BACKGROUND

Experimental and clinical studies have brought evidence that surgical trauma markedly affects the immune system, including both the specific and the non-specific immune response.

METHODS

This report reviews the present knowledge on the mechanisms of surgical trauma-induced immune dysfunction and outlines experimental and clinical approaches to find effective treatment strategies.

RESULTS

Major surgical trauma induces an early hyperinflammatory response, which is characterized by (1) pro-inflammatory tumour necrosis factor <em>alpha</em> (TNF), interleukin (IL)-1, and IL-6 cytokine release and (2) neutrophil activation and microvascular adherence, as well as (<em>3</em>) uncontrolled polymorphonuclear (PMN) and macrophage oxidative burst. The massive and continuous IL-6 release induces an acute phase response, but, more importantly, also accounts for the up-regulation of major anti-inflammatory mediators, such as prostaglandin (PG) E2, IL-10 and transforming growth factor (TGF)-ss. This results in surgical, trauma-induced, immunosuppression, as indicated by (1) monocyte deactivation, reflected by the lack of monocytic TNF- production upon lipopolysaccharide (LPS) stimulation, and (2) a shift of the Th1/Th2 ratio towards a Th2-dominated cytokine pattern. The imbalance between pro-inflammatory and anti-inflammatory cytokines and immuno-competent cells determines the phenotype of disease and should help the physician to compose the therapeutic strategy. In fact, recent clinical studies have shown that both the initial uncontrolled hyperinflammation and the continued cell-mediated immunosuppression represent primary targets to counteract post-surgery immune dysfunction. The balance between inflammatory and anti-inflammatory forces may be restored by <em>interferon</em> gamma (IFN-gamma) to counteract monocyte deactivation; the anti-inflammatory PGE2 may be inhibited by indomethacin to attenuate immunosuppression; or the initial hyperinflammation may be targeted by administration of anti-inflammatory substances, such as granulocyte colony-stimulating factor (G-CSF), hydoxyethyl starch, or pentoxifylline.

CONCLUSIONS

When drawing up the therapeutic regimen the physician should not consider hyperinflammation versus immunosuppression, but hyperinflammation and immunosuppression, aiming at restoring an appropriate mediator- and immune cell-associated balance.

C-X-C motif chemokine receptor <em>3</em> (CXCR<em>3</em>) and CXCR4 expressed on immunoglobulin G (IgG)-plasma-cell precursors formed in memory immune responses are crucial modulators of the homing of these cells. Here, we studied the regulation of the expression of these chemokine receptors during the differentiation of human memory B cells into plasma cells. We show that CXCR<em>3</em> is absent on CD27- naive B cells but is expressed on a fraction of memory B cells, preferentially on those coexpressing IgG1. On differentiation into plasma-cell precursors, CXCR<em>3</em>+ memory B cells maintain the expression of this chemokine receptor. CXCR<em>3</em>- memory B cells up-regulate CXCR<em>3</em> and migrate toward concentration gradients of its ligands only when costimulated with <em>interferon</em> gamma (IFN-gamma), but not interleukin 4 (IL-4), IL-1beta, IL-6, IFN-<em>alpha</em>, IFN-beta, or tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>). In contrast, the differentiation of CXCR4- B cells into plasma cells is generally accompanied by the induction of CXCR4 expression. These results show that lack of CXCR4 expression on plasma-cell precursors is not a limiting factor for plasma-cell homing and that the expression of CXCR<em>3</em> on memory B cells and plasma-cell precursors is induced by IFN-gamma, provided in human T helper type 1 (Th1)-biased immune responses. Once induced in memory B cells, CXCR<em>3</em> expression remains part of the individual cellular memory.

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to <em>interferon</em>-<em>alpha</em>/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme. The three-dimensional structure of recombinant ISG15C78S was determined at 2.4-A resolution. The ISG15 structure comprises two beta-grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 A (N-terminal) and 1.0 A (C-terminal). The beta-grasp domains pack across two conserved <em>3</em>(10) helices to bury 627 A2 that accounts for 7% of the total solvent-accessible surface area. The distribution of ISG15 surface charge forms a ridge of negative charge extending nearly the full-length of the molecule. Additionally, the N-terminal domain contains an apolar region comprising almost half its solvent accessible surface. The C-terminal domain of ISG15 was superimposed on the structure of Nedd8 (r.m.s.d. = 0.84 A) bound to its AppBp1-Uba<em>3</em> activating enzyme to model ISG15 binding to UbE1L. The docking model predicts several key side-chain interactions that presumably define the specificity between the ubiquitin and ISG15 ligation pathways to maintain functional integrity of their signaling.

The mechanisms of action of dietary fish oil (FO) on osteoporosis are not fully understood. This study showed FO decreased bone loss in ovariectomized mice because of inhibition of osteoclastogenesis. This finding supports a beneficial effect of FO on the attenuation of osteoporosis.

BACKGROUND

Consumption of fish or n-<em>3</em> fatty acids protects against cardiovascular and autoimmune disorders. Beneficial effects on bone mineral density have also been reported in rats and humans, but the precise mechanisms involved have not been described.

METHODS

Sham and ovariectomized (OVX) mice were fed diets containing either 5% corn oil (CO) or 5% fish oil (FO). Bone mineral density was analyzed by DXA. The serum lipid profile was analyzed by gas chromatography. Receptor activator of NF-kappaB ligand (RANKL) expression and cytokine production in activated T-cells were analyzed by flow cytometry and ELISA, respectively. Osteoclasts were generated by culturing bone marrow (BM) cells with 1,25(OH)2D<em>3</em>. NF-kappaB activation in BM macrophages was measured by an electrophoretic mobility shift assay.

CONCLUSIONS

Plasma lipid C16:1n6, C20:5n<em>3</em>, and C22:6n<em>3</em> were significantly increased and C20:4n6 and C18:2n6 decreased in FO-fed mice. Significantly increased bone mineral density loss (20% in distal left femur and 22.6% in lumbar vertebrae) was observed in OVX mice fed CO, whereas FO-fed mice showed only 10% and no change, respectively. Bone mineral density loss was correlated with increased RANKL expression in activated CD4+ T-cells from CO-fed OVX mice, but there was no change in FO-fed mice. Selected n-<em>3</em> fatty acids (docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]) added in vitro caused a significant decrease in TRACP activity and TRACP+ multinuclear cell formation from BM cells compared with selected n-6 fatty acids (linoleic acid [LA] and arachidonic acid [AA]). DHA and EPA also inhibited BM macrophage NF-kappaB activation induced by RANKL in vitro. TNF-alpha, interleukin (IL)-2, and interferon (IFN)-gamma concentrations from both sham and OVX FO-fed mice were decreased in the culture medium of splenocytes, and interleukin-6 was decreased in sham-operated FO-fed mice. In conclusion, inhibition of osteoclast generation and activation may be one of the mechanisms by which dietary n-<em>3</em> fatty acids reduce bone loss in OVX mice.

Interleukin-10 (IL-10)-deficient (IL-10(-/-)) mice infected with Plasmodium chabaudi (AS) suffer a more severe disease and exhibit a higher rate of mortality than control C57BL/6 mice. Here, we show that a drop in body temperature to below 28 degrees C and pronounced hypoglycemia of below <em>3</em> mM are reliable indicators of a lethal infection. Elevated inflammatory responses have been shown to accompany pathology in infected IL-10(-/-) mice. We show that neutralization of tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) in IL-10(-/-) mice abolishes mortality and ameliorates the hypothermia, weight loss, and anemia but does not affect the degree of hypoglycemia. These data suggest that TNF-<em>alpha</em> is involved in some of the pathology associated with a P. chabaudi infection in IL-10(-/-) mice but other factors play a role. IL-10(-/-) mice that survive a primary infection have been shown to control gamma <em>interferon</em> (IFN-gamma) and TNF-<em>alpha</em> production, indicating that other cytokines or mechanisms may be involved in their down-regulation. Significantly higher levels of transforming growth factor beta (TGF-beta), a cytokine with such properties, are present in the plasma of infected IL-10(-/-) mice at a time that coincides with the disappearance of IFN-gamma and TNF-<em>alpha</em> from the blood. Neutralization of TGF-beta in IL-10(-/-) mice resulted in higher circulating amounts of TNF-<em>alpha</em> and IFN-gamma, and all treated IL-10(-/-) mice died within 12 days with increased pathology but with no obvious increase in parasitemia. Our data suggest that a tight regulation of the balance between regulatory cytokines such as IL-10 and TGF-beta and inflammatory cytokines such as IFN-gamma and TNF-<em>alpha</em> is critical for survival in a mouse malaria infection.

Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-<em>3</em> dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-<em>3</em> transcriptionally up regulates <em>alpha</em>/beta <em>interferon</em> genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF-<em>3</em>(5D)] and dominant negative (IRF-<em>3</em> DeltaN) forms of IRF-<em>3</em> that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-<em>3</em>, we observed that IRF-<em>3</em> is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-<em>3</em> is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-<em>3</em>(5D) alone is sufficient to induce apoptosis in human embryonic kidney 29<em>3</em> and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-<em>3</em> expression augments paramyxovirus-induced apoptosis, while expression of IRF-<em>3</em> DeltaN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and <em>3</em> in IRF-<em>3</em>-induced apoptosis. These results suggest that IRF-<em>3</em>, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the <em>3</em>a accessory protein, are pro-apoptotic. Since the <em>3</em>a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the <em>3</em>a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in <em>3</em>a-expressing cells. Only the PERK pathway was found to be activated in <em>3</em>a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 <em>alpha</em> (eIF2<em>alpha</em>) and inhibitory effects of a dominant-negative form of eIF2<em>alpha</em> on GRP78 promoter activity, (2) increased translation of activating transcription factor 4 (ATF4) mRNA, and (<em>3</em>) ATF4-dependent activation of the C/EBP homologous protein (CHOP) gene promoter. Activation of PERK affects innate immunity by suppression of type 1 <em>interferon</em> (IFN) signaling. The <em>3</em>a protein was found to induce serine phosphorylation within the IFN <em>alpha</em>-receptor subunit 1 (IFNAR1) degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in <em>3</em>a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV <em>3</em>a protein, and suggest a potential role for it in attenuating <em>interferon</em> responses and innate immunity.

OBJECTIVE

Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells.

METHODS

Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, <em>interferon</em>-gamma, and/or tumor necrosis factor-<em>alpha</em>). Activation of Bad was determined by Ser1<em>3</em>6 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -<em>3</em>, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively.

RESULTS

We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser1<em>3</em>6, mitochondrial stress, cytochrome c release, activation of caspase-9 and -<em>3</em>, and DNA fragmentation. Inhibition of Bad Ser1<em>3</em>6 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.

CONCLUSIONS

Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser1<em>3</em>6 dephosphorylation and Bax activity in cytokine-induced apoptosis.

The acute stages of infection with swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive-respiratory syndrome virus (PRRSV) were shown to differ in terms of clinical and lung inflammatory effects and proinflammatory cytokine profiles in bronchoalveolar lavage (BAL) fluids. Caesarian-derived colostrum-deprived pigs were inoculated intratracheally with one of the three viruses. SIV infection was followed within 1 day post inoculation (d PI) by characteristic respiratory and general signs, and excessive lung epithelial desquamation and neutrophil infiltration (<em>3</em>8 to 56 per cent of BAL cells at 1 d PI vs 0 to 1 per cent in controls). High concentrations of bioactive <em>interferon</em>-<em>alpha</em> (IFN -<em>alpha</em>), tumour necrosis factor-<em>alpha</em> (TNF -<em>alpha</em>) and interleukin-1 (IL -1) coincided with peak symptoms and neutrophil infiltration. PRCV infection was asymptomatic and produced a mild bronchointerstitial pneumonitis and neutrophil infiltration (1<em>3</em> to 22 per cent of BAL cells at 4 d PI). IFN -<em>alpha</em> titres parallelled those found during SIV infection, TNF -<em>alpha</em> was negligible and IL -1 undetectable. PRRSV infection induced anorexia and lethargy between <em>3</em> and 5 d PI. There was marked infiltration with mononuclear cells in alveolar septa and BAL fluids between 7 and 10 d PI, while neutrophils remained at less than 11 per cent of BAL cells at any time. IL -1 was produced from three throughout 10 d PI, while IFN -<em>alpha</em> production was minimal and TNF -<em>alpha</em> undetectable. These data strongly suggest that proinflammatory cytokines can be important mediators of viral respiratory disease.

Psychiatric disorders or drug addiction are often regarded as contraindications against the use of <em>interferon</em> alfa (IFN-<em>alpha</em>) in patients with chronic hepatitis C. Our aim was to obtain prospective data on adherence to as well as efficacy and mental side effects of treatment with IFN-<em>alpha</em> in different psychiatric risk groups compared with controls. In a prospective trial, 81 patients with chronic hepatitis C (positive hepatitis C virus[HCV] RNA and elevated alanine aminotransferase[ALT] level) and psychiatric disorders (n = 16), methadone substitution (n = 21), former drug addiction (n = 21), or controls without a psychiatric history or drug addiction (n = 2<em>3</em>) were treated with a combination of IFN-<em>alpha</em>-2a <em>3</em> MU <em>3</em> times weekly and ribavirin (1,000-1,200 mg/d). Sustained virologic response (overall, <em>3</em>7%) did not differ significantly between subgroups. No significant differences between groups were detected with respect to IFN-<em>alpha</em>-related development of depressions during treatment. However, in the psychiatric group, significantly more patients received antidepressants before and during treatment with IFN-<em>alpha</em> (P <.001). Most of those who dropped out of the study were patients with former drug addiction (4<em>3</em>%; P =.04) compared with 14% in the methadone group, 1<em>3</em>% in the control group, and 18% in the psychiatric group. No patient in the psychiatric group had to discontinue treatment because of psychiatric deterioration. In conclusion, our data do not confirm the supposed increased risk for IFN-<em>alpha</em>-induced mental side effects and dropouts in psychiatric patients if interdisciplinary care and antidepressant treatment are available. Preexisting psychiatric disorders or present methadone substitution should no longer be regarded as contraindications to treatment of chronic hepatitis C with IFN-<em>alpha</em> and ribavirin in an interdisciplinary setting.

Greater than 95% of all cervical carcinomas have been found to be associated with "high-risk" human papillomavirus (mainly types 16 and 18) infections, with the viral E6 and E7 oncoproteins essential for neoplastic development and maintenance. <em>Interferon</em>-<em>alpha</em> (IFN<em>alpha</em>) is used in the treatment of HPV infections yet both in vivo and in vitro data suggest that the virus has developed mechanisms to avoid the effects of <em>interferon</em>. Here we show that the HPV16 E7 oncoprotein is able to inhibit the induction of IFN<em>alpha</em>-inducible genes but has no effect of IFNgamma-inducible genes. Expression of E7 correlates with the loss of formation of the <em>interferon</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>) transcription complex. Moreover, in the presence of E7, p48, the DNA-binding component of ISGF<em>3</em>, was unable to translocate to the nucleus upon IFN<em>alpha</em> stimulation. A direct protein-protein interaction was identified between E7 and p48 with the site of interaction within E7 defined as the region between amino acids 17-<em>3</em>7, a domain that includes the binding site for the retinoblastoma protein, pRb. These results suggest that HPV, via E7, targets p48, resulting in the loss of IFN<em>alpha</em>-mediated signal transduction and may provide a means by which HPV can avoid the innate immune system.

<em>Interferon</em> <em>alpha</em> induction of transcription operates through <em>interferon</em>-stimulated-gene factor <em>3</em> (ISGF), a transcription factor two components of which are members of the newly characterized Stat family of transcription factors. <em>Interferon</em> <em>alpha</em> induces tyrosine phosphorylation of Stat1 and Stat2 proteins that associate and, together with a 48-kDa protein, form ISGF<em>3</em>. Evidence is presented that a heterodimer of Stat1 and Stat2 is present in ISGF<em>3</em> and that Stat1 and the 48-kDa protein make precise contact, while Stat2 makes general contact, with the <em>interferon</em>-stimulated response element, the binding site of the ISGF<em>3</em>.

Mycobacterium tuberculosis infection is accompanied by acute and chronic inflammatory infiltrates associated with necrotizing granulomas in lung tissue. The cellular infiltrate is characterized by inflammatory cells which include neutrophils, lymphocytes, and macrophages. In animal and in vitro models of mycobacterial infection, cytokines including tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), <em>interferon</em> gamma (IFN-gamma), and interleukin-1 beta (IL-1 beta) participate in granulomatous inflammation. We hypothesized that interleukin-<em>3</em>, a potent chemoattractant for neutrophils and lymphocytes, could be released by activated alveolar macrophages after exposure to M. tuberculosis or its components and contribute to granulomatous lung inflammation. A quantitative immunoassay revealed that IL-8 protein release was significantly elevated in supernatants of macrophages and in lavage fluid obtained from patients with pulmonary tuberculosis compared to normal controls. In addition, Northern blots demonstrated striking up-regulation of IL-8 mRNA in macrophages from these patients. M. tuberculosis and its cell wall components lipoarabinomannan (LAM), lipomannan (LM), and phosphoinositolmannoside (PIM) stimulated IL-8 protein release and mRNA expression in vitro from alveolar macrophages, but deacylated LAM did not. Neutralizing antibodies to TNF-<em>alpha</em> and/or IL-1-<em>alpha</em> and beta blocked 8<em>3</em>% of the stimulation. IL-8 synthesis and release is an early response of macrophages after phagocytosis of M. tuberculosis. Its production serves to attract both acute and chronic inflammatory cells of active infection and thus participates in the process of containment of the pathogen.

The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q46<em>3</em>H and E<em>3</em>20Q alleles, are intrinsically deleterious for both <em>interferon</em> gamma (IFNG)-induced gamma-activating factor-mediated immunity and <em>interferon</em> <em>alpha</em> (IFNA)-induced <em>interferon</em>-stimulated genes factor <em>3</em>-mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q46<em>3</em>H and E<em>3</em>20Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor-mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced <em>interferon</em>-stimulated genes factor <em>3</em>-mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding.

BACKGROUND

Several studies found a high incidence rate of neuro-psychiatric complications during long-term therapy with interferon alpha (IFNalpha), e.g. slowness, severe fatigue, hypersomnia, lethargy, depressed mood, mnemonic troubles, irritability, short temper, emotional lability, social withdrawal, and lack of concentration. The aim of this study was to examine the incidence of depressed mood and major depression in patients who were treated with IFNalpha.

METHODS

30 patients, affected by chronic active C-hepatitis, have been evaluated at baseline and 3 months after IFNalpha treatment. The evaluation consisted of psychometric assessments employing the DSM-IV criteria and the Montgomery Asberg Depression Rating Scale (MADRS).

RESULTS

At end-point, 40.7% of the patients suffered from a full blown major depression, according to the DSM-IV criteria for major depression. IFNalpha treatment induced a significant increase in the MADRS score from baseline to 3 months later. The MADRS items which were significantly increased at end-point were: expressed and unexpressed sadness; irritability; insomnia; loss of appetite; and asthenia.

CONCLUSIONS

The results show that prolonged IFNalpha treatment may induce depressive symptoms and major depression in a considerable number of subjects.

OBJECTIVE

Both infliximab (chimeric anti-tumor necrosis factor [TNF]-alpha antibody) and etanercept (p75 TNF-alpha receptor/immunoglobulin G fusion protein) are effective against rheumatoid arthritis, but only infliximab induces clinical remission in Crohn's disease. To clarify this difference in clinical efficacy, we investigated reverse signaling through transmembrane TNF-alpha (mTNF) by these 2 anti-TNF agents.

METHODS

We stably transfected wild-type and cytoplasmic serine-replaced mutant forms of mTNF in human Jurkat T cells. Cells were stimulated with infliximab and etanercept and then analyzed for E-selectin expression, reactive oxygen species accumulation, apoptosis, and cell cycle distribution by flow cytometry. Interleukin-10 and interferon-gamma were measured by enzyme-linked immunosorbent assay. Phospho-c-Jun NH2-terminal kinase, Bax, Bak, p21(WAF1/CIP1), caspase-8, and caspase-3 were examined by immunoblotting.

RESULTS

Both anti-TNF agents induced E-selectin expression, but only infliximab induced interleukin-10 production, apoptosis, and G0/G1 cell cycle arrest. Apoptosis and cell cycle arrest were abolished by substitution of all 3 cytoplasmic serine residues of mTNF by alanine residues. Infliximab induced accumulation of reactive oxygen species and up-regulation of Bax, Bak, and p21(WAF1/CIP1) proteins, suggesting the involvement of p53 activation. Moreover, phosphorylation of c-Jun NH2-terminal kinase was necessary for infliximab-induced apoptosis and cell cycle arrest.

CONCLUSIONS

We revealed the mTNF motifs and the downstream intracellular molecular events essential for reverse signaling through mTNF. The biologic effects of mTNF elicited by infliximab should be important action mechanisms of this potent anti-inflammatory agent in addition to the neutralization of soluble TNF-alpha. These observations will provide insight into the novel role of mTNF in inflammation.

The pathogenesis of neurological dysfunction associated with human immunodeficiency (HIV)-1 infection is uncertain. However, the presence of macrophage infiltrates in the central nervous system is a key feature of HIV encephalitis and is correlated with HIV-associated dementia. Moreover, it has been demonstrated that HIV-infected monocyte/macrophages can produce toxic substances that may play a critical role in the development of HIV-associated dementia. However, the exact mechanisms responsible for HIV infection and leukocyte recruitment to the central nervous system remain speculative. Similar to HIV-infected patients, simian immunodeficiency virus (SIV)-infected macaque monkeys develop immunosuppression and acquired immune deficiency syndrome (AIDS)-related inflammatory disorders, including AIDS encephalitis. In this study, we demonstrate that encephalitic brain from SIV-infected animals has elevated immunohistochemical expression of the C-C chemokines, macrophage inflammatory protein-1 <em>alpha</em> and -beta, RANTES, and monocyte chemotactic protein-<em>3</em>, and the C-X-C chemokine <em>interferon</em>-inducible protein-10. These findings suggest that one or all of of these chemokines could be involved in leukocyte recruitment to the brain in SIV-infected macaque monkeys.

<em>Interferons</em> (IFN) are potent components of the innate immune response to microbial infection. The genes for type I IFN (IFN-<em>alpha</em> and IFN-beta) are rapidly induced in response to viral infection through a mechanism that involves latent cellular transcription factors that are activated in response to innate recognition of viral components. IFN regulatory factor (IRF) proteins are key to this regulation, and their conversion from latent to active involves virus-induced serine phosphorylation. Differential utilization of distinct IRF proteins by different members of the type I IFN gene family produces a graded induction of gene expression, resulting in tight control of these cytokines through a positive feedback mechanism. Early response to virus causes secretion of a subset of IFN genes through the action of IRF-<em>3</em> in conjunction with additional transcription factors, such as NF-kappaB and activator protein-1 (AP-1) (c-jun/ATF). This early IFN acts in an autocrine manner to stimulate production of IRF-7, a transcription factor capable of activating the many additional members of the IFN-<em>alpha</em> gene family. The dependence of IRF-7 on virus-induced phosphorylation for its activity insures that IFN production is limited to virus-infected cells. Characterization of the cellular components involved in viral detection and IRF activation will further delineate this vital mechanism of innate immune response.

OBJECTIVE

Various side effects have been reported in patients treated with alpha interferon, but their incidence and prognosis remain unknown.

METHODS

Nine hundred and eighty-seven patients with chronic active hepatitis C received 6 to 10 MU of alpha interferon per day for 2 weeks and 3 times per week for 22 weeks. Autoantibodies, thyroid function tests, and fasting plasma glucose concentrations were evaluated prior to alpha interferon therapy.

RESULTS

Of the 987 patients, 310 were required reduction in the dose of alpha interferon to 3 MU/day or cessation of alpha interferon therapy because of adverse reactions such as flu-like symptoms, leukopenia, and thrombocytopenia. Of the remaining 677, five developed diabetes mellitus, 12 had hyperthyroidism, and six acquired hypothyroidism. Of the 18 with thyroid disorders, five demonstrated antimicrosomal antibodies before therapy. Forty-four patients revealed high or low concentrations of thyroid stimulating hormone at the end of alpha interferon therapy. Three patients developed interstitial pneumonia, one acquired systemic lupus erythematosus-like syndrome, two had autoimmune hepatitis, two developed rheumatoid arthritis, and one developed autoimmune thrombocytopenic purpura. No patients had a history of an autoimmune disorder. One patient experienced sudden hearing impairment and one had retinal detachment. Melena was seen in three patients; two of these cases were compatible with ischemic colitis. Symptoms of depression were seen in 23 patients, and one patient manifested memory loss.

CONCLUSIONS

High-dose alpha interferon therapy induces various adverse effects. Most of the side effects cannot be predicted, but are reversible.

Transient or long-term quiescence, the latter referred to as dormancy are fundamental features of at least some adult stem cells. The status of dormancy is likely a critical mechanism for the observed resistance of normal HSCs and leukemic stem cells (LSCs) to anti-proliferative chemotherapy. Recent studies have revealed cytokines such as <em>Interferon</em>-<em>alpha</em> (IFNα) and G-CSF as well as arsenic trioxide (As(2)O(<em>3</em>)) to be efficient agents for promoting cycling of dormant HSCs and LSCs. Most interestingly, such cell cycle activated stem cells become exquisitely sensitive to killing by different chemotherapeutic agents, suggesting that dormant LSCs in patients may be targeted by a sequential two-step protocol involving an initial activation by IFNα, G-CSF or As(2)O(<em>3</em>), followed by targeted chemotherapy.

Viral infections and local production of cytokines probably contribute to the pathogenesis of Type 1 diabetes. The viral replicative intermediate double-stranded RNA (dsRNA, tested in the form of polyinosinic-polycytidylic acid, PIC), in combination with the cytokine <em>interferon</em>-gamma (IFN-gamma), triggers beta-cell apoptosis. We have previously observed by microarray analysis that PIC induces expression of several mRNAs encoding for genes downstream of Toll-like receptor <em>3</em> (TLR<em>3</em>) signaling pathway. In this report, we show that exposure of beta-cells to dsRNA in combination with IFN-<em>alpha</em>, -beta, or -gamma significantly increases apoptosis. Moreover, dsRNA induces TLR<em>3</em> mRNA expression and activates NF-kappaB and the IFN-beta promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN-beta mRNA expression, and blocking IFN-beta with a specific antibody partially prevents PIC plus IFN-gamma-induced beta-cell death. On the other hand, dsRNA plus IFN-gamma does not induce apoptosis in INS-1E cells, and expression of TLR<em>3</em> and type I IFNs mRNAs is not detected in these cells. Of note, disruption of the STAT-1 signaling pathway protects beta-cells against dsRNA plus IFN-gamma-induced beta-cell apoptosis. This study suggests that dsRNA plus IFN-gamma triggers beta-cell apoptosis by two complementary pathways, namely TLR<em>3</em>-TRIF-NF-kappaB and STAT-1.

In this study, we examined the effect of injecting various cytokines. We report here that tumour necrosis factor (TNF)<em>alpha</em>, gamma-<em>interferon</em> and interleukin 2 (IL-2) can, in some circumstances, increase fetal resorption rates in abortion-prone (CBA/J x DBA/2) and non-abortion prone (CBA/J x BALB/c,C<em>3</em>H x DBA/2) matings: 1000 units TNF enhanced resorptions from 4<em>3</em> to 79% in CBA x DBA/2, from 7 to 89% in CBA x BALB/c, from 5 to 47% in C<em>3</em>H x DBA/2. The effect was both gestational age- and dose-dependent. Gamma <em>interferon</em> and R-IL-2 enhanced resorptions from <em>3</em>8 to 68% and 76% respectively in the CBA/J x DBA/2 mating combination, whereas the rates in CBA/J x BALB/c matings were enhanced from 6 to 44% and 55%. Lipopolysaccharide (LPS), which is known to lead to the release of TNF-<em>alpha</em>, had a similar effect, leading to gestational age- and dose-dependent enhancement of resorptions up to 100%. However, cytokines of the CSF family, including IL-<em>3</em> and GM-CSF, increased the chances of fetal survival when injected into abortion-prone mice, e.g. reducing resorption rates in the abortion-prone CBA/J x DBA/2 mating combination from 55 to 22% (IL-<em>3</em>), and 47 to 8% (GM-CSF). They also increased fetal and placental weight and, in particular, expanded the spongiotrophoblast zone in the placenta. The latter observations may be due to a direct trophic influence on placental cells, perhaps through a cytokine cascade, or an indirect effect due to inhibition of natural killer (NK)-like cells, or both. Whatever the mechanism, these results may find practical application in influencing reproductive outcome in women and other species.

Human blood contains 2 populations of dendritic cells (DCs): plasmacytoid and myeloid (mDC). mDCs are subdivided into <em>3</em> subsets using the surface markers CD16, CD1c, and BDCA-<em>3</em>. Their role as pathogen sentinels and adjuvant targets was tested by phenotypic and functional analysis. We show that mDC subsets are immature and express mRNA for most toll-like receptors (TLRs), except for TLR<em>3</em> in CD16-mDCs. The most represented subsets, CD16- and CD1c-mDCs, are similarly responsive to all TLR agonists. Among <em>3</em>1 cytokines tested, both subsets produce CXCL8 (IL-8)/tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>)/IL-6/CCL<em>3</em> (MIP-1 <em>alpha</em>)/CCL4 (MIP-1beta)/IL-1 beta. CXCL8 (IL-8) is the predominant cytokine produced by CD1c-mDCs on TLR engagement, whereas all other cytokines, particularly TNF-<em>alpha</em>, are secreted in 10-fold to 100-fold higher amounts by CD16-mDCs. CD16-mDCs cocultured with human umbilical vein endothelial cells induce a significantly higher production of CXCL10 (IP-10), granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor than CD1c-mDCs. In addition, interleukin-<em>3</em> and type I <em>interferons</em> are stimuli specifically for DC maturation rather than cytokine secretion, whereas TNF-<em>alpha</em> is almost ineffective in inducing either function, suggesting a mechanism of T-cell-DC crosstalk and of rapid induction of antigen-presenting cell function during viral infection rather than inflammation. In conclusion, CD16-mDCs show strong proinflammatory activity, whereas CD1c-mDCs appear to be mainly inducers of chemotaxis.

OBJECTIVE

To study the efficacy of topical application of alpha-linolenic acid (ALA) and linoleic acid (LA) for dry eye treatment.

METHODS

Formulations containing ALA, LA, combined ALA and LA, or vehicle alone, were applied to dry eyes induced in mice. Corneal fluorescein staining and the number and maturation of corneal CD11b(+) cells were determined by a masked observer in the different treatment groups. Real-time polymerase chain reaction was used to quantify expression of inflammatory cytokines in the cornea and conjunctiva.

RESULTS

Dry eye induction significantly increased corneal fluorescein staining; CD11b(+) cell number and major histocompatibility complex Class II expression; corneal IL-1alpha and tumor necrosis factor alpha (TNF-alpha) expression; and conjunctival IL-1alpha, TNF-alpha, interferon gamma, IL-2, IL-6, and IL-10 expression. Treatment with ALA significantly decreased corneal fluorescein staining compared with both vehicle and untreated controls. Additionally, ALA treatment was associated with a significant decrease in CD11b(+) cell number, expression of corneal IL-1alpha and TNF-alpha, and conjunctival TNF-alpha.

CONCLUSIONS

Topical ALA treatment led to a significant decrease in dry eye signs and inflammatory changes at both cellular and molecular levels.

CONCLUSIONS

Topical application of ALA omega-3 fatty acid may be a novel therapy to treat the clinical signs and inflammatory changes accompanying dry eye syndrome.