em>'-deoxythymidine and 2',<em>3</em>'-dideoxycytidine), three biological response modifiers (recombinant <em>interferons</em> <em>alpha</em> and beta and mismatched double-stranded RNA), a direct inactivator of HIV virions (amphotericin B), and neutralizing antibodies from two HIV-positive human subjects. Evaluation of data was facilitated by computer-assisted analysis. This assay provides a means for rapid, sensitive, and inexpensive large-scale in vitro testing of potential anti-HIV therapeutic regimens and quantitation of HIV-neutralizing antibody titers.

OBJECTIVE

alpha-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression.

METHODS

A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis muscle was conducted. Control sides received saline. A 3-day course of methylprednisolone accompanied gene transfer without further immune suppression.

RESULTS

No adverse events were encountered. SGCA gene expression increased 4-5-fold over control sides when examined at 6 weeks (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (AAV1)-neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found, but showed features of programmed cell death. Enzyme-linked immunospot (ELISpot) showed no interferon-gamma response to alpha-SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 subjects successfully achieved study aims, precluding the need for additional enrollment.

CONCLUSIONS

The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials.

C-type lectin receptors (CLRs) fulfill multiple functions within the immune system by recognition of carbohydrate moieties on foreign or (altered) self-structures. CLRs on myeloid dendritic cells (DCs) have been well characterized as pattern-recognition receptors (PRRs) combining ligand internalization with complex signaling events. Much less is known about CLR expression and function in human plasmacytoid DCs (pDCs), the major type I <em>interferon</em> (IFN) producers. In this study, we demonstrate that, next to the CLR BDCA-2, human pDCs express DC immunoreceptor (DCIR), a CLR with putative immune-inhibitory function, but not dectin-1, mannose receptor, or DC-specific ICAM-<em>3</em>-grabbing nonintegrin. DCIR surface levels are reduced on pDC maturation after TLR9 triggering. Interestingly, DCIR triggering inhibits TLR9-induced IFN-<em>alpha</em> production while leaving up-regulation of costimulatory molecule expression unaffected. Furthermore, DCIR is readily internalized into pDCs after receptor triggering. We show that DCIR internalization is clathrin-dependent because it can be inhibited by hypertonic shock and dominant-negative dynamin. Importantly, antigens targeted to pDCs via DCIR are presented to T cells. These findings indicate that targeting DCIR on pDCs not only results in efficient antigen presentation but also affects TLR9-induced IFN-<em>alpha</em> production. Collectively, the data show that targeting of DCIR can modulate human pDC function and may be applied in disease prevention and treatment.

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma <em>interferon</em> (IFN-gamma), IFN-<em>alpha</em>/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with <em>3</em> immunoglobulin domains that functions as a soluble receptor for IFN-<em>alpha</em>/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-<em>alpha</em>/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-<em>alpha</em>/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-<em>alpha</em>, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-<em>alpha</em>/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-<em>alpha</em>/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-<em>alpha</em>/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-<em>alpha</em>/beta against virus infections.

TAK-242, a small-molecule antisepsis agent, has shown to suppress lipopolysaccharide (LPS)-induced inflammation. In this study, we demonstrate that TAK-242 is a selective inhibitor of Toll-like receptor (TLR)-4 signaling. TAK-242 almost completely suppressed production of nitric oxide (NO) or tumor necrosis factor (TNF)-<em>alpha</em> induced by a TLR4-specific ligand, ultra-pure LPS, in mouse RAW264.7, human U-9<em>3</em>7 and P<em>3</em>1/FUJ cells, whereas this agent showed little effect on other TLR ligands, Pam(<em>3</em>)CSK(4) (TLR1/2), peptidoglycan (TLR2/6), double strand RNA (TLR<em>3</em>), R-848 (TLR7) and CpG oligonucleotide (TLR9). Furthermore, TAK-242 potently inhibited nuclear factor (NF)-kappaB activation induced by ultra-pure LPS in HEK29<em>3</em> cells transiently expressing TLR4 and co-receptors, myeloid differentiation protein-2 (MD2) and CD14, whereas this agent showed little effect on other TLRs, TLR1/2, TLR2/6, TLR<em>3</em>, TLR5, TLR7 and TLR9. TAK-242 also inhibited ligand-independent NF-kappaB activation resulting from over-expression of TLR4. Although chimera receptors, which are consist of the extracellular domain of CD4 and the intracellular domain of human or mouse TLR4, showed constitutive NF-kappaB activation, TAK-242 potently inhibited the signaling from CD4-TLR4 chimera receptors. In contrast, the NF-kappaB activation mediated by TLR4 adaptors, myeloid differentiation factor 88 (MyD88), TIR-associated protein (TIRAP), Toll/IL-1R homology (TIR)-domain-containing adaptor protein-inducing <em>interferon</em>-beta (TRIF) or TRIF-related adaptor molecule (TRAM) was not affected by TAK-242. TAK-242 is therefore a selective inhibitor of signaling from the intracellular domain of TLR4 and represents a novel therapeutic approach to the treatment of TLR4-mediated diseases.

Infection with Mycobacterium tuberculosis is controlled by an efficacious immune response in about 90% of infected individuals who do not develop disease. Although essential mediators of protection, e.g., <em>interferon</em>-gamma, have been identified, these factors are insufficient to predict the outcome of M. tuberculosis infection. As a first step to determine additional biomarkers, we compared gene expression profiles of peripheral blood mononuclear cells from tuberculosis patients and M. tuberculosis-infected healthy donors by microarray analysis. Differentially expressed candidate genes were predominantly derived from monocytes and comprised molecules involved in the antimicrobial defense, inflammation, chemotaxis, and intracellular trafficking. We verified differential expression for <em>alpha</em>-defensin 1, <em>alpha</em>-defensin 4, lactoferrin, Fcgamma receptor 1A (cluster of differentiation 64 [CD64]), bactericidal permeability-increasing protein, and formyl peptide receptor 1 by quantitative polymerase chain reaction analysis. Moreover, we identified increased protein expression of CD64 on monocytes from tuberculosis patients. Candidate biomarkers were then assessed for optimal study group discrimination. Using a linear discriminant analysis, a minimal group of genes comprising lactoferrin, CD64, and the Ras-associated GTPase <em>3</em><em>3</em>A was sufficient for classification of (1) tuberculosis patients, (2) M. tuberculosis-infected healthy donors, and (<em>3</em>) noninfected healthy donors.

<em>Interferon</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>), the primary transcription factor induced by <em>interferon</em> <em>alpha</em>, is a complex of four (11<em>3</em>, 91, 84, and 48 kd) proteins. This paper reports that the 11<em>3</em>, 91, and 84 kd (ISGF<em>3</em> <em>alpha</em>) proteins of ISGF<em>3</em> contain conserved SH2 and SH<em>3</em> domains. A specific <em>interferon</em> <em>alpha</em>-induced cytoplasmic protein tyrosine kinase(s) can form a transient complex with ISGF<em>3</em> <em>alpha</em> proteins. These ISGF<em>3</em> <em>alpha</em> proteins can be immunoprecipitated by anti-phosphotyrosine antibodies only after <em>interferon</em> <em>alpha</em> treatment. Phosphoamino acid analyses of <em>3</em>2P-labeled ISGF<em>3</em> <em>alpha</em> proteins confirm that ISGF<em>3</em> <em>alpha</em> proteins are directly tyrosine phosphorylated both in vitro and in vivo in response to <em>interferon</em> <em>alpha</em>, and this tyrosine phosphorylation can be inhibited by staurosporine and genistein. Phosphatase treatment of these ISGF<em>3</em> <em>alpha</em> proteins results in inhibition of ISGF<em>3</em> complex formation in vitro. These observations indicate that <em>interferon</em> <em>alpha</em>-induced direct tyrosine phosphorylation of ISGF<em>3</em> <em>alpha</em> proteins is necessary for activation of the transcription factor ISGF<em>3</em>.

OBJECTIVE

To determine the toxicity and the therapeutic efficacy of the combination of the recombinant tumor necrosis factor alpha (rTNF alpha), recombinant interferon gamma (rIFN-gamma), and melphalan, we designed a protocol using isolation limb perfusion (ILP) with hyperthermia for in-transit metastases of melanoma and recurrent sarcoma. The triple combination was chosen because of the reported synergistic antitumor effect of rTNF alpha with IFN-gamma and of rTNF alpha with alkylating agents.

METHODS

Twenty-three patients received a total of 25 ILPs with the triple combination. There were 19 females and four males with either multiple progressive in-transit melanoma metastases of the extremities (stage IIIa or IIIab; 19 patients) or recurrent soft tissue sarcoma (five). The rTNF alpha was injected as a bolus in the arterial line, and total dose ranged between 2 and 4 mg, under hyperthermic conditions (40 degrees C to 40.5 degrees C) for 90 minutes. The rIFN-gamma was given subcutaneously (SC) on days -2 and -1 and in the perfusate, with rTNF alpha at the dose of 0.2 mg. Melphalan (Alkeran; Burroughs Wellcome Co, London, England) was administered in the perfusate at 40 micrograms/mL.

RESULTS

Toxicity observed during three ILPs in a pilot study with rTNF alpha included only two severe toxicities: one severe hypotension with tachycardia and transient oliguria and one moderate hypotension for 4 hours followed by severe kidney failure with complete recovery on day 29. In all 18 ILPs performed in the triple combination protocol, the patients received continuous infusion dopamine at 3 micrograms/kg/min from the start of ILP and for 72 hours and showed only mild hypotension and transient chills and temperature. Regional toxicity attributable to rTNF alpha was minimal. There have been 11 cases with hematologic toxicity consisting of neutropenia (one grade 4 and one grade 3) and neutropenia with thrombocytopenia (one grade 4 and three grade 2). Twelve patients had been previously treated with melphalan in ILP (11) or with cisplatin (one). The 23 patients are assessable: there have been 21 complete responses (CRs; range, 4 to 29 months; 89%), two partial responses (PRs; range, 2 to 3 months), and no failures. Overall disease-free survival and survival have been 70% and 76%, respectively, at 12 months. In all cases, softening of the nodules was obvious within 3 days after ILP and time to definite response ranged between day 5 and 30.

CONCLUSIONS

This preliminary analysis of a phase II study suggests that high-dose rTNF alpha can be administered with acceptable toxicity by ILP with dopamine and hyperhydration. Tumor responses can be evidenced in melanoma and sarcoma. Furthermore, combination of rTNF alpha, rIFN-gamma, and melphalan seems to achieve high efficacy with minimal toxicity, even after failure of prior therapy with melphalan alone.

The genes encoding nearly all of the serologically defined class II antigens of the major histocompatibility complex have been isolated. Three class II loci have been studied in great detail. The DR region contains a single <em>alpha</em> gene and <em>3</em> beta chain genes, 1 of which is a pseudogene. The DR <em>alpha</em> chain gene has been linked to a DR beta gene which encodes a beta protein which contains the serological determinant MT<em>3</em>. A second cosmid cluster contains 2 beta genes, 1 of which encodes the DR4 allospecificity. The identification of these genes has been made by the comparison of amino terminal sequences of DR molecules obtained from a DR4 cell line and the deduced protein sequences of the beta 1 exons from cosmid and phage clones. A conserved element including the promoter and signal sequence is found at the 5' end of each of the <em>3</em> DR beta genes. Additionally, this element occurs three more times in the DR region, raising the question of whether additional beta chain genes might be found. The DQ region contains 2 pairs of genes, 1 of which encodes the DQ antigen. The 2nd pair of genes, called DX <em>alpha</em> and beta, appears to be capable of expressing a DQ-related product, although, to date, there is no evidence for its expression. The DP region also contains 2 pairs of genes. One pair encodes the DP antigen while the 2nd <em>alpha</em>-beta pair is shown to be composed of pseudogenes. The location of polymorphic regions in these genes and aspects of their relationship to the serology, evolution, and function of the class II MHC are discussed. The control of expression of class II genes by gamma-<em>interferon</em> has been examined. The promoters of class II genes are characterized by two conserved sequences common to all <em>alpha</em> and beta chain genes as well as by conserved sequences specific for either <em>alpha</em> or beta chain genes. In addition to studies of expression by DNA-mediated gene transformation, a system for the gene transfer of MHC antigens utilizing transmissible retrovirus vectors is described. Retrovirus vectors have been used to transmit DR <em>alpha</em>, DR beta, and the invariant chain (gamma) sequences to recipient cells with resultant expression of these proteins.

We have previously shown that Toll-like receptor (TLR)-activated murine nonparenchymal liver cells [(NPC); Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC)] can suppress hepatitis B virus (HBV) replication. Therefore, the aim of this study was to investigate whether HBV has the ability to counteract the TLR-mediated control of its replication. Freshly purified murine hepatocytes and NPCs obtained from C57BL6 mice were stimulated by TLR 1-9 ligands in the presence or absence of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), HBV virions, or supernatants from HBV-producing HBV-Met cells, and HBV replication was suppressed by anti- hepatitis B virus X protein (HBx) small interfering RNA (siRNA) in HBV-Met cells. Supernatants were collected and tested for antiviral cytokines by viral protection assay. HBV gene expression and replication was analyzed by southern blot. RNA and proteins were analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) or western blot and enzyme-linked immunosorbent assay, respectively. Pretreatment of hepatocytes and NPCs with HBV-Met cells supernatants, HBsAg, HBeAg, or HBV virions almost completely abrogated TLR-induced antiviral activity, which correlated with suppression of <em>interferon</em> beta (IFN-beta) production and subsequent <em>interferon</em>-stimulated gene induction as well as suppressed activation of <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>), nuclear factor kappa B (NF-kappaB), and extracellular signal-regulated kinase (ERK) 1/2. In HBV-infected HBV-Met cells, TLR stimulation did not induce antiviral cytokines in contrast to primary hepatocytes. TLR-stimulated expression of proinflammatory cytokines [tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), interleukin-6 (IL-6)], and activation of IRF-<em>3</em> was suppressed after up-regulation of HBV replication in HBV-Met cells. Accordingly, suppression of HBV replication by siRNA led to activation or expression of proinflammatory transcription factors and cytokines.

CONCLUSIONS

Our data indicate that HBV can suppress the TLR-induced antiviral activity of liver cells. This has major implications for the interaction between HBV and the immune system.

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-<em>3</em>-hydroxyphthioceranoyl-2'-sulfate-<em>alpha</em>-<em>alpha</em>'-D-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release <em>interferon</em> gamma, efficiently recognize M. tuberculosis-infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

OBJECTIVE

The authors assessed the relationship between the hypothalamic-pituitary-adrenal (HPA) axis response to interferon-alpha (IFN-alpha) and the development of major depression during IFN-alpha treatment.

METHODS

Adrenocorticotropic hormone (ACTH), cortisol, and interleukin-6 (IL-6) plasma concentrations were measured in 14 patients with malignant melanoma at regular intervals during the first 12 weeks of IFN-alpha therapy, both immediately before and 1, 2, and 3 hours after IFN-alpha administration. Symptom criteria for major depression were also evaluated at each visit.

RESULTS

ACTH and cortisol responses but not IL-6 responses to the initial administration of IFN-alpha were significantly higher in the seven patients who subsequently developed symptom criteria for major depression than in those who did not. No differences in hormonal or cytokine responses were found between these two groups during chronic IFN-alpha administration.

CONCLUSIONS

The HPA axis response to the acute administration of IFN-alpha reveals a vulnerability to IFN-alpha-induced depression, possibly due to sensitization of corticotropin-releasing factor pathways.

Production of <em>alpha</em>/beta <em>interferons</em> (IFN-<em>alpha</em>/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-<em>alpha</em>/beta, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-kappaB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-<em>3</em>. Furthermore, both in cultured cells and in mice lacking a functional IFN-<em>alpha</em>/beta system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-<em>alpha</em>/beta in order to increase the virulence of Bunyamwera virus.