OBJECTIVE

Interleukin-6 (IL-6) facilitates cancer cell survival via pleotrophic effects. We conducted a multicenter phase II study of CNTO328 (siltuximab) as second-line therapy for men with castration-resistant prostate cancer.

METHODS

Eligible men had castration-resistant prostate cancer treated with one prior chemotherapy. Subjects were treated with 6 mg/kg CNTO328 i.v. every 2 weeks for 12 cycles. Response was assessed after every three cycles. Primary end point was prostate-specific antigen (PSA) response rate defined as a 50% reduction. Accrual was planned in two stages, with 20 eligible patients in the first stage and 40 overall. Plasma cytokines and growth factors were measured by Luminex.

RESULTS

Fifty-three eligible subjects had all received prior taxane therapy. Two (3.8%; 95% CI, 0.5-13.0%) had PSA response. None of the 31 patients with measurable disease had a RECIST (Response Evaluation Criteria in Solid Tumors) response but 7 (23%) had stable disease. With median follow-up of 14.8 months, median progression-free survival was 1.6 months (95% CI, 1.6-1.7) and median overall survival was 11.6 months (95% CI, 7.5-19.0). Grade 3/4 toxicities included disseminated intravascular coagulation (1), central nervous system ischemia (1), elevated aspartate aminotransferase (1), gastritis/esophagitis (2), thrombocytopenia (2), pain (2), leukopenia (1), and neuropathy (2). Median baseline IL-6 levels were 12.5 pg/mL (interquartile range, 2.5-41.5). Patients with IL-6 >12.5 pg/mL had worse survival than those with levels <12.5 pg/mL (53% versus 94%; P = 0.02). After treatment, IL-6 levels were >250-fold higher. Thirty-two of 38 patients had a decline in C-reactive protein plasma levels at 6 weeks.

CONCLUSIONS

CNTO328 resulted in a PSA response rate of 3.8% and a RECIST stable disease rate of 23%. Declining C-reactive protein levels during treatment may reflect biological activity. Despite evidence of CNTO-mediated IL-6 inhibition, elevated baseline IL-6 levels portended a poor prognosis.

Streptococcus suis, an important swine and human pathogen, causes septic shock and meningitis. The pathogenesis of both systemic and CNS infections caused by S. suis is poorly understood. A hematogenous model of infection in CD1 mice was developed to study the systemic release of cytokines during the septic shock phase and the proinflammatory events in the CNS associated with this pathogen. Using a liquid array system, high levels of systemic TNF-alpha, <em>IL</em>-6, <em>IL</em>-12, IFN-gamma, CCL2, CXCL1, and CCL5 were observed 24 h after infection and might be responsible for the sudden death of <em>20</em>% of animals. Infected mice that survived the early sepsis later developed clinical signs of meningitis and exhibited lesions in the meninges and in numerous regions of the brain, such as the cortex, hippocampus, thalamus, hypothalamus, and corpus callosum. Bacterial Ags were found in association with microglia residing only in the affected zones. In situ hybridization combined with immunocytochemistry showed transcriptional activation of TLR2 and TLR3 as well as CD14, NF-kappaB, <em>IL</em>-1beta, CCL2, and TNF-alpha, mainly in myeloid cells located in affected cerebral structures. Early transcriptional activation of TLR2, CD14, and inflammatory cytokines in the choroid plexus and cells lining the brain endothelium suggests that these structures are potential entry sites for the bacteria into the CNS. Our data indicate an important role of the inflammatory response in the pathogenesis of S. suis infection in mice. This experimental model may be useful for studying the mechanisms underlying sepsis and meningitis during bacterial infection.

OBJECTIVE

This prospective study quantified cytokine and chemokine levels in seminal plasma of patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and benign prostatic hyperplasia (BPH), to evaluate inflammatory mediators as possible surrogate markers for diagnosis and treatment efficacy.

METHODS

Seminal plasma levels of eight cytokines and nine chemokines were evaluated by multiplex arrays in 83 men: <em>20</em> healthy controls and 9 men with CP/CPPS IIIA, 31 with CP/CPPS IIIB, and 23 with BPH. Prostate samples obtained by transurethral resection of the prostate from 13 patients with BPH were analysed by immunohistochemistry to detect interleukin 8 (<em>IL</em>-8)-producing cells and characterise inflammatory infiltrates.

RESULTS

Significantly increased levels of cytokines (<em>IL</em>-1alpha, <em>IL</em>-1beta, <em>IL</em>-6, <em>IL</em>-10, <em>IL</em>12p70) and chemokines (CCL1, CCL3, CCL4, CCL17, CCL22, CXCL8/<em>IL</em>-8) were observed in seminal plasmas from patients with CP/CPPS or BPH. However, only <em>IL</em>-8 was significantly elevated compared to controls (median [quartiles] 1984 [1164-2444] pg/ml), in patients with CP/CPPS IIIA (15,240 [10,630-19,501] pg/ml; p<0.0001), CP/CPPS IIIB (2983 [<em>20</em>33-5287] pg/ml; p=0.008), and BPH (5044 [3063-11,795] pg/ml, p<0.0001), discriminating CP/CPPS IIIA versus IIIB (accuracy=0.882+/-0.078; p=0.001). Inflammatory infiltrates were detected in prostate samples from 13 of 13 BPH patients, and <em>IL</em>-8-producing prostate cells in 11 of 13 samples. <em>IL</em>-8 concentration in seminal plasma was positively correlated with symptom score and prostate-specific antigen levels both in CP/CPPS and BPH patients.

CONCLUSIONS

IL-8 is expressed in situ by epithelial and stromal prostate cells and is functional, as shown by recruitment of cells expressing cognate receptors in BPH prostate tissue, indicating its involvement in disease pathogenesis. Among all the cytokines and chemokines analysed, IL-8 appears to be the most reliable and predictive surrogate marker to diagnose prostate inflammatory conditions, such as CP/CPPS and BPH.

Microglia-mediated inflammatory responses have been implicated in the pathogenesis of neuritic plaques in Alzheimer's disease. The strong age association of Alzheimer's disease incidence suggests that events in normal aging may promote such responses. We used immunohistochemistry and computerized image analysis to determine the numbers, size, activation state, and immunoreactive intensity of interleukin-1 alpha-immunoreactive (<em>IL</em>-1 alpha +) microglia in mesial temporal lobe of <em>20</em> neurologically normal individuals, 2-80 years of age. We also used Northern analysis to determine tissue levels of <em>IL</em>-1 alpha mRNA in an additional 11 neurologically normal individuals aged 1 day to 78 years. <em>IL</em>-1 alpha + microglia were characterized as primed, enlarged, or phagocytic (enlarged with heterogeneous cytoplasmic contents) based on morphology. These three microglial subtypes showed significant differences in size [27 +/- 1 58 +/- 2 114 +/- 6 (mean +/- SEM) micron 2/cell, respectively, P < 0.001 for each comparison] and in immunoreactive intensity [60 +/- 1 68 +/- 2 79 +/- 2 (arbitrary units), respectively, P < 0.001 or better for each comparison]. There were significant age-associated increases in the total numbers of activated <em>IL</em>-1 alpha + microglia. Among microglial subtypes, there were significant increases in the numbers of enlarged (threefold) and especially phagocytic (elevenfold), but not primed, microglia. Tissue <em>IL</em>-1 alpha mRNA levels were higher in individuals over 60 than in those less than 60 (P < 0.05). The age-associated increases in microglial activation were independent of postmortem interval, patient sex, and the presence of Alzheimer-type 'senile' changes. Age-associated increases in microglial activation and <em>IL</em>-1 expression may contribute to the age-associated increased risk of Alzheimer's disease.

The skin has long been recognized as a major producer of cytokines, but the keratinocyte as principal epidermal cell has received less attention as potential source and target of cytokines. Nevertheless, keratinocytes produce a plethora of cytokines including interleukin (<em>IL</em>)-1, -6, -7, -8, -10, -12, -15, -18, and -<em>20</em>, and tumor necrosis factor alpha (TNF). The production by keratinocytes of pro-inflammatory (<em>IL</em>)-1, -6, -8, and TNF was recognized early and is well studied. Keratinocyte-derived <em>IL</em>-7 and -15 are considered to be significant in T-cell trafficking, possibly even in the pathogenesis of cutaneous T-cell lymphoma. Immunomodulatory <em>IL</em>-10 and -12 originating from keratinocytes are considered to be responsible for systemic effects, and <em>IL</em>-18 perhaps has a similar action. Keratinocytes were fairly recently recognized as being source or target of other <em>IL</em>-10 family members like <em>IL</em>-<em>20</em> and <em>IL</em>-24 and the role of these cytokines in specific diseases is under investigation. In addition, a variety of cytokine receptors are present on keratinocytes like those for <em>IL</em>-4, -13, and -17 and to lesser degree <em>IL</em>-2. The ability to study the expression of cytokines by keratinocytes in vivo and in vitro using primary cells, immortalized cells or even organotypic culture systems offers many possibilities to further investigate the role of cytokine production in keratinocyte biology and disease.

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-3 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)2D3. NF-kappaB activation in BM macrophages was measured by an electrophoretic mobility shift assay.

CONCLUSIONS

Plasma lipid C16:1n6, C<em>20</em>:5n3, and C22:6n3 were significantly increased and C<em>20</em>:4n6 and C18:2n6 decreased in FO-fed mice. Significantly increased bone mineral density loss (<em>20</em>% 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-3 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 (<em>IL</em>)-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-3 fatty acids reduce bone loss in OVX mice.

To examine the hypothesis that tumor necrosis factor (TNF) alpha is an essential cytokine in carcinogenesis, we conducted two-stage carcinogenesis experiments with an initiator, 7,12-dimethylbenz(a)anthracene (DMBA), plus either of two tumor promoters, okadaic acid and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the skin of TNF-alpha-deficient (TNF-/-) mice. TNF-/- mice treated with DMBA plus okadaic acid developed no tumors for up to 19 weeks, and at <em>20</em> weeks, the percentage of tumor-bearing TNF-/- mice was 10%, whereas the percentage of tumor-bearing TNF+/+ mice was 100%. In TNF-/- mice treated with DMBA plus TPA, tumor onset was delayed 4 weeks, and the time to development of small tumors in 100% of mice was 9 weeks later than that seen in TNF+/+ CD-1 mice. The average number of tumors in TPA-treated TNF-/- mice was 2.8, compared with 11.8 for TNF+/+ CD-1 mice. To understand the residual tumor-promoting activity in TNF-/- mice, we also investigated the possible significance of interleukin (<em>IL</em>) 1 as an additional cytokine in tumor promotion. A single application of TPA and okadaic acid increased <em>IL</em>-1alpha and <em>IL</em>-1beta gene expression in TNF-/- mice. All of our results demonstrate that TNF-alpha is the key cytokine for tumor promotion in mouse skin and, very possibly, for carcinogenesis in humans as well.

The role of <em>IL</em>-6 in experimental autoimmune encephalomyelitis (EAE) provoked by myelin oligodendrocyte glycoprotein (MOG) was investigated using <em>IL</em>-6-deficient mice. We show here that <em>IL</em>-6-deficient mice were resistant to the MOG-induced EAE as compared to wild-type mice (one out of 18 versus 17 out of <em>20</em>). The delayed-type hypersensitivity response, lymphocyte proliferation response and antibody reactivity to MOG in <em>IL</em>-6-deficient mice were significantly lower than those in wild-type mice. Furthermore, the histological examination revealed that no infiltration of inflammatory cells was observed in the central nervous system of <em>IL</em>-6-deficient mice. These results indicate that <em>IL</em>-6 may play a crucial role in the induction phase of EAE. Given the potential relevance of this animal model for multiple sclerosis (MS), it is possible that anti-<em>IL</em>-6 therapy may be useful in the prevention of relapses of MS.

OBJECTIVE

To determine the relative efficacy and complications of the Ahmed glaucoma valve (AGV) model FP7 (New World Medical, Ranchos Cucamonga, CA) and the Baerveldt glaucoma implant (BGI) model 101-350 (Abbott Medical Optics, Abbott Park, IL) in refractory glaucoma.

METHODS

Multicenter, randomized, controlled clinical trial.

METHODS

Two hundred seventy-six patients, including 143 in the AGV group and 133 in the BGI group.

METHODS

Patients 18 to 85 years of age with refractory glaucoma having intraocular pressure (IOP) of 18 mmHg or more in whom an aqueous shunt was planned were randomized to undergo implantation of either an AGV or a BGI.

METHODS

The primary outcome was failure, defined as IOP >21 mmHg or not reduced by 20% from baseline, IOP ≤5 mmHg, reoperation for glaucoma or removal of implant, or loss of light perception vision. Secondary outcomes included mean IOP, visual acuity, use of supplemental medical therapy, and complications.

RESULTS

Preoperative IOP (mean±standard deviation [SD]) was 31.2±11.2 mmHg in the AGV group and 31.8±12.5 mmHg in the BGI group (P = 0.71). At 1 year, mean±SD IOP was 15.4±5.5 mmHg in the AGV group and 13.2±6.8 mmHg in the BGI group (P = 0.007). The mean±SD number of glaucoma medications was 1.8±1.3 in the AGV group and 1.5±1.4 in the BGI group (P = 0.071). The cumulative probability of failure was 16.4% (standard error [SE], 3.1%) in the AGV group and 14.0% (SE, 3.1%) in the BGI group at 1 year (P = 0.52). More patients experienced early postoperative complications in the BGI group (n = 77; 58%) compared with the AGV group (n = 61; 43%; P = 0.016). Serious postoperative complications associated with reoperation, vision loss of ≥2 Snellen lines, or both occurred in 29 patients (20%) in the AGV group and in 45 patients (34%) in the BGI group (P = 0.014).

CONCLUSIONS

Although the average IOP after 1 year was slightly higher in patients who received an AGV, there were fewer early and serious postoperative complications associated with the use of the AGV than the BGI.

BACKGROUND

Acute trauma involving the anterior cruciate ligament is believed to be a major risk factor for the development of post-traumatic osteoarthritis 10 to <em>20</em> years post-injury. In this study, to better understand the early biological changes which occur after acute injury, we investigated synovial fluid and serum biomarkers.

METHODS

We collected serum from 11 patients without pre-existing osteoarthritis from a pilot intervention trial (5 placebo and 6 drug treated) using an intra-articular interleukin-1 receptor antagonist (IL-1Ra) therapy, 9 of which also supplied matched synovial fluid samples at presentation to the clinic after acute knee injury (mean 15.2 ± 7.2 days) and at the follow-up visit for reconstructive surgery (mean 47.6 ± 12.4 days). To exclude patients with pre-existing osteoarthritis (OA), the study was limited to individuals younger than 40 years of age (mean 23 ± 3.5) with no prior history of joint symptoms or trauma. We profiled a total of 21 biomarkers; <em>20</em> biomarkers in synovial fluid and 13 in serum with 12 biomarkers measured in both fluids. Biomarkers analyzed in this study were found to be independent of treatment (P>> 0.05) as measured by Mann-Whitney and two-way ANOVA.

RESULTS

We observed significant decreases in synovial fluid (sf) biomarker concentrations from baseline to follow-up for (sf)C-Reactive protein (CRP) (P = 0.039), (sf)lubricin (P = 0.008) and the proteoglycan biomarkers: (sf)Glycosaminoglycan (GAG) (P = 0.019), and (sf)Alanine-Arginine-Glycine-Serine (ARGS) aggrecan (P = 0.004). In contrast, we observed significant increases in the collagen biomarkers: (sf)C-terminal crosslinked telopeptide type II collagen (CTxII) (P = 0.012), (sf)C1,2C (P = 0.039), (sf)C-terminal crosslinked telopeptide type I collagen (CTxI) (P = 0.004), and (sf)N-terminal telopeptides of type I collagen (NTx) (P = 0.008). The concentrations of seven biomarkers were significantly higher in synovial fluid than serum suggesting release from the signal knee: IL-1β (P < 0.0001), fetal aggrecan FA846 (P = 0.0001), CTxI (P = 0.0002), NTx (P = 0.012), osteocalcin (P = 0.012), Cartilage oligomeric matrix protein (COMP) (P = 0.0001) and matrix metalloproteinase (MMP)-3 (P = 0.0001). For these seven biomarkers we found significant correlations between the serum and synovial fluid concentrations for only CTxI (P = 0.0002), NTx (P < 0.0001), osteocalcin (P = 0.0002) and MMP-3 (P = 0.038).

CONCLUSIONS

These data strongly suggest that the biology after acute injury reflects that seen in cartilage explant models stimulated with pro-inflammatory cytokines, which are characterized by an initial wave of proteoglycan loss followed by subsequent collagen loss. As the rise of collagen biomarkers in synovial fluid occurs within the first month after injury, and as collagen loss is thought to be irreversible, very early treatment with agents to either reduce inflammation and/or reduce collagen loss may have the potential to reduce the onset of future post-traumatic osteoarthritis.

BACKGROUND

The samples used in this study were derived from a clinical trial NCT00332254 registered with ClinicalTrial.gov.

Objectives: Studies reported associations of inflammatory markers with the severity of COVID-19, but conclusions were inconsistent. We aimed to provide an overview of the association of inflammatory markers with the severity of COVID-19.

<strong class="sub-title"> Methods: </strong> We searched PubMed, Embase, Cochrane Library, Wanfang and China National Knowledge Infrastructure (CNKI) database until March <em>20</em>, <em>20</em><em>20</em>. Weighted mean difference (WMD) and 95% confidence intervals (CIs) were pooled using random or fixed-effects models.

<strong class="sub-title"> Results: </strong> A total of 16 studies comprising 3962 patients with COVID-19 were included in our analysis. Random-effect results demonstrated that patients with COVID-19 in the nonsevere group had lower levels for CRP (WMD = -41.78 mg/l, 95% CI = [-52.43, -31.13], P < 0.001), PCT (WMD = -0.13 ng/ml, 95% CI = [-0.<em>20</em>, -0.05], P < 0.001), IL-6 (WMD = -21.32 ng/l, 95% CI = [-28.34, -14.31], P < 0.001), ESR (WMD = -8 mm/h, 95% CI = [-14, -2], P = 0.005), SAA (WMD = -43.35 μg/ml, 95% CI = [-80.85, -5.85], P = 0.0<em>20</em>) and serum ferritin (WMD = -398.80 mg/l, 95% CI = [-625.89, -171.71], P < 0.001), compared with those in the severe group. Moreover, survivors had a lower level of IL-6 than non-survivors (WMD = -4.80 ng/ml, 95% CI = [-5.87, -3.73], P < 0.001). These results were consistent through sensitivity analysis and publication bias assessment.

Conclusions: The meta-analysis highlights the association of inflammatory markers with the severity of COVID-19. Measurement of inflammatory markers might assist clinicians to monitor and evaluate the severity and prognosis of COVID-19.

Keywords: COVID-19; Inflammatory markers; Meta-analysis; SARS-CoV-2; Severity.

Keratinocytes produce an <em>IL</em>-1 like factor termed epidermal cell-derived thymocyte-activating factor (ETAF). In this study, we show that ETAF and <em>IL</em>-1 are identical by the following criteria: Both normal and malignant human keratinocytes contain mRNAs identical to monocytic <em>IL</em>-1 alpha and <em>IL</em>-1 beta mRNA, as determined by an S1 nuclease protection assay; and <em>IL</em>-1 activity in medium conditioned by these cells can be neutralized by antibodies specific for human <em>IL</em>-1. The <em>IL</em>-1 alpha and <em>IL</em>-1 beta mRNAs can be identified in cultured human keratinocytes in the absence of identifiable stimulation; this basal level of mRNA can be further induced to accumulate with certain defined stimuli. Cultured normal human keratinocytes (HFKs) contain 2-4 times more <em>IL</em>-1 alpha than <em>IL</em>-1 beta mRNA; in contrast, human peripheral blood monocytes contain 10-<em>20</em> times more <em>IL</em>-1 beta than <em>IL</em>-1 alpha mRNA. The <em>IL</em>-1 activity released by these HFK can be neutralized by an antibody that neutralizes both alpha and beta <em>IL</em>-1, but not by an antibody that neutralizes only <em>IL</em>-1 beta. While human monocytes produce a large excess of <em>IL</em>-1 beta after appropriate stimulation, these data suggest that <em>IL</em>-1 alpha is a major (and may be the predominant) form of <em>IL</em>-1 produced by human keratinocytes.

Signaling via the CD3/TCR complex induces programmed cell death (apoptosis) in immature thymocytes and transformed T lymphocytes (hybridomas or leukemic cells). Accumulating evidence indicates, however, that apoptosis can be triggered also in mature peripheral T cells. Here we show that a significant fraction of cells of a given <em>IL</em>-2-dependent TCR-alpha beta + clone or polyclonal short term line is killed when cultured for <em>20</em> h in the presence of PHA, anti-CD3 (OKT3), or anti-TCR (BMA031) mAb. Apoptosis can be triggered by these stimuli in CD4+, CD8+, and CD4-CD8- (double-negative) TCR-alpha beta+ clones. Activation-driven cell death (as quantified by propidium iodide staining and FACS analysis) is associated with fragmentation of DNA into oligonucleosomal bands of approximately <em>20</em>0 bp. Although freshly isolated peripheral blood T cells are largely resistant to apoptosis, the sensitivity to anti-CD3/TCR mAb or PHA-triggered cell death gradually increases upon activation and <em>IL</em>-2-dependent culture of T cells, and reaches a plateau level after 15 to <em>20</em> days. These data indicate that stimuli that activate resting T cells initiate death by apoptosis in activated T cells. The implications of these results for the regulation of cellular immune responses and the establishment of peripheral tolerance will be discussed.

Exposure of renal mesangial cells to sphingosine 1-phosphate (S1P) leads to a rapid and transient activation of the mitogen- and stress-activated protein kinases but also the protein kinase B. Here, we show that S1P also induces phosphorylation of Smad proteins, which are members of the transforming growth factor-beta (TGF-beta) signaling device. However, Smad phosphorylation occurred more slowly with a maximal effect after <em>20</em>-30 min of S1P stimulation when compared with the rapid activation of the MAPKs. Interestingly, Smad phosphorylation is increased by pertussis toxin, which is in contrast to the complete inhibition of S1P-induced MAPK phosphorylation by pertussis toxin. TGF-beta is a potent anti-inflammatory cytokine, which in mesangial cells attenuates the expression of (i) inducible nitricoxide synthase (iNOS) caused by interleukin (<em>IL</em>)-1beta, (ii) secreted phospholipase A(2) (sPLA(2)), and (iii) matrix metalloproteinase-9 (MMP-9). These gene products are also down-regulated by S1P in a concentration-dependent manner. Furthermore, the expression of connective tissue growth factor is enhanced by both TGF-beta(2) and S1P. These effects of S1P are not mediated by the MAPK cascade as neither pertussis toxin nor the MAPK cascade inhibitor U0126 are able to reverse this inhibition. Overexpression of the inhibitory Smad-7 or down-regulation of co-Smad-4 lead to a reversal of the blocking effect of S1P on <em>IL</em>-1beta-induced NO release. Moreover, down-regulating the TGF-beta receptor type II by the siRNA technique or antagonizing the S1P(3) receptor subtype with suramin abrogates S1P-stimulated Smad phosphorylation. In summary, our data show that S1P trans-activates the TGF-beta receptor and triggers activation of Smads followed by activation of connective tissue growth factor gene transcription and inhibition of <em>IL</em>-1beta-induced expression of iNOS, sPLA(2), and MMP-9.

The roles of CXC chemokine-mediated host responses were examined with an A/J mouse model of Legionella pneumophila pneumonia. After intratracheal inoculation of 10(6) CFU of L. pneumophila, the bacterial numbers in the lungs increased 10-fold by day 2; this increase was accompanied by the massive accumulation of neutrophils. Reverse transcription-PCR data demonstrated the up-regulation of CXC chemokines, such as keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Consistent with these data, increased levels of KC, MIP-2, and LIX proteins were observed in the lungs and peaked at days 1, 2, and 2, respectively. Although the administration of anti-KC or anti-MIP-2 antibody resulted in an approximately <em>20</em>% decrease in neutrophil recruitment on day 2, no increase in mortality was observed. In contrast, the blockade of CXC chemokine receptor 2 (CXCR2), a receptor for CXC chemokines, including KC and MIP-2, strikingly enhanced mortality; this effect coincided with a 67% decrease in neutrophil recruitment. Interestingly, anti-CXCR2 antibody did not affect bacterial burden by day 2, even in the presence of a lethal challenge of bacteria. Moreover, a significant decrease in interleukin-12 (<em>IL</em>-12) levels, in contrast to the increases in KC, MIP-2, and LIX levels, was demonstrated for CXCR2-blocked mice. These data indicated that CXCR2-mediated neutrophil accumulation may play a crucial role in host defense against L. pneumophila pneumonia in mice. The increase in lethality without a change in early bacterial clearance suggested that neutrophils may exert their protective effect not through direct killing but through more immunomodulatory actions in L. pneumophila pneumonia. We speculate that a decrease in the levels of the protective cytokine <em>IL</em>-12 may explain, at least in part, the high mortality in the setting of reduced neutrophil recruitment.

OBJECTIVE

Cytokines contribute to pancreatic beta-cell death in type 1 diabetes. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells.

METHODS

Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h, and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and small-interfering RNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis. RESULTS Nearly 16,000 transcripts were detected as present in beta-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of >50% of the cytokine-modified genes.

CONCLUSIONS

The present study doubles the number of known genes expressed in primary beta-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in beta-cells. It also shows that cytokines modify alternative splicing in beta-cells, opening a new avenue of research for the field.

OBJECTIVE

The 14 amino acid sequence (aa(450-463)) TKDNNLLGRFELSG (TKD) of heat shock protein 70 (Hsp70) was identified as a tumor-selective recognition structure for natural killer (NK) cells. Incubation of peripheral blood lymphocyte cells with TKD plus low-dose interleukin 2 (IL-2) enhances the cytolytic activity of NK cells against Hsp70 membrane-positive tumors, in vitro and in vivo. These data encouraged us to test tolerability, feasibility, and safety of TKD-activated NK cells in a clinical Phase I trial.

METHODS

Patients with metastatic colorectal cancer (n = 11) and non-small cell lung cancer (n = 1) who had failed standard therapies were enrolled. After ex vivo stimulation of autologous peripheral blood lymphocytes with Hsp70-peptide TKD (2 microg/ml) plus low-dose IL-2 (100 units/ml), TKD was removed by extensive washing, and activated cells were reinfused i.v. The procedure was repeated for up to six cycles, applying a dose escalation schedule in 4 patients.

RESULTS

The percentage of activated NK cells in the reinfused leukapheresis products ranged between 8 and 20% of total lymphocytes, corresponding to total NK cell counts of 0.1 up to 1.5 x 10(9). Apart from restless feeling in 1 patient and itching in 2 patients, no negative side effects were observed. Concomitant with an enhanced CD94 cell surface density, the cytolytic activity of NK cells against Hsp70 membrane-positive colon carcinoma cells was enhanced after TKD/IL-2 stimulation in 10 of 12 patients. Concerning tumor response, 1 patient was in stable disease during therapy by formal staging criteria and another patient showed stable disease in one metastases and progression in another.

CONCLUSIONS

Reinfusion of Hsp70-activated autologous NK cells is safe. Immunological results warrant additional studies in patients with lower tumor burden.

During pregnancy, infection or immune responses induce cytokine release, which might influence fetal neurodevelopment, leading to neurodegenerative disease in adulthood. Because the hippocampus is a key area for learning and memory, we evaluated 4- and 24-wk-old rats for the effects of early and late prenatal exposure to interleukin-6 (<em>IL</em>-6) on hippocampal morphology, expression of mRNA for <em>IL</em>-6, the gamma-aminobutyric acid receptor (GABA(Aalpha5)), the NR1 subunit of the N-methyl-D-aspartate receptor, and glial fibrillary acidic protein (GFAP), caspase-3 protein and mRNA levels, and learning abilities. Late exposure increased serum <em>IL</em>-6 and hippocampal expression of <em>IL</em>-6 mRNA at 4 and 24 wk. All adult rats showed neuronal loss in the hilus and astrogliosis; males had losses mainly in the CA2 and CA3 regions, and females in CA1. Expression of GABA(Aalpha5), NR1, and GFAP mRNA increased in late-exposed males and females at 4 and 24 wk. mRNA and protein levels of the apoptosis marker caspase-3 were increased in all late-exposed rats except males at 4 wk. Evaluation of hippocampus-dependent working memory in the Morris water maze at <em>20</em> wk of age showed increases in escape latency and time spent near the pool wall in all <em>IL</em>-6 adult rats, especially females. These findings suggest that fetal <em>IL</em>-6 exposure, especially in late pregnancy, leads to increased <em>IL</em>-6 levels in the circulation and hippocampus, abnormalities of hippocampal structural and morphology, and decreased learning during adulthood.

Human Toll-like receptor 2 (TLR2) is a receptor for a variety of microbial products and mediates activation signals in cells of the innate immune system. We have investigated expression and regulation of the TLR2 protein in human blood cells and tissues by using two anti-TLR2 mAbs. Only myelomonocytic cell lines expressed surface TLR2. In tonsils, lymph nodes, and appendices, activated B-cells in germinal centers expressed TLR2. In human blood, CD14+ monocytes expressed the highest level of TLR2 followed by CD15+ granulocytes, and CD19+ B-cells, CD3+ T-cells, and CD56+ NK cells did not express TLR2. The level of TLR2 on monocytes was after <em>20</em> h up-regulated by LPS, GM-CSF, <em>IL</em>-1, and <em>IL</em>-10 and down-regulated by <em>IL</em>-4, IFN-gamma, and TNF. On purified granulocytes, LPS, GM-CSF, and TNF down-regulated, and <em>IL</em>-10 modestly increased TLR2 expression after 2 h. These data suggest that TLR2 protein expression in innate immune cells is differentially regulated by inflammatory mediators.

OBJECTIVE

To investigate whether molecular markers of inflammation and endothelial injury are associated with early growth of intracerebral hemorrhage (ICH).

METHODS

In a multicenter prospective study, we determined concentrations of interleukin-6 (<em>IL</em>-6), tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinase-9 (MMP-9), and cellular fibronectin (c-Fn) in blood samples obtained on admission from 183 patients with primary hemispheric ICH of <12 hours' duration. Patients had a neurological evaluation and a computed tomography (CT) scan performed at baseline and at 48+/-6 hours. Early growth of the ICH was defined as a volume increase >33% between the 2 CT examinations for ICH with a baseline volume (<em>20</em> mL and >10% for ICH>> or =<em>20</em> mL. Clinical, radiological, and biochemical predictive factors of ICH enlargement were analyzed by logistic regression analysis.

RESULTS

Fifty-four (29.5%) patients showed a relevant early growth of ICH. High leukocyte count and fibrinogen levels, low platelet count, and intraventricular bleeding were associated with early ICH growth in bivariate analyses. Plasma concentrations of <em>IL</em>-6 (median [quartiles]: 19.6 [13.6; 29.9] versus 15.9 [11.5; 19.8] pg/mL), TNF-alpha (13.5 [8.4; 30.5] versus 8.7 [4.7; 13.5] pg/mL), MMP-9 (153.3 [117.7; <em>20</em>4.7] versus 70.6 [47.8; 103.8] ng/mL), and c-Fn (8.8 [6.2; 12.5] versus 2.8 [1.6; 4.2] microg/mL) were significantly higher in patients with early growth of ICH (all P<0.001). C-Fn levels >6 microg/mL (OR, 92; 95%CI, 22 to 381; P<0.0001) and <em>IL</em>-6>24 pg/mL (OR, 16; 95%CI, 2.3 to 119; P=0.005) were independently associated with ICH enlargement in the logistic regression analysis.

CONCLUSIONS

Molecular signatures of vascular injury and inflammatory markers in the early acute phase of ICH are associated with subsequent enlargement of the hematoma.

BACKGROUND

Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects biologically active salivary components that favorably change the environment at the feeding site. Exposure to bites or to salivary proteins results in immunity specific to these components. Mice immunized with Phlebotomus papatasi salivary gland homogenate (SGH) or pre-exposed to uninfected bites were protected against Leishmania major infection delivered by needle inoculation with SGH or by infected sand fly bites. Immunization with individual salivary proteins of two sand fly species protected mice from L. major infection. Here, we analyze the immune response to distinct salivary proteins from P. papatasi that produced contrasting outcomes of L. major infection.

RESULTS

DNA immunization with distinct DTH-inducing salivary proteins from P. papatasi modulates L. major infection. PpSP15-immunized mice (PpSP15-mice) show lasting protection while PpSP44-immunized mice (PpSP44-mice) aggravate the infection, suggesting that immunization with these distinct molecules alters the course of anti-Leishmania immunity. Two weeks post-infection, 31.5% of CD4(+) T cells produced IFN-gamma in PpSP15-mice compared to 7.1% in PpSP44-mice. Moreover, <em>IL</em>-4-producing cells were 3-fold higher in PpSP44-mice. At an earlier time point of two hours after challenge with SGH and L. major, the expression profile of PpSP15-mice showed over 3-fold higher IFN-gamma and <em>IL</em>-12-Rbeta2 and <em>20</em>-fold lower <em>IL</em>-4 expression relative to PpSP44-mice, suggesting that salivary proteins differentially prime anti-Leishmania immunity. This immune response is inducible by sand fly bites where PpSP15-mice showed a 3-fold higher IFN-gamma and a 5-fold lower <em>IL</em>-4 expression compared with PpSP44-mice.

CONCLUSIONS

Immunization with two salivary proteins from P. papatasi, PpSP15 and PpSP44, produced distinct immune profiles that correlated with resistance or susceptibility to Leishmania infection. The demonstration for the first time that immunity to a defined salivary protein (PpSP44) results in disease enhancement stresses the importance of the proper selection of vector-based vaccine candidates.

Toll-like receptor (TLR) 2 is a member of the vertebrate protein family of TLRs that has been studied in substantial detail over the last years. The extracellular domain of the type I receptor molecule TLR2 contains 18 to <em>20</em> leucine rich repeat (LRR) and LRR like motives. The intracellular domain of TLR2 contains a Toll/<em>IL</em>-1 receptor/resistance protein typical TIR domain. After the first implication of TLR4 in immunity thereinafter followed by the discovery of the lipopolysaccharide signal transducer function of TLR4, TLR2 was the first of ten mammalian TLRs proven to be directly involved in recognition of pathogen associated molecular patterns (PAMPs). Among the TLR2 specific agonists are microbial products representing broad groups of species such as Gram-positive and Gram-negative bacteria, as well as mycobacteria, spirochetes, and mycoplasm. PAMP induced phagosomal localization of TLR2 and TLR2 dependent apoptosis have been shown. Complex formation with other molecules involved in pattern recognition such as CD14, MD2, TLR1, and TLR6 has been implicated for TLR2. Surprisingly even proteinaceous host material such as heat shock protein (HSP) 60 has been demonstrated to activate cells through TLR2. Thus, TLR2 may be a sensor and inductor of specific defense processes, including oxidative stress and cellular necrosis initially spurred by microbial compounds. Here we summarize the current knowledge on the structure and function of TLR2, which is far from being complete. Detailed understanding of the biology of TLR2 will probably contribute to the characterization of a number of infectious diseases and potentially help in the development of novel intervention strategies.

The CD4 (T4) antigen was originally described as a phenotypic marker specific for helper T cells, and has recently been shown to be the receptor for the human immunodeficiency virus (HIV). Functional studies using monoclonal antibodies directed at CD4 and major histocompatibility complex (MHC) class II molecules led to the suggestion that CD4 binds to the MHC class II molecules expressed on stimulator cells, enhancing T-cell responsiveness by increasing the avidity of T cell-stimulator cell interaction and/or by transmitting a positive intracellular signal. But recent evidence that antibodies to CD4 inhibit T-cell responsiveness in the absence of any putative ligand for CD4 has been interpreted as suggesting that antibody-mediated inhibition may involve the transmission of a negative signal via the CD4 molecule instead. We have infected a murine T-cell hybridoma that produces interleukin 2 (<em>IL</em>-2) in response to human class II HLA-DR antigens with a retroviral vector containing CD4 cDNA. The resulting CD4-expressing hybridoma cell lines produce 6- to <em>20</em>-fold more <em>IL</em>-2 in response to HLA-DR antigens than control cell lines. Furthermore, when antigen levels are suboptimal, the response of the cell lines is entirely CD4-dependent. The data presented here clearly demonstrate that CD4 can enhance T-cell responsiveness and may be crucial in the response to suboptimal levels of antigen.

BACKGROUND

Despite significant advances in understanding the biology of renal cell carcinoma (RCC) during the past decade, metastatic disease remains nearly incurable and a major medical challenge. Because RCC is known to be immunogenic, immunotherapeutic agents such as recombinant human interleukin-2 (rIL-2) and interferon-alpha (IFN-alpha) have represented encouraging treatment modalities.

METHODS

A review of the natural history of and therapeutic approaches to RCC was examined. Studies involving rIL-2 alone and in combination with other adjuvant therapies were critically evaluated.

RESULTS

Overall response rates for metastatic RCC patients treated with r<em>IL</em>-2 were similar (i.e., in the range of 15-<em>20</em>%), regardless of whether r<em>IL</em>-2 was administered as monotherapy or in combination with IFN-alpha. Recombinant <em>IL</em>-2 monotherapy response rates were similar to those of IFN-alpha, but with an increased frequency of complete responses and enhanced response duration. Subcutaneous administration generally resulted in lower toxicity than intravenous administration. The roles of chemotherapy or adoptive immunotherapy in combination with r<em>IL</em>-2 and IFN-alpha therapy remain unclear and require further study. The importance of patient performance status as a predictor of response and survival in r<em>IL</em>-2 therapy was demonstrated.

CONCLUSIONS

The use of rIL-2 with or without IFN-alpha may represent the most useful therapeutic approach currently available for patients with good performance status. In patients with borderline performance status or severe comorbid disease, therapeutic approaches depend on patient factors and outcome expectation and may involve cytokine therapy. However, regardless of performance status, palliative measures and/or observation are important choices, because the majority of patients with metastatic RCC are incurable.