Inflammatory stimuli and lipid peroxidation activate nuclear factor kappa B (NF-kappaB) and upregulate proinflammatory cytokines and chemokines. The present study evaluated the relationship between pathological liver injury, endotoxemia, lipid peroxidation, and NF-kappaB activation and imbalance between pro- and anti-inflammatory cytokines. Rats (5 per group) were fed ethanol and a diet containing saturated fat, palm oil, corn oil, or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. Pathological analysis was performed and measurements of endotoxin were taken, lipid peroxidation, NF-kappaB, and messenger RNA (mRNA) levels of proinflammatory cytokines (tumor necrosis factor-alpha [TNFalpha], interleukin-1 beta [IL-1beta], interferon-gamma, [IFN-gamma], and IL-12), C-C chemokines (regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein [MCP]-1, macrophage inflammatory protein [MIP]-1alpha), C-X-C chemokines (cytokine induced neutrophil chemoattractant (CINC), MIP-2, IP-10, and epithelial neutrophil activating protein [ENA]-78), and anti-inflammatory cytokines (IL-10, IL-4, and IL-13). Activation of NF-kappaB and increased expression of proinflammatory cytokines C-C and C-X-C chemokines was seen in the rats exhibiting necroinflammatory injury (fish oil-ethanol [FE] and corn oil-ethanol[CE]). These groups also had the highest levels of endotoxin and lipid peroxidation. Levels of IL-10 and IL-4 mRNA were lower in the group exhibiting inflammatory liver injury. Thus, activation of NF-kappaB occurs in the presence of proinflammatory stimuli and results in increased expression of proinflammatory cytokines and chemokines. The Kupffer cell is probably the major cell type showing activation of NF-kappaB although the contribution of endothelial cells and hepatocytes cannot be excluded. Downregulation of anti-inflammatory cytokines may additionally exacerbate liver injury.

A majority of angiogenic factors has been shown to be produced by macrophages. This review will give a concise description of their biochemical nature, their isolation from macrophages and their angiogenic activity. Among the factors with mitogenic effects on endothelial cells are basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF-alpha) and very probably insulin-like growth factor-1 (IGF-1). Other secretory products such as angiotropin and human angiogenic factor (HAF) are nonmitogenic but promote angiogenesis by inducing migration of endothelial cells. Prostaglandins, platelet-derived growth factor (PDGF), granulocyte-macrophage- and granulocyte-colony stimulating factor (GM-CSF, G-CSF), interleukin 6 (IL-6) and angiotensin converting enzyme (ACE) have also been shown to be angiogenic, but their mode of action is still to be clearly defined. As the extracellular matrix appears to be involved in the control of angiogenesis, macrophage-derived factors that can alter this structure via degradation or via the clotting system will also be discussed. Tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and transforming growth factor-beta (TGF-beta) have complex actions on endothelial cells, and can partially inhibit angiogenesis. Among the factors which solely inhibit neovascularization are the interferons. As it is not known whether all of these factors play a role in angiogenesis in vivo attempts to detect them in situ during the course of neovascularization will be described. Finally macrophages will be discussed as cells that may not be mandatory for each phase of the angiogenic process but whose angiogenic capabilities are comprehensive and unsurpassed by any other cell.

Ly-1 B cells have the distinctive property of continuous self-replenishment and, as we have shown previously, can be further distinguished from conventional B cells on the basis of greatly elevated constitutive and inducible production of the recently described cytokine interleukin 10 (IL-10). To test the possibility that IL-10 acts as either an autocrine or paracrine growth factor for Ly-1 B cells, we treated mice continuously from birth to 8 wk of age with a monoclonal rat IgM antibody that specifically neutralizes mouse IL-10. Mice treated in this way lacked peritoneal-resident Ly-1 B cells, contained greatly reduced serum immunoglobulin M levels, and were unable to generate significant in vivo antibody responses to intraperitoneal injections of alpha 1,3-dextran or phosphorylcholine, antigens for which specific B cells reside in the Ly-1 B cell subset. In contrast, conventional splenic B cells of anti-IL-10-treated mice were normal with respect to total numbers, phenotype, and in vitro responsiveness to B cell mitogens and the thymus-dependent antigen trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). The mechanism of Ly-1 B cell depletion appeared to be related to elevation of endogenous interferon gamma (IFN-gamma) levels in anti-IL-10-treated mice, since coadministration of neutralizing anti-IFN-gamma antibodies substantially restored the number of peritoneal-resident Ly-1 B cells in these mice. These results implicate IL-10 as a regulator of Ly-1 B cell development, and identify a procedure to specifically deplete Ly-1 B cells, thereby allowing further evaluation of the role of these cells in the immune system.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV; also called human herpesvirus 8) is believed to be the etiologic agent of Kaposi's sarcoma, multicentric Castleman's disease, and AIDS-associated primary effusion lymphoma. KSHV infection of human dermal microvascular endothelial cells (DMVEC) in culture results in the conversion of cobblestone-shaped cells to spindle-shaped cells, a characteristic morphological feature of cells in KS lesions. All spindle-shaped cells in KSHV-infected DMVEC cultures express the latency-associated nuclear protein LANA1, and a subfraction of these cells undergo spontaneous lytic cycle induction that can be enhanced by tetradecanoyl phorbol acetate (TPA) treatment. To study the cellular response to infection by KSHV, we used two different gene array screening systems to examine the expression profile of either 2,350 or 9,180 human genes in infected compared to uninfected DMVEC cultures in both the presence and absence of TPA. In both cases, between 1.4 and 2.5% of the genes tested were found to be significantly upregulated or downregulated. Further analysis by both standard and real-time reverse transcription-PCR procedures directly confirmed these results for 14 of the most highly upregulated and 13 of the most highly downregulated genes out of a total of 37 that were selected for testing. These included strong upregulation of interferon-responsive genes such as interferon response factor 7 (IRF7) and myxovirus resistance protein R1, plus upregulation of exodus 2 beta-chemokine, RDC1 alpha-chemokine receptor, and transforming growth factor betaalpha(4)-integrin and fibronectin plus downregulation of bone morphogenesis protein 4, matrix metalloproteinase 2, endothelial plasminogen activator inhibitor 1, connective tissue growth factor, and interleukin-8. Significant dysregulation of several other cytokine-related genes or receptors, as well as endothelial cell and macrophage markers, and various other genes associated with angiogenesis or transformation was also detected. Western immunoblot and immunohistochemical analyses confirmed that the cellular IRF7 protein levels were strongly upregulated during the early lytic cycle both in KSHV-infected DMVEC and in the body cavity-based lymphoma BCBL1 PEL cell line.

Expression of the high-affinity receptor for IgG (Fc gamma RI) is restricted to cells of myeloid lineage and is induced by gamma-interferon (IFN-gamma) but not by IFN-alpha/beta. The organization of the human Fc gamma RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-gamma induction are reported here. A 39-nucleotide sequence (IFN-gamma response region, or GRR) is defined that is both necessary and sufficient for IFN-gamma inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class II genes: i.e., X, H, and gamma-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-gamma indicates that the presence of these elements is a general feature of IFN-gamma-responsive genes. Our studies suggest that the combination of X, H, and gamma-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Five of the 10 human Toll-like receptors (TLRs) (TLR3, TLR4, TLR7, TLR8, and TLR9), and four of the 12 mouse TLRs (TLR3, TLR4, TLR7, TLR9) can trigger interferon (IFN)-alpha, IFN-beta, and IFN-lambda, which are critical for antiviral immunity. Moreover, TLR3, TLR7, TLR8, and TLR9 differ from TLR4 in two particularly important ways for antiviral immunity: they can be activated by nucleic acid agonists mimicking compounds produced during the viral cycle, and they are typically present within the cell, along the endocytic pathway, where they sense viral products in the intraluminal space. Investigations in mice have demonstrated that the TLR7/9-IFN and TLR3-IFN pathways are different and critical for protective immunity to various experimental viral infections. Investigations in humans with interleukin-1 receptor-associated kinase-4 (IRAK-4) deficiency (unresponsive to TLR7, TLR8, and TLR9), UNC-93B deficiency (unresponsive to TLR3, TLR7, TLR8, and TLR9), and TLR3 deficiency have recently shed light on the role of these two pathways in antiviral immunity in natural conditions. UNC-93B- and TLR3-deficient patients appear to be specifically prone to herpes simplex virus 1 (HSV-1) encephalitis, although clinical penetrance is incomplete, whereas IRAK-4-deficient patients appear to be normally resistant to most viruses, including HSV-1. These experiments of nature suggest that the TLR7-, TLR8-, and TLR9-dependent induction of IFN-alpha, IFN-beta, and IFN-lambda is largely redundant in human antiviral immunity, whereas the TLR3-dependent induction of IFN-alpha, IFN-beta, and IFN-lambda is critical for primary immunity to HSV-1 in the central nervous system in children but redundant for immunity to most other viral infections.

Despite advances in critical care medicine, mortality from sepsis in ICU patients remains high. In response to several infectious and non-infectious stimuli, monocytes/ macrophages release a number of mediators, including cytokines, involved in the proinflammatory response that underlies sepsis. The excessive release of these mediators results in the development of whole body inflammation, and plays an important role in the pathogenesis of sepsis and septic shock. In addition, patients with sepsis also undergo an anti-inflammatory phase (the compensatory anti-inflammatory response syndrome) and at times, a mixed response with both pro-and anti-inflammatory components (the mixed antagonistic response syndrome). The initial systemic hyperinflammation is caused by production of inflammatory cytokines, especially tumour necrosis factor-a (TNF-alpha), and also interleukin-1 (IL-1), IL-6, and interferon gamma, which act synergistically with TNF-alpha in inducing shock in animal models. However, clinical trials aimed at downregulating these mediators using antibodies against endotoxin, TNF-alpha, antagonists of IL-1 or platelet activating factor have proved to be uniformly disappointing. Not only have these agents been found to have no effect, but they may also increase mortality. One of the reasons for such failure may be the lack of precise immunological monitoring during the course of sepsis. We have recently demonstrated that sepsis shows a biphasic immunological pattern during the initial and later phase: the early hyperinflammatory phase is counterbalanced by an anti-inflammatory response which may lead to a hypoinflammatory state. The latter is associated with immunodeficiency that is characterised by monocytic deactivation, so-called immunoparalysis. Interferon gamma-1 b has an immunoregulatory effect in patients with immunoparalysis during the compensatory anti-inflammatory response syndrome, not only restoring levels of HLA-DR expression but also reestablishing the ability of monocytes to secrete cytokines such as TNF-alpha. By monitoring immune status in septic patients, targeted intervention may lead to more success in immunomodulation of sepsis.

OBJECTIVE

The aims of alpha-interferon treatment for chronic viral liver infections are clearance of the virus and healing of the disease. Hepatocellular carcinoma is a complication of viral cirrhosis; but it is not yet known whether treatment of viral cirrhosis with alpha-interferon prevents this complication.

METHODS

The incidence and the risk (Cox regression analysis) of developing hepatocellular carcinoma were calculated in 347 patients with hepatic cirrhosis; 227 (34 hepatitis B virus and 193 hepatitis C virus related) were treated with alpha-interferon and 120 (28 hepatitis B virus and 92 hepatitis C virus) did not receive this treatment, in order to evaluate the efficacy of alpha-interferon in the prevention of hepatocellular carcinoma. In all patients, the cirrhosis was well compensated (Child A).

RESULTS

Over mean follow-up periods of 49 months for hepatitis B virus and 32 months for hepatitis C virus, 20/347 patients (6/62 hepatitis B virus and 14/285 hepatitis C virus) developed hepatocellular carcinoma. The risk of developing this tumor was significantly greater in males (p < 0.007) and in patients not treated with alpha-interferon (p < 0.01). The Relative Risk of developing hepatocellular carcinoma increased significantly (p < 0.0002) with each passing year. In patients with hepatic cirrhosis secondary to hepatitis B virus infections, the risk did not seem to be modified by alpha-interferon treatment, even though a greater, but not significant risk (Relative Risk = 4.9; p = 0.3) was calculated for untreated patients; in contrast, in hepatitis C virus-related cirrhosis, this risk was reduced by a factor of 4.0 (p = 0.04). The tumor developed only in non-responder patients regardless of virus type. After adjustment for confounding factors (sex, age, alcohol consumption, cigarette smoking), a statistically significant (p < 0.025) effect of interferon treatment in preventing hepatocellular carcinoma was still demonstrated when responders were matched with controls, but not when responders were compared with non-responders.

CONCLUSIONS

These results show that, in addition to its ability to halt the progression of viral-induced liver disease, alpha-interferon is also of benefit in patients with hepatitis C virus cirrhosis who respond to this treatment by lowering their risk of developing hepatocellular carcinoma.

The lymphokine profiles were determined in the skin lesions of the three distinct clinical forms of American cutaneous leishmaniasis (ACL), using a reverse transcriptase polymerase chain reaction (RT-PCR) and primers for various lymphokines. The message for interferon-gamma (IFN-gamma), tumour necrosis factor-beta (TNF-beta), and IL-8 was expressed in the three clinical forms of ACL. IL-1 beta mRNA was expressed in most localized (LCL) and mucocutaneous (MCL) leishmaniasis, but in only few of the diffuse cutaneous leishmaniasis (DCL). IL-2 mRNA was detected in about half of the lesions, with more prominent values for MCL. IL-4 mRNA was present in most lesions from the three clinical forms, but markedly increased in DCL. IL-5 and IL-10 mRNAs were expressed in all MCL and in half of the DCL lesions and weakly expressed in LCL lesions. IL-10 mRNA was more abundant in MCL lesions. In contrast, IL-6 and TNF-alpha mRNAs were expressed in a large number of LCL. In MCL, IL-6 mRNA was expressed in most cases and TNF-alpha mRNA in all the cases. In DCL, IL-6 mRNA was absent and TNF-alpha mRNA was weakly expressed. These results suggest that most T cells present in the MCL and DCL lesions secrete a mixture of type 1 and type 2 cytokine patterns, but in DCL granulomas type 2 cytokines predominate. In LCL the cytokine patterns show a mixture of type 1 and type 0 with a preponderance of IFN-gamma over IL-4, and low levels of IL-5 and IL-10. The lack of IL-6 and TNF-alpha mRNAs, and the low expression of IL-1 beta in DCL lesions suggest a defect in the antigen-processing cells that may account for the state of unresponsiveness in these patients.

Treatment of human diploid fibroblastic cells with interferon induces the synthesis of two guanylate binding proteins (GBP) with molecular weights of 67,000 and 56,000. The Mr = 67,000 protein (67K GBP) is synthesized upon treatment with either alpha-, beta-, or gamma-interferon. Among these interferons, gamma-interferon induces a higher level of 67K GBP synthesis. The 67K GBP synthesized in either beta- or gamma-interferon-treated cells has two charge forms with isoelectric points of 6.0 and 5.8, respectively. The synthesis of the Mr = 56,000 protein is induced by the treatment using either alpha- or beta-interferon, but its synthesis in gamma-interferon-treated cells is undetectable. The amounts of the radioactive GBPs synthesized in human fibroblasts are proportional to the amounts of the purified beta-interferon used for the inductions. Syntheses of GBPs require the transcription of cellular genes because their syntheses are completely blocked by actinomycin D treatments. The mRNA for the 67K GBP is found in fibroblasts that are treated by either alpha-, beta-, or gamma-interferon, but it is not detected in untreated cells. More 67K GBP mRNA is accumulated in the gamma-interferon-treated than in alpha- or beta-interferon-treated fibroblasts. This is consistent with more 67K GBP synthesis found in gamma-interferon-treated fibroblasts.

The long-term effect of interferon alfa (IFN-alpha) in Chinese patients with chronic hepatitis B infection is unknown. A total of 411 chronic hepatitis B patients (208 treated with IFN-alpha and 203 as control) were followed up for hepatitis B serology and the development of hepatoma and other cirrhosis-related complications. The hepatitis B e antigen (HBeAg) seroconversion rate in the IFN-alpha-treated group, though significantly greater at 6 and 24 months, was comparable with the control group on subsequent follow-up, irrespective of pretreatment alanine transaminase (ALT) levels. HBeAg seroreversion rate was higher in the IFN-alpha group compared with the control group (21.1% vs. 2.2%; P =.001). Loss of hepatitis B surface antigen (HBsAg) occurred in 2.4% of the IFN-alpha-treated patients and 0.49% of the control patients (P = NS). Around 90% of the anti-HBe-positive patients in both groups were still hepatitis B virus (HBV)-DNA-positive by polymerase chain reaction (PCR) assay. Two patients suffered from hepatic reactivation during the course of treatment. Nine (4.3%) patients in the IFN-alpha group and 2 (1.0%) in the control group developed complications of cirrhosis and hepatoma (P =.062). In Chinese HBsAg carriers, IFN-alpha was of no long-term benefit in inducing HBeAg seroconversion or in the prevention of hepatoma and other cirrhosis-related complications.

The role of innate, alpha/beta interferon (IFN-alpha/beta)-dependent protection versus specific antibody-mediated protection against vesicular stomatitis virus (VSV) was evaluated in IFN-alpha/beta receptor-deficient mice (IFN-alpha/beta R0/0 mice). VSV is a close relative to rabies virus that causes neurological disease in mice. In contrast to normal mice, IFN-alpha/beta R0/0 mice were highly susceptible to infection with VSV because of ubiquitous high viral replication. Adoptive transfer experiments showed that neutralizing antibodies against the glycoprotein of VSV (VSV-G) protected these mice efficiently against systemic infection and against peripheral subcutaneous infection but protected only to a limited degree against intranasal infection with VSV. In contrast, VSV-specific T cells or antibodies specific for the nucleoprotein of VSV (VSV-N) were unable to protect IFN-alpha/beta R0/0 mice against VSV. These results demonstrate that mice are extremely sensitive to VSV if IFN-alpha/beta is not functional and that under these conditions, neutralizing antibody responses mediate efficient protection, but apparently only against extraneuronal infection.

Interferon regulatory factor-3 (IRF-3) activation directs alpha/beta interferon production and interferon-stimulated gene (ISG) expression, which limits virus infection. Here, we examined the distribution of hepatitis C virus (HCV) nonstructural 3 protein, the status of IRF-3 activation, and expression of IRF-3 target genes and ISGs during asynchronous HCV infection in vitro and in liver biopsies from patients with chronic HCV infection, using confocal microscopy and functional genomics approaches. In general, asynchronous infection with HCV stimulated a low-frequency and transient IRF-3 activation within responsive cells in vitro that was associated with cell-to-cell virus spread. Similarly, a subset of HCV patients exhibited the nuclear, active form of IRF-3 in hepatocytes and an associated increase in IRF-3 target gene expression in hepatic tissue. Moreover, ISG expression profiles formed disease-specific clusters for HCV and control nonalcoholic fatty liver disease patients, with increased ISG expression among the HCV patients. We identified the presence of T cell and plasmacytoid dendritic cell infiltrates within all biopsy specimens, suggesting they could be a source of hepatic interferon in the setting of hepatitis C and chronic inflammatory condition.

CONCLUSIONS

These results indicate that HCV can transiently trigger IRF-3 activation during virus spread and that in chronic HCV, IRF-3 activation within infected hepatocytes occurs but is limited.

OBJECTIVE

Little is known about the long-term effects of interferon alpha on clinical outcome and survival of patients with chronic hepatitis D.

METHODS

Thirty-six patients with chronic hepatitis D who participated in a randomized controlled trial of a 48-week course of high (9 million units) or low (3 million units) doses of interferon alpha or no treatment were followed for an additional 2 to 14 years.

RESULTS

Long-term survival was significantly longer in the high-dose group than in untreated controls (P = 0.003) or in the low-dose group (P = 0.019) but did not differ between patients treated with 3 million units and controls. Among surviving patients at 12 years of follow-up, a biochemical response was present in 7 of 12 treated with 9 million units, in 2 of 4 who received 3 million units, and in none of 3 controls. Long-term alanine aminotransferase (ALT) normalization correlated with improved hepatic function and loss of IgM antibody to hepatitis delta antigen (anti-HD). Patients in the high-dose group had a sustained decrease in HDV replication (P = 0.008), leading to clearance of HDV RNA and, eventually, hepatitis B virus (HBV) in some patients, as well as a dramatic improvement in liver histology with respect to activity grade (P = 0.0004) and fibrosis stage (P = 0.007). Strikingly, we documented an absence of fibrosis in the final biopsy of 4 patients with a long-term biochemical response and an initial diagnosis of active cirrhosis.

CONCLUSIONS

High doses of interferon alpha-2a significantly improved the long-term clinical outcome and survival of patients with chronic hepatitis D, even though the majority had active cirrhosis before the onset of therapy.

This work aims to demonstrate that CD4(+)CD56(+) malignancies arise from transformed cells of the lymphoid-related plasmacytoid dendritic cell (pDC) subset. The analysis of malignant cells from 7 patients shows that in all cases, like pDCs, leukemic cells are negative for lineage markers CD3, CD19, CD13, CD33, and CD11c but express high levels of interleukin-3 receptor alpha chain (IL-3Ralpha), HLA-DR, and CD45RA. Tumor cells produce interferon-alpha in response to influenza virus, while upon maturation with IL-3 they become a powerful inducer of naive CD4(+) T-cell proliferation and promote their T-helper 2 polarization. As pDCs, leukemic cells also express pre-Talpha and lambda-like 14.1 transcripts, arguing in favor of a lymphoid origin. In addition, malignant cells express significant levels of CD56 and granzyme B. Overall, those observations suggest that CD4(+)CD56(+) leukemic cells could represent the malignant counterpart of pDCs, both of which are closely related to B, T, and NK cells.

Recent findings from animal models suggest that the bone loss induced by estrogen deficiency may stem in large measure from a pathological upregulation of the adaptive immune response. While the role of activated T cells in the osteoporosis driven by inflammatory conditions and infection has been well documented, only recently has the role of T cells in the bone destruction associated with estrogen deficiency begun to be appreciated. In vivo and in vitro models of postmenopausal osteoporosis demonstrate that estrogen deficiency leads to an increase in the adaptive immune function that culminates in an increased production of tumor necrosis factor alpha (TNF) by activated T cells. TNF increases osteoclast (OC) formation and bone resorption both directly and by augmenting the sensitivity of maturing OCs to the essential osteoclastogenic factor receptor activator of nuclear factor kappaB ligand. The activation and expansion of TNF-producing T cells are key steps in estrogen deficiency-driven bone loss and are regulated by multiple interacting cytokines including transforming growth factor-beta, interleukin-7, and interferon-gamma, as well as by the process of antigen presentation. Herein, we review the experimental evidence that suggests estrogen prevents bone loss by regulating T-cell function and immune cell bone interactions.

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers maturation and release of the pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) in monocytes and macrophages. We report that interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) synergistically induce P2X7R mRNA and functional responses in the human THP-1 monocytic cell line. Induction was dose dependent, with maximal functional activity requiring 1000 units/mL IFN-gamma and 10 ng/mL TNF-alpha and incubations of 36-72 h. The up-regulation of P2X7R function by lipopolysaccharide (LPS)/IFN-gamma and TNF-alpha/IFN-gamma was markedly attenuated by coincubation with prostaglandin E2 or the cell permeant cyclic AMP analog dibutryl cAMP (Bt2cAMP). Bt2cAMP did not significantly alter P2X7 function in HEK-293 cells stably transfected with the human P2X7 cDNA, indicating that Bt2cAMP does not exert a generalized effect on P2X7R synthesis or downstream signal transduction. These studies demonstrate that elevated cAMP negatively modulates P2X7R expression.

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.

The therapeutic option for patients with chronic hepatitis delta virus infection (CHD) is limited to interferon alpha with rare curative outcome. Myrcludex B is a first-in-class entry inhibitor inactivating the hepatitis B virus (HBV) and hepatitis D virus (HDV) receptor sodium taurocholate co-transporting polypeptide. We report the interim results of a pilot trial on chronically infected HDV patients treated with myrcludex B, or pegylated interferon alpha (PegIFNα-2a) or their combination.

Twenty-four patients with CHD infection were equally randomized (1:1:1) to receive myrcludex B, or PegIFNα-2a or their combination. Patients were evaluated for virological and biochemical response and tolerability of the study drugs at weeks 12 and 24.

Myrcludex B was well tolerated and no serious adverse event occurred. Although hepatitis B surface antigen levels remained unchanged, HDV RNA significantly declined at week 24 in all cohorts. HDV RNA became negative in two patients each in the Myrcludex B and PegIFNα-2a cohorts, and in five patients of the Myrcludex B+PegIFNα-2a cohort. ALT decreased significantly in the Myrcludex B cohort (six of eight patients), and HBV DNA was significantly reduced at week 24 in the Myrcludex B+PegIFNα-2a cohort. Virus kinetic modeling suggested a strong synergistic effect of myrcludex B and PegIFNα-2a on both HDV and HBV.

Myrcludex B showed a strong effect on HDV RNA serum levels and induced ALT normalization under monotherapy. Synergistic antiviral effects on HDV RNA and HBV DNA in the Myr-IFN cohort indicated a benefit of the combination of entry inhibition with PegIFNα-2a to treat CHD patients.

Myrcludex B is a new drug to treat hepatitis B and D infection. After 24weeks of treatment with myrcludex B and/or pegylated interferon α-2a, HDV R NA, a relevant marker for hepatitis D infection, decreased in all patients with chronic hepatitis B and D. Two of eight patients which received either myrcludex B or pegylated interferon α-2a, became negative for HDV RNA, and five of seven patients who received both drugs at the same time became negative. The drug was well tolerated.

Since Toll-like receptor (TLR) signaling was found crucial for the activation of innate and adaptive immunity, it has been the focus of immunological research. There are at least 13 identified mammalian TLRs, to date, that share similarities in their extracellular and intracellular domains. A vast number of ligands have been identified that are specifically recognized by different TLRs. As a response the TLRs dimerize and their signaling is initiated. The molecular basis of that signaling depends on the conserved part of their intracellular domain; namely the Toll/IL-1 receptor (TIR) domain. Upon TLR dimerization a TIR-TIR structure is formed that can recruit TIR-containing intracellular proteins that mediate their signaling. For this reason these proteins are named adapters. There are five adapters identified so far named myeloid differentiation primary response protein 88 (MyD88), MyD88-adapter like (Mal) or TIR domain-containing adapter (TIRAP), TIR domain-containing adapter inducing interferon-beta (IFN-beta) (TRIF) or TIR-containing adapter molecule-1 (TICAM-1), TRIF-related adapter molecule (TRAM) or TICAM-2, and sterile alpha and HEAT-Armadillo motifs (SARM). The first four play a fundamental role in TLR-signaling, defining which pathways will be activated, depending on which of these adapters will be recruited by each TLR. Among these adapter proteins MyD88 and TRIF are now considered as the signaling ones and hence the TLR pathways can be categorized as MyD88-dependent and TRIF-dependent.

Type I interferons (IFN-I) limit viral spread by inducing antiviral genes in infected target cells and by shaping the adaptive response through induction of additional cytokines. Vesicular stomatitis virus (VSV) efficiently triggers the production of IFN-I in mice, and it is suggested that IFN-alpha is induced after binding of VSV to TLR7 in infected cells. Our study with virus-specific B cell receptor-transgenic mice demonstrates here that IFN-I directly fuel early humoral immune responses in vivo. VSV-specific B cells that lacked IFN-alpha/beta receptors were considerably impaired in plasma cell formation and in generating antiviral IgM. At low viral titers, production of IFN-alpha following VSV infection was independent of TLR7-mediated signals. Interestingly, however, TLR7 ligation in B cells increased the formation of early antiviral IgM. These findings indicate that IFN-alpha-mediated augmentation of specific B cell responses is a partially TLR7- and virus dose-dependent mechanism.

Experimental allergic encephalomyelitis (EAE) was generated in SJL and B10.PL mice by using the synthetic myelin basic protein peptides. Inflammation in brain and spinal cord preceded clinical signs of disease. Infiltrating lymphocytes were predominantly Lyt1+ (CD5+), L3T4+ (CD4+) T cells, until day 18. After that, F4/80+ monocyte/macrophages outnumbered T cells. Ia+ cells were microglia, macrophages, and endothelial cells, but Ia was not detectable on astrocytes in this EAE model. Ia+ endothelial cells appeared later in the disease than Ia+ microglia and macrophages, suggesting that antigen presentation at the blood-brain barrier is not initially responsible for inflammation. Cells staining for interferon gamma, interleukin 2 (IL-2), and IL-2 receptors were more prominent than IL-4, IL-5, lymphotoxin (LT), and tumor necrosis factor alpha (TNF-alpha), which occurred transiently in the second week and were associated with fewer cells. TNF-alpha and LT were never seen in spinal cord, suggesting that these cytokines are not responsible for initiation of clinical disease. Few or no cells stained for IL-6, IL-1, or transforming growth factor beta. Control animals injected with complete Freund's adjuvant in saline or control antigen demonstrated no inflammatory cell infiltration or cytokine production. Thus, our findings suggest a peptide-induced EAE model in which Th1 T-cell-macrophage interactions result in the disease process.

Monocyte chemoattractant protein-1 (MCP-1), formerly termed JE, is a member of the beta-chemokine (C-C chemokine) family and has been shown to be produced by a variety of cell types. Recently, mRNA of JE/MCP-1 was detected in astrocytes during the acute phase of experimental allergic encephalomyelitis (EAE). In addition, supernatants collected from human cultured astrocytes have recently been found to be chemotactic for monocytes. However, chemokine production and function in glial cells has not been fully examined. Using a sandwich ELISA assay, we have now quantitated MCP-1 levels and assessed MCP-1 function on murine glial cells. Lipopolysaccharide (LPS), interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha induced MCP-1 secretion by astrocytes, but not microglia. In addition, pretreatment with interferon (IFN)-gamma significantly augmented MCP-1 production by either LPS or the above cytokines. In contrast, LPS preferentially induced production of another beta-chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha) from microglial cells. MCP-1 induced chemotaxis of microglial cells and macrophages. Similarly, another beta-chemokine, TCA3, which is produced by encephalitogenic T lymphocytes, also induced chemotaxis of microglia and macrophages. These findings suggested that astrocytes and microglial cells differentially produce chemokines in the central nervous system, and that both astrocytes and T cells may facilitate recruitment and activation of microglial cells via production of beta-chemokines.