The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of <em>15</em>3 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and <em>15</em>N chemical shifts by a 3D triple-resonance NH-<em>15</em>N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-<em>15</em>N(i)-13C alpha (i) and some weaker interresidue NH(i)-<em>15</em>N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, <em>15</em>N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn <em>15</em>N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser <em>15</em>N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of <em>15</em>) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly <em>15</em>N/13C-labeled 1.7 mM sample of <em>interleukin</em>-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming growth factor beta (TGF-beta) has potent down-regulating effects on macrophages and is thus capable of influencing the fate of intramacrophage parasites, including leishmanias. We report the development of a mouse model for the study of the human pathogen Leishmania braziliensis and demonstrate, both in vitro and in vivo, a key regulatory role for TGF-beta in the pathogenesis of infection with this parasite. Recombinant TGF-beta added to cultures of murine peritoneal macrophages led to increased intracellular L. braziliensis replication, whereas addition of neutralizing anti-TGF-beta monoclonal antibody decreased levels of infection. Macrophages infected with L. braziliensis produced biologically active TGF-beta, with a direct correlation between amounts of TGF-beta induced by two parasite isolates and their relative virulence. In vivo, treatment with recombinant TGF-beta rendered avirulent parasites virulent and activated latent L. braziliensis infection. Activation of parasite replication was observed in mice which had been infected with L. braziliensis <em>15</em> weeks previously but had not developed lesions or had healed lesions, depending on the parasite isolate used to infect the mice. The exacerbation of L. braziliensis infection in vivo was associated with an increase of <em>interleukin</em> 10 mRNA in the draining lymph node. These results demonstrate that TGF-beta is able to alter the course of in vitro and in vivo infections with L. braziliensis, the latter being characterized by an increase in <em>interleukin</em> 10, an important Th2 helper-T-cell cytokine.

Emerging evidence has implicated reactive oxygen species (ROS) in the pathogenesis of inflammatory bowel disease (IBD). Although intestinal epithelial cells produce the ROS-neutralizing enzyme superoxide dismutase (SOD), the protein and activity levels of copper/zinc (Cu/Zn) and manganese (Mn) SOD are perturbed in inflamed tissues of IBD patients. Thus we investigated the ability of MnSOD from Streptococcus thermophilus to reduce colitis symptoms in <em>interleukin</em> (IL) 10-deficient mice using Lactobacillus gasseri as a delivery vehicle. Cohorts of 13-<em>15</em> IL-10-deficient mice were left untreated or supplemented with native L. gasseri or L. gasseri expressing MnSOD for 4 wk. Colonic tissue was collected and inflammation was histologically scored. The presence of innate immune cells was investigated by immunohistochemistry and the host antioxidant response was determined by quantitative PCR. It was demonstrated that L. gasseri was stably maintained in mice for at least 3 days. L. gasseri producing MnSOD significantly reduced inflammation in IL-10-deficient mice compared with untreated controls (P < 0.05), whereas the anti-inflammatory effects of both native and MnSOD producing L. gasseri were more pronounced in males. The anti-inflammatory effects of L. gasseri were associated with a reduction in the infiltration of neutrophils and macrophages. Transcripts of antioxidant genes were equivalent in colonic tissues obtained from control and probiotic-treated IL-10-deficient mice. This study demonstrates that L. gasseri producing MnSOD has significant anti-inflammatory activity that reduces the severity of colitis in the IL-10-deficient mouse.

Acute liver failure (ALF) shares striking similarities with septic shock where a decrease in HLA-DR expression on monocytes is associated with disease severity and predicts outcome. We investigated monocyte HLA-DR expression in ALF in relation to inflammatory mediator levels and clinical outcome. Monocyte HLA-DR expression was determined in 50 patients with acetaminophen-induced ALF (AALF) and 20 non-acetaminophen-induced ALF (NAALF). AALF patients were divided into dead/transplanted (AALF-NS, n = 26) and spontaneous survivors (AALF-S, n = 24). Fifty patients with chronic liver disease (CLD) and 50 healthy volunteers served as controls. Monocyte HLA-DR expression was determined by double-color flow-cytometry with monoclonal antibodies detecting HLA-DR and monocyte specific CD14. Serum levels of <em>interleukin</em> (IL) -4, -6, -10, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were concomitantly measured by ELISA. Compared to healthy volunteers (75%) and CLD (67%) monocyte HLA-DR percentage expression was lower in AALF (<em>15</em>%, P < .001) and NAALF (22 %, P < .001). Compared to AALF-S, AALF-NS had lower monocyte HLA-DR % (11% vs. 36%, P < .001) and higher levels of IL-4, IL-6, IL-10 and TNF-alpha (P < .001). HLA-DR percentage negatively correlated with INR, blood lactate, pH and levels of encephalopathy (r = -0.8 to -0.5, P < .01), IL-10 (r = -0.8, P < .0001), TNF-alpha (r = -0.4, P = .02). HLA-DR percentage level <or=<em>15</em>% has a 96% sensitivity and 100% specificity and 98% accuracy in predicting poor prognosis. In conclusion, the strong relationship of monocyte HLA-DR expression with indices of disease severity, mediators of inflammation and outcome indicates a key role for this molecule as a biomarker of disease severity and prognosis.

The rapid increase in incidence of malignant melanoma has not been associated with better therapeutic options over the years. Single-agent chemotherapy or immunotherapy remain the treatments of choice when systemic therapy is offered. Dacarbazine (DTIC) is the chemotherapy of choice with a response rate of 16%. Other chemotherapies, including cisplatinum, paclitaxel, docetaxel and the DTIC analogue temozolomide, have shown activity in this disease. Based on their single-agent activity, several combination chemotherapies have been investigated with preliminary results that appeared promising. However, in randomized phase III trials the two most active chemotherapy combination regimens, cisplatin, vinblastine, and DTIC (CVD) and the Dartmouth regimen (DTIC, cisplatin, bischloroethylnitrosourea , and tamoxifen), did not prove to be superior to single-agent DTIC for overall survival. Immunotherapy with either <em>interleukin</em> (IL)-2 or interferon (IFN) has demonstrated response rates of 10% to <em>15</em>% in appropriately selected patients. In patients who achieve a complete response, responses can be of greater durability than those with chemotherapy. However, IL-2 and IFN administration are associated with multiple side effects, and only physicians experienced in the management of such therapies should administer them. The potential benefit of combining chemotherapy with immunotherapy has led to multiple phase II trials of biochemotherapy that appeared to be associated with higher response rates and longer median survivals. However, several phase III trials have been completed that have not consistently demonstrated an improvement in either response rates or overall survival, and these approaches to therapy cannot be routinely recommended outside the context of a clinical trial. The surgical resection of isolated metastatic disease has demonstrated an important palliative benefit in those patients who present with solitary single-organ disease with the exception of the liver. Radiation has an important role in the palliative management of brain metastasis and symptomatic bony metastasis. Both stereotactic radiosurgery and whole brain radiotherapy have been used alone and in combination to benefit patients in this troubling clinical circumstance. Isolated limb perfusion and a newer technique, isolated limb infusion have demonstrated high response rates for those uncommon patients who develop recurrent disease isolated to a limb. In our opinion, if complete metastasectomy is not feasible and in the absence of brain metastases, single-agent IL-2 is a good initial treatment choice in appropriately selected patients. Single-agent chemotherapy with DTIC is the treatment of choice for patients who are not candidates for IL-2. Adoptive immunotherapy combining nonmyeloablative chemotherapy with high-dose IL-2 is a potentially promising therapeutic strategy under investigation. Targeted therapy is also an area of promising development as single agents, in combination, and combined with chemotherapy. The latter will be the focus of at least one upcoming cooperative group phase III trial.

A 2-year-old girl with recurrent severe varicella infections had a fatal outcome. Studies of cellular and humoral immunity were normal. No natural killer (NK) cells were detected, and NK activity was markedly decreased. The <em>interleukin</em> (IL)<em>15</em>/IL<em>15</em>R signaling pathway was intact. This case emphasizes the role of NK cells in controlling herpes viral infection.

BACKGROUND

Olive plant leaves (Olea europaea L.) have been used for centuries in folk medicine to treat diabetes, but there are very limited data examining the effects of olive polyphenols on glucose homeostasis in humans.

OBJECTIVE

To assess the effects of supplementation with olive leaf polyphenols (51.1 mg oleuropein, 9.7 mg hydroxytyrosol per day) on insulin action and cardiovascular risk factors in middle-aged overweight men.

METHODS

Randomized, double-blinded, placebo-controlled, crossover trial in New Zealand. 46 participants (aged 46.4 ± 5.5 years and BMI 28.0 ± 2.0 kg/m(2)) were randomized to receive capsules with olive leaf extract (OLE) or placebo for 12 weeks, crossing over to other treatment after a 6-week washout. Primary outcome was insulin sensitivity (Matsuda method). Secondary outcomes included glucose and insulin profiles, cytokines, lipid profile, body composition, 24-hour ambulatory blood pressure, and carotid intima-media thickness.

RESULTS

Treatment evaluations were based on the intention-to-treat principle. All participants took >96% of prescribed capsules. OLE supplementation was associated with a <em>15</em>% improvement in insulin sensitivity (p = 0.024) compared to placebo. There was also a 28% improvement in pancreatic β-cell responsiveness (p = 0.013). OLE supplementation also led to increased fasting <em>interleukin</em>-6 (p = 0.014), IGFBP-1 (p = 0.024), and IGFBP-2 (p = 0.0<em>15</em>) concentrations. There were however, no effects on <em>interleukin</em>-8, TNF-α, ultra-sensitive CRP, lipid profile, ambulatory blood pressure, body composition, carotid intima-media thickness, or liver function.

CONCLUSIONS

Supplementation with olive leaf polyphenols for 12 weeks significantly improved insulin sensitivity and pancreatic β-cell secretory capacity in overweight middle-aged men at risk of developing the metabolic syndrome.

<em>Interleukin</em>-<em>15</em> (IL-<em>15</em>) is a cytokine which is highly expressed in skeletal muscle tissue, and which has anabolic effects on skeletal muscle protein dynamics both in vivo and in vitro. Additionally, administration of IL-<em>15</em> to rats and mice inhibits white adipose tissue deposition. To determine if the action of IL-<em>15</em> on adipose tissue is direct, the capacity of cultured murine 3T3-L1 preadipocytes and adipocytes to respond to IL-<em>15</em> was examined. IL-<em>15</em> administration inhibited lipid accumulation in differentiating 3T3-L1 preadipocytes, and stimulated secretion of the adipocyte-specific hormone adiponectin by differentiated 3T3-L1 adipocytes. The latter observation constitutes the first report of a cytokine or growth factor which stimulates adiponectin production. IL-<em>15</em> mRNA expression by cultured 3T3-L1 adipogenic cells and C2C12 murine skeletal myogenic cells was also examined. Quantitative real-time PCR indicated IL-<em>15</em> mRNA was expressed by C2C12 skeletal myogenic cells, and was upregulated more than 10-fold in differentiated skeletal myotubes compared to undifferentiated myoblasts. In contrast, 3T3-L1 cells expressed little or no IL-<em>15</em> mRNA at either the undifferentiated preadipocyte or differentiated adipocyte stages. These findings provide support for the hypothesis that IL-<em>15</em> functions in a muscle-to-fat endocrine axis which modulates fat:lean body composition and insulin sensitivity.

OBJECTIVE

IL-<em>15</em> decreases lipid deposition in preadipocytes and decreases the mass of white adipose tissue in rats, indicating that IL-<em>15</em> may take part in regulating this tissue. IL-<em>15</em> is expressed in human skeletal muscle and skeletal muscle may be a source of plasma IL-<em>15</em> and in this way regulate adipose tissue mass.

METHODS

The relation between skeletal muscle IL-<em>15</em> mRNA expression, plasma IL-<em>15</em>, and adipose tissue mass was studied in 199 humans divided into four groups on the basis of obesity and type 2 diabetes. Furthermore, using a DNA electrotransfer model, we assessed the effect of IL-<em>15</em> overexpression in skeletal muscle of mice.

RESULTS

In humans, multiple regression analysis showed a negative association between plasma IL-<em>15</em> and total fat mass (P<0.05), trunk fat mass (P<0.01), and percent fat mass (P<0.05), independent of type 2 diabetes. Negative associations were also found between muscle IL-<em>15</em> mRNA and obesity parameters. IL-<em>15</em> overexpression in skeletal muscle of mice reduced trunk fat mass but not sc fat mass.

CONCLUSIONS

Our results indicate that IL-<em>15</em> may be a regulator of trunk fat mass.

How inflammation causes cancer is unclear. <em>Interleukin</em>-<em>15</em> (IL-<em>15</em>) is a pro-inflammatory cytokine elevated in human large granular lymphocyte (LGL) leukemia. Mice overexpressing IL-<em>15</em> develop LGL leukemia. Here, we show that prolonged in vitro exposure of wild-type (WT) LGL to IL-<em>15</em> results in Myc-mediated upregulation of aurora kinases, centrosome aberrancies, and aneuploidy. Simultaneously, IL-<em>15</em> represses miR-29b via induction of Myc/NF-κBp65/Hdac-1, resulting in Dnmt3b overexpression and DNA hypermethylation. All this is validated in human LGL leukemia. Adoptive transfer of WT LGL cultured with IL-<em>15</em> led to malignant transformation in vivo. Drug targeting that reverses miR-29b repression cures otherwise fatal LGL leukemia. We show how excessive IL-<em>15</em> initiates cancer and demonstrate effective drug targeting for potential therapy of human LGL leukemia.

The purinergic P2X(7) receptor is a ligand-gated ion channel found on peripheral macrophages and microglia in the nervous system. Activation of P2X(7) receptors results in the rapid release of <em>interleukin</em>-1 beta (IL-1 beta). Cytokines like IL-1 beta are suggested to be involved in the pathophysiology of depression. The aim of this study was to behaviorally profile P2X(7) receptor knockout (KO) mice in behavioral models of depression- and anxiety-like behaviors. P2X(7) receptor KO and wild type (WT) mice were tested in multiple models including; forced swim test, tail suspension test, elevated plus maze, novelty suppressed feeding, spontaneous locomotor activity, and food intake. P2X(7) receptor KO mice exhibited an antidepressant-like profile in tail suspension test and forced swim test; an effect that was not associated with changes in spontaneous locomotor activity. In addition, P2X(7) receptor KO mice showed higher responsivity to a subefficacious dose of the antidepressant drug imipramine (<em>15</em> mg/kg) in forced swim test. No significant differences between genotypes were observed in models of anxiety. These data support the relevance of pro-inflammatory cytokines in depressive-like states, and suggest that P2X(7) receptor antagonists could be of potential interest for the treatment of affective disorders.

<em>Interleukin</em>-<em>15</em> is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of <em>interleukin</em>-<em>15</em> overlap that of <em>interleukin</em>-2. Using murine models, the present studies have shown that <em>interleukin</em>-<em>15</em>, in vivo, is three to four times more potent than <em>interleukin</em>-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as <em>interleukin</em>-2 in inducing specific cytolytic cells that lyse allogeneic target cells. <em>Interleukin</em>-<em>15</em> is approximately half as potent as <em>interleukin</em>-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of <em>interleukin</em>-<em>15</em> required to induce pulmonary vascular leak in mice is six times higher than that required for <em>interleukin</em>-2. These results support the view that <em>interleukin</em>-<em>15</em> exhibits a therapeutic index that is superior to <em>interleukin</em>-2.

OBJECTIVE

Tofacitinib, an orally administered Janus kinase inhibitor, blocks signaling through γ-chain-containing cytokines (<em>interleukins</em> 2, 4, 7, 9, <em>15</em>, and 21). We performed a phase 2 trial to measure its efficacy in patients with moderate-to-severe active Crohn's disease.

METHODS

Patients (N = 139; age, ≥18 y) with moderate-to-severe active Crohn's disease were assigned randomly to groups given 1 mg (n = 36), 5 mg (n = 34), or <em>15</em> mg (n = 35) tofacitinib or placebo (n = 34), twice daily for 4 weeks, at 48 centers in 12 countries. The primary end point was the proportion of clinical responders at week 4 (decrease from baseline in the Crohn's Disease Activity Index score of ≥70 points [Response-70]). Secondary end points included clinical remission (Crohn's Disease Activity Index score of (<em>15</em>0 points) at week 4.

RESULTS

A clinical response was observed in 36% (P = .467), 58% (P = .466), and 46% (P ≥ .999) of patients given the 1-, 5-, and <em>15</em>-mg doses of tofacitinib, compared with 47% of patients given placebo. Clinical remission was observed in 31% (P = .417), 24% (P = .776), and 14% (P = .540) of patients given the 1-, 5-, and <em>15</em>-mg doses of tofacitinib, compared with 21% of patients given placebo. The <em>15</em>-mg dose of tofacitinib reduced levels of C-reactive protein and fecal calprotectin from baseline. Adverse and serious adverse events were similar among groups. Dose-dependent increases in low- and high-density lipoprotein cholesterol were observed in patients given the 5- or <em>15</em>-mg doses of tofacitinib.

CONCLUSIONS

There were no significant differences in the percentage of patients with moderate-to-severe active Crohn's disease who achieved clinical responses (Response-70) or clinical remission after 4 weeks' administration of tofacitinib (1, 5, or <em>15</em> mg) or placebo twice daily. However, a large percentage of patients given placebo achieved Response-70 or remission. Reductions in C-reactive protein and fecal calprotectin levels among patients given the <em>15</em>-mg dose of tofacitinib indicate its biologic activity. ClinicalTrials.gov number: NCT006<em>15</em>199.

OBJECTIVE

Longer-term effects of prolonged selective interleukin-1β blockade with canakinumab were evaluated in the largest cohort of cryopyrin-associated periodic syndrome (CAPS) patients studied to date.

METHODS

Adult and paediatric CAPS patients (n=166, including canakinumab-naive and pretreated patients from previous studies) received canakinumab subcutaneously 150 mg or 2 mg/kg (≤40 kg) every 8 weeks for up to 2 years. Response and relapse was assessed using scores for disease activity, skin rash and C-reactive protein (CRP) and/or serum amyloid A (SAA) levels.

RESULTS

Complete response was achieved in 85 of 109 canakinumab-naive patients (78%; 79/85 patients within 8 days, and five patients between days 10 and 21). Of 141 patients with an available relapse assessment, 90% did not relapse, their CRP/SAA levels normalised (<10 mg/l) by day 8, and remained in the normal range thereafter. Median treatment duration was 414 days (29-687 days). Upward adjustments of dose or frequency were needed in 24.1% patients; mostly children and those with severe CAPS. Predominant adverse events (AE) were infections (65.7%) of mostly mild-to-moderate severity. Serious AE reported in 18 patients (10.8%) were mainly infections and were responsive to standard treatment. The majority of patients (92%) reported having no injection-site reactions and only 8% patients reported mild-to-moderate reactions. Patients receiving vaccination (15%) showed normal immune response.

CONCLUSIONS

Subcutaneous canakinumab 150 mg every 8 weeks was well tolerated and provided substantial disease control in children and adults across all CAPS phenotypes. Higher canakinumab doses in younger patients and more severe CAPS disease were efficacious in achieving complete responses without evidence of increased AE.

BACKGROUND

NCT00685373 (clinicaltrials.gov).

<em>Interleukin</em> 1beta-converting enzyme-like (ICE-like) proteases are important mediators of apoptosis in diverse cell types and organisms. However, the role of these proteases in apoptosis cannot be satisfactorily explained on the basis of the physiological functions of their known substrates. Here we show that the C-terminal 42 amino acid peptide of the retinoblastoma (Rb) protein, an important cell cycle regulator with a known anti-apoptotic function, is specifically cleaved off by an ICE-like protease in tumour necrosis factor (TNF)- and staurosporine-induced apoptosis. Cleavage of Rb induced by TNF was blocked in vivo and in vitro by two specific inhibitors of ICE-like proteases, and in vitro by a point mutation (Asp886 to Ala) within the ICE-like protease cleavage site of Rb, (883)DEAD(886). An antibody raised against the C-terminal <em>15</em> amino acid peptide of Rb recognized the full-length but not the cleaved form of Rb. The extent of Rb cleavage correlated directly with TNF-induced apoptosis in all tumour cell lines examined. Cleaved Rb bound cyclin D3 and inhibited the transcriptional activity of E2F-1, but failed to bind to the regulatory protein MDM2, which has been implicated in apoptosis. As Rb suppresses cell death and its C-terminus has important regulatory functions, our results suggest that Rb cleavage is an important event in apoptosis.

A new human breast cancer cell line, KPL-4, was recently isolated from the malignant pleural effusion of a breast cancer patient with an inflammatory skin metastasis. This cell line can be cultured under serum-free conditions and is tumorigenic in female athymic nude mice. Flow cytometric analysis revealed the expression of Erb B-1, -2 and -3. Dot blot hybridization showed a <em>15</em>-fold amplification of the erb B-2. Reverse transcription-polymerase chain reaction analysis showed a detectable level of mRNA expression of all the Erb B family receptors. In addition, all the receptors were autophosphorylated under a serum-supplemented condition. Unexpectedly, transplanted KPL-4 tumours induced cachexia of recipient mice. A high concentration of <em>interleukin</em>-6 (IL-6) was detected in both the culture medium and the serum of mice. The weight of tumours significantly correlated with the serum IL-6 level. The antiproliferative effect of a humanized anti-Erb B-2 monoclonal antibody, rhuMAbHER2, was investigated. This antibody significantly inhibited the growth of KPL-4 cells in vitro but modestly in vivo. Loss of mouse body weight was partly reversed by rhuMAbHER2. These findings suggest that KPL-4 cells may be useful in the development of new strategies against breast cancer overexpressing the Erb B family receptors and against IL-6-induced cachexia.

OBJECTIVE

We conducted a systematic review of the literature with meta-analysis to determine whether complex regional pain syndrome (CRPS) is associated with a specific inflammatory profile and whether this is dependent on the duration of the condition.

METHODS

Comprehensive searches of the literature using MEDLINE, Embase, Scopus, Web of Science, and reference lists from published reviews identified articles that measured inflammatory factors in CRPS. Two independent investigators screened titles and abstracts, and performed data extraction and risk of bias assessments. Studies were subgrouped by medium (blood, blister fluid, and CSF) and duration (acute and chronic CRPS). Where possible, meta-analyses of inflammatory factor concentrations were performed and pooled effect sizes were calculated using random-effects models.

RESULTS

Twenty-two studies were included in the systematic review and <em>15</em> in the meta-analysis. In acute CRPS, the concentrations of <em>interleukin</em> (IL)-8 and soluble tumor necrosis factor receptors I (sTNF-RI) and II (sTNF-RII) were significantly increased in blood. In chronic CRPS, significant increases were found in 1) TNFα, bradykinin, sIL-1RI, IL-1Ra, IL-2, sIL-2Ra, IL-4, IL-7, interferon-γ, monocyte chemoattractant protein-1 (MCP-1), and sRAGE (soluble receptor for advanced glycation end products) in blood; 2) IL-1Ra, MCP-1, MIP-1β, and IL-6 in blister fluid; and 3) IL-1β and IL-6 in CSF. Chronic CRPS was also associated with significantly decreased 1) substance P, sE-selectin, sL-selectin, sP-selectin, and sGP130 in blood; and 2) soluble intercellular adhesion molecule-1 (sICAM-1) in CSF. Most studies failed to meet 3 or more of our quality criteria.

CONCLUSIONS

CRPS is associated with the presence of a proinflammatory state in the blood, blister fluid, and CSF. Different inflammatory profiles were found for acute and chronic cases.

OBJECTIVE

To compare the conjunctival epithelial cell expressions of inflammatory cytokines in normal subjects and in glaucoma patients treated over the long term.

METHODS

Case-control study.

METHODS

A total of 69 glaucoma patients treated over the long term and <em>15</em> normal subjects with no ocular abnormality or topical treatment.

METHODS

Amongst the 69 glaucoma patients, 27 were treated with preserved beta-blockers, 24 with unpreserved 0.5% timolol, and the other 18 patients with an association of>> or =2 preserved drugs. All patients were treated for more than 1 year with the same treatment, with no significant differences between groups for mean ages and durations of treatment at the time of the study. Impression cytology specimens were taken and processed for immunofluorescence techniques. Conjunctival cell expressions of HLA DR, as a standard for inflammatory level, and the interleukins IL-6, IL-8, and IL-10 were obtained and quantified using flow cytometry.

METHODS

Immune markers and proinflammatory cytokines in impression cytology specimens.

RESULTS

We found a significantly increased expression of all immunoinflammatory markers and mediators in the conjunctival epithelium of glaucoma patients compared with normal eyes. Human leukocyte antigen DR was significantly higher in the 2 groups receiving preserved drugs than in the unpreserved timolol group. The 3 interleukins were similarly overexpressed in all glaucoma groups, with no significant between-groups differences except for the expression level of IL-8, which was significantly higher in the multitreatment group than in the preservative-free one.

CONCLUSIONS

The present study confirms the increased expression of immunoinflammatory markers by the conjunctival epithelium of glaucoma patients treated over the long term. The development of nontoxic preservatives or preservative-free solutions is therefore of great interest.

OBJECTIVE

The pathologic changes within the intestinal muscle layer may be at the origin of the cytokines that account for acute radiation-induced inflammation. We were specifically interested in evaluating the efficacy of an inhibitor of nuclear transcription factor kappa B (NF-kappaB) activation that is involved in regulating cytokine expression.

METHODS

Cytokine expression was analyzed in the ileal muscularis layer by reverse transcriptase-polymerase chain reaction (RT-PCR) at 3 h, 6 h, and 3 days after a 10-Gy gamma whole-body irradiation of rats. Caffeic acid phenethyl ester (CAPE) was injected intraperitoneally (30 mg/kg) <em>15</em> min before irradiation and once a day for 3 days.

RESULTS

Interleukin (IL)-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNA increased at 3 h and 6 h after irradiation, and expression of IL-6 and IL-8 was elevated at 3 days. On the other hand, levels of the anti-inflammatory cytokine IL-10 were markedly lower on Day 3. Overexpression of IL-6 on Day 3 was combined with upregulation of the IL-6 receptors (gp130/gp80) and suppressor of cytokine signaling-3 (SOCS3) genes. CAPE treatment did not significantly change IL-1beta or TNF-alpha expressions in the irradiated rats; it increased IL-10 expression at 6 h but had no effect on it on Day 3. CAPE treatment inhibited the radiation-induced expression of IL-6, IL-6 receptors (IL-6rs), and SOCS3 at 3 days.

CONCLUSIONS

In vivo, irradiation induced a cascade of inflammatory responses that involved the transcription factor NF-kappaB; this inflammation was reduced by CAPE treatment.

This study determined the presence of <em>interleukin</em> 1 (IL-1), <em>interleukin</em> 6 (IL-6), tumour necrosis factor alpha (TNF alpha), tumour necrosis factor beta (TNF beta), interferon gamma (IFN gamma), transforming growth factor beta 2 (TGF beta 2) and fibroblast proliferation activity (FPA) in vitreous aspirates from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy (PVR) or uncomplicated retinal detachment (RD). Cadaveric vitreous from normal subjects were used as controls. The results showed that IL-1 and IL-6 predominated in vitreous from eyes with PVR or RD, and that concentrations of IL-6 greater than 20 pg/ml were more frequently found in PVR than in RD (p = 0.031) or control specimens (p = 0.006). Low levels of TNF alpha were observed in 4/18 eyes with PVR, 1/<em>15</em> eyes with RD and 1/<em>15</em> control vitreous, and small concentrations of TNF alpha were seen in 3/18 eyes with PVR, 1/<em>15</em> eyes with RD and 2/<em>15</em> control vitreous. IFN gamma was detected in 12/18 eyes with PVR, but only in 5/<em>15</em> eyes with RD (p = 0.048) and 6/<em>15</em> control specimens. TGF beta 2 was present in all vitreous samples at concentrations ranging from 100 to 4,500 pg/ml with no significant differences among the three groups. Control vitreous possessed the greatest FPA when compared with vitreous from eyes with PVR (p = 0.031) or RD (p = 0.048). These observations provide further evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of PVR and point to the possible involvement of IL-1, IL-6 and IFN gamma in cellular interactions leading to chronicity.

OBJECTIVE

We studied the expression of cytokines and inflammatory cells in normal and inflamed esophageal mucosa of children with the aim of furthering our understanding of the pathophysiology of allergic eosinophilic esophagitis (AEE).

METHODS

Controls and AEE patients >>or=<em>15</em> eosinophils/high-power field on esophageal mucosal biopsies) between the ages of 1 and 18 years were recruited. Esophageal biopsies were obtained for histologic examination, immunohistochemical studies, and cytokine analysis.

RESULTS

Eight controls (4 males; mean age 9.99 years) and 11 AEE patients (8 males; mean age 7.<em>15</em> years) were studied. mRNA expression of interferon (IFN)-gamma, interleukin (IL)-4, IL-5, IL-13, eotaxin-1, eotaxin-2, eotaxin-3, and RANTES was studied. IFN-gamma and IL-5 expressions were significantly up-regulated in AEE patients compared with controls. Expressions of IL-4 and IL-13 were similar between AEE patients and controls. Eotaxin-1 expression was significantly up-regulated in AEE patients, whereas eotaxin-2 was up-regulated in controls. Expression of RANTES and eotaxin-3 was similar between the two groups. There was increased staining for mast cells in AEE patients compared with controls.

CONCLUSIONS

Our data suggests that AEE is primarily an IL-5 selective TH2 response, with a possible TH1 component, and a differential role of eosinophilic chemoattractants. The role of mast cells in the pathogenesis of AEE needs additional study.

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold, P < 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with <em>interleukin</em> 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- +/- 2-fold, myeloid colony-forming cells increased 140- +/- 36-fold, and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with <em>15</em> Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is strongly implicated in graft-versus-host disease (GVHD) and other acute bone marrow transplant (BMT) complications. The antiinflammatory <em>interleukin</em>-10 (IL-10) antagonizes TNF-alpha and reduces GVHD. We previously showed association of recipient TNF (TNFd) and IL-10 (IL-10(-1064)) gene polymorphisms with acute GVHD severity in matched sibling BMT using only cyclosporin A monotherapy. The current study tested association of GVHD with TNFd and IL-10(-1064/-1082) polymorphisms in a large cohort (144 matched sibling donor/recipient pairs) given both cyclosporine A (CyA) and methotrexate (MTX) prophylaxis. Genotype results were correlated with acute and chronic GVHD and mortality. Patients homozygous for the TNFd microsatellite allele 3 had higher early mortality: 23.7% of TNFd3/d3 homozygotes died before day 30, compared with 6.80% of non-d3/d3 recipients (P =.013). Recipients possessing longer IL-10(-1064) microsatellite alleles developed more severe acute GVHD: 22.3% of recipients possessing alleles 12 to <em>15</em> developed grade III to IV GVHD, versus 3.92% of those with smaller alleles (P <.01). Other recipient or donor genotypes tested did not significantly affect GVHD or mortality. We conclude that recipient TNFd and IL-10(-1064) polymorphisms associate with early mortality and severe acute GVHD in matched sibling BMT with dual prophylaxis. This supports the hypothesis of genetic predisposition towards GVHD and other BMT complications other than histocompatibility antigen disparity.

It is not known whether one or both of the <em>interleukin</em> 1 (IL1) receptors mediates the induction of the DNA-binding protein NF-kappa B. Nuclear extracts of the murine lines EL4.NOB.1 and 70Z/3, which bear the type I (80 kDa) and type II (67 kDa) IL1 receptor, respectively, were analyzed by an electrophoretic mobility shift assay. A 265-base pair sequence of the human serum amyloid A gene or a synthetic oligonucleotide each containing the NF-kappa B site were used as the DNA probes. IL1 induction of NF-kappa B was rapid (optimal at <em>15</em>-30 min) and transient in both cell types. The IL1 receptor antagonist (IL1ra), which binds strongly to the type I receptor, inhibited the NF-kappa B response in both cell lines. IL1ra did not bind to the type II receptor on 70Z/3 cells as judged by competition for binding with 125I-IL1 alpha. When 125I-IL1ra binding to 70Z/3 cells was measured, a small number (10/cell) of high affinity sites (Kd = 5 x 10(-12) M) were detected. These were likely to have been type I receptor because an antibody to this inhibited the NF-kappa B induction in 70Z/3 cells (as well as EL4). Potential signal transduction mechanisms involving protein kinase C or oxygen radicals were studied. Phorbol 12-myristate 13-acetate induced NF-kappa B with a similar time course to IL1 in 70Z/3 but only after 4 h in EL4.IL1 was unaffected by a protein kinase C inhibitor (staurosporine). H2O2 did not mimic IL1, and IL1 was not inhibited by an antioxidant. The type I receptor mediates the induction of NF-kappa B in response to IL1 via a signaling mechanism that still remains to be identified.