B-1 cells temper endotoxemic inflammatory responses

Sepsis syndrome is caused by inappropriate immune activation due to bacteria and bacterial components released during infection. This syndrome is the leading cause of death in intensive care units. Specialized B-lymphocytes located in the peritoneal and pleural cavities are known as B-1 cells. These cells produce IgM and IL-10, both of which are potent regulators of cell-mediated immunity. It has been suggested that B-1 cells modulate the systemic inflammatory response in sepsis. In this study, we conducted in vitro and in vivo experiments in order to investigate a putative role of B-1 cells in a murine model of LPS-induced sepsis. Macrophages and B-1 cells were studied in monocultures and in co-cultures. The B-1 cells produced the anti-inflammatory cytokine IL-10 in response to LPS. In the B-1 cell-macrophage co-cultures, production of proinflammatory mediators (TNF-alpha, IL-6 and nitrite) was lower than in the macrophage monocultures, whereas that of IL-10 was higher in the co-cultures. Co-culture of B-1 IL-10(-/-) cells and macrophages did not reduce the production of the proinflammatory mediators (TNF-alpha, IL-6 and nitrite). After LPS injection, the mortality rate was higher among Balb/Xid mice, which are B-1 cell deficient, than among wild-type mice (65.0% vs. 0.0%). The Balb/Xid mice also presented a proinflammatory profile of TNF-alpha, IL-6 and nitrite, as well as lower levels of IL-10. In the early phase of LPS stimulation, B-1 cells modulate the macrophage inflammatory response, and the main molecular pathway of that modulation is based on IL-10-mediated intracellular signaling. (C) 2010 Elsevier GmbH. All rights reserved.

HDL-C concentration is related to markers of absorption and of cholesterol synthesis: Study in subjects with low vs. high HDL-C

Background: The antiatherogenic functions of high density lipoprotein (HDL-C) include its role in reverse cholesterol transport, but to what extent the concentration of HDL-C interferes with the whole-body cholesterol metabolism is unknown. Therefore, we measured markers of body cholesterol synthesis (desmosterol and lathosterol) and of intestinal cholesterol absorption (campesterol and beta-sitosterol) in healthy subjects that differ according to their plasma HDL-C concentrations. Methods: Healthy participants presented either low HDL-C (<40 mg/dl, n = 33,17 male and 16 female) or high HDL-C (>60 mg/dl, n = 33, 17 male and 16 female), BMI <30 kg/m(2), were paired according to age and gender, without secondary factors that might interfere with their plasma lipid concentrations. Plasma concentrations of non-cholesterol sterols were measured by the combined GC-MS analysis. Results: Plasma desmosterol did not differ between the two groups; however, as compared with the high HDL-C participants, the low HDL-C participants presented higher concentration of lathosterol and lower concentration of the intestinal cholesterol absorption markers campesterol and beta-sitosterol. Conclusion: Plasma concentrations of HDL, and not the activities of LCAT and CETP that regulate the reverse cholesterol transport system, correlate with plasma sterol markers of intestinal cholesterol absorption directly, and of cholesterol synthesis reciprocally. (C) 2010 Elsevier B.V. All rights reserved.

Anti-inflammatory effect of bee venom on antigen-induced arthritis in rabbits: Influence of endogenous glucocorticoids

Aim of the study: This study assessed the involvement of endogenous glucocorticoids (GCs) in the anti-arthritic properties of bee venom (BV) on antigen-induced arthritis (AIA) in rabbits. Materials and methods: BV (1.5-6 mu g/kg/day) was injected for 7 days before AIA induction, whereas the control group received sterile saline. The total and differential leukocyte count. PGE(2) levels in synovial fluid and synovial membrane cell infiltrate were evaluated. The contribution of GCs to BV action was assessed in rabbits treated with BV plus metyrapone, an inhibitor of GC synthesis, or RU-38 486, a steroid antagonist. Results: Treatment with BV (1.5 mu g/kg/day) reduced the leukocyte count and PGE2 level (18571 +/- 1909 cells/mm(3) and 0.49 +/- 0.05 ng/mL, respectively) as well as the cellular infiltrate compared with the control group (40968 +/- 5248 cells/mm(3) and 2.92 +/- 0.68 ng/mL, p < 0.05). The addition of metyrapone to BV treatment completely reversed the inhibition of AIA, whereas RU-38 486 was ineffective. Conclusion: Our data show that bee venom treatment prevents the development of antigen-induced arthritis in rabbits through the action of GCs. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

LOSS OF CD40 ENDOGENOUS S-NITROSYLATION DURING INFLAMMATORY RESPONSE IN ENDOTOXEMIC MICE AND PATIENTS WITH SEPSIS

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1 beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

Small volume resuscitation with 3% hypertonic saline solution decrease inflammatory response and attenuates end organ damage after controlled hemorrhagic shock

BACKGROUND: Recently, studies have been conducted examining the efficacy of 3% hypertonic saline solution (HS) for the treatment of traumatic brain injury; however, few studies have analyzed the effects of 3% hemorrhagic shock during hemorrhagic shock. The aim of this study was to test the potential immunomodulatory benefits of 3% hemorrhagic shock resuscitation over standard fluid resuscitation. METHODS: Wistar rats were bled to a mean arterial pressure of 35 mm Hg and then randomized into 3 groups: those treated with lactated Ringer`s solution (LR; 33 mL/kg, n = 7), 3% HS (10 mL/kg, n = 7), and 7.5% HS (4 mL/kg, n = 7). Half of the extracted blood was reinfused after fluid resuscitation. Animals that did not undergo shock served as controls (n = 5). Four hours after hemorrhagic shock, blood was collected for the evaluation of tumor necrosis factor-a and interleukin-6 by enzyme immunoassay. Lung and intestinal samples were obtained for histopathologic analysis. RESULTS: Animals in the HS groups had significantly higher mean arterial pressure than those in the LR group 1 hour after treatment. Osmolarity and sodium levels were markedly elevated in the HS groups. Tumor necrosis factor-alpha and interleukin-6 levels were similar between the control and HS groups but significantly higher in the LR group (P < .05). The lung injury score was significantly higher in the LR group compared with the 7.5% HS and 3% HS groups (5.7 +/- 0.7, 2.1 +/- 0.4, and 2.7 +/- 0.5, respectively). Intestinal injury was attenuated in the 7.5% HS and 3% HS groups compared with the LR group (2.0 +/- 0.6, 2.3 +/- 0.4, and 5.9 +/- 0.6, respectively). CONCLUSIONS: A small-volume resuscitation strategy modulates the inflammatory response and decreases end-organ damage after HS. Three percent HS provides immunomodulatory and metabolic effects similar to those observed with conventional concentrations of HS. (C) 2009 Elsevier Inc. All rights reserved.

Severe Periodontitis Is Associated With Diastolic Blood Pressure Elevation in Individuals With Heterozygous Familial Hypercholesterolemia: A Pilot Study

Background: This pilot study evaluates the association of severe periodontitis with pulse wave velocity (PWV), carotid artery intima-medial thickness (IMT), and clinical, metabolic, and atherogenic inflammatory markers in 79 subjects with heterozygous familial hypercholesterolemia (hFH). All subjects were free of previous vascular disease manifestations. Methods: The body mass index (in kilograms per square meter), plasma lipids, glucose, C-reactive protein, and white blood cell counts were evaluated. After full-mouth periodontal examinations, patients were categorized into the severe periodontitis group (SPG) or non-severe periodontitis group (NSPG). Results: The SPG showed significantly higher values of cholesterol-year scores, triglycerides, glucose, PWV, IMT, and diastolic blood pressure (DBP) (P <= 0.05) than the NSPG. After adjustment for traditional risk factors for atherosclerosis, only the association between severe periodontitis and DBP (odds ratio: 3.1; 95% CI: 1.1 to 8.5; P = 0.03) was confirmed. Conclusion: In individuals with hFH, severe periodontitis was associated with a higher DBP, which suggests that severe periodontitis, itself, may contribute to the increased cardiovascular risk profile in this population. J Periodontol 2011;82:683-688.

Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis

Elevated concentrations of plasma tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 have been detected in patients with alcoholic hepatitis and have been implicated in the pathogenesis of hepatocyte necrosis. The present study used a rat model to conduct a detailed histological and biochemical examination of the expression of various pro-inflammatory cytokines and associated liver pathology in ethanol-potentiated lipopolysaccharide (LPS)-induced liver injury. Male Wistar rats were pair-fed either the control or ethanol-containing (36% of caloric intake as ethanol) form of the Lieber-DeCarli liquid diet for 6 weeks. Liver injury was induced by the i.v. injection of LPS (1 mu g/g bodyweight), with animals being killed at O, 1, 3, 6, 12 and 24 h after injection. At the later time points, plasma transaminase and transpeptidase activities were significantly elevated in ethanol-fed LPS-treated rats compared with control-fed LPS-treated animals. At these times after LPS treatment, hepatocytes in ethanol-fed animals displayed fatty change and necrosis with an associated neutrophil polymorph infiltrate. Time course analysis revealed that plasma TNF-alpha (1-3 h post-LPS) and IL-6 (3 h post-LPS) bioactivity was significantly elevated in ethanol-fed compared with control-fed animals. No difference was seen in plasma IL-1 alpha concentration (maximal in both groups 6 h post-LPS). The expression of TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 mRNA were elevated between 1 and 6 h post-LPS in the livers of both control and ethanol-fed rats. However, ethanol-fed LPS-treated animals exhibited significantly higher maximal expression of IL-1 and IL-6 mRNA. Comparison of the appearance of cytokine mRNA and plasma bioactivity indicated an effect of ethanol feeding on post-transcriptional processing and/or the kinetics of the circulating cytokines. Elevated levels of both hepatic cytokine mRNA expression and the preceding plasma cytokines are presumably a necessary prerequisite for hepatic injury seen in this model and, therefore, possibly for the damage seen in human alcoholics. Further studies using this model may lead to significant advances in our understanding of the pathogenic mechanisms of alcoholic liver disease in humans.

Azithromycin does not prevent six-month myointimal proliferation but attenuates the transient systemic inflammation occurring after coronary stenting

Stent implantation produces a systemic increase of inflammatory markers that correlates with Chlamydophila pneumoniae infection in atherosclerotic plaque. We performed a clinical intervention study to investigate the effect of antibiotic treatment on 6-month follow-up angiographic minimal luminal diameter after stenting. Ninety patients were randomly assigned to oral azithromycin or placebo in a double-blinded and randomized fashion. Medication was initiated 2 weeks before a pre-scheduled stenting procedure and maintained 12 weeks thereafter. Angiographic outcomes were evaluated by a six-month follow-up angiography and laboratorial parameters were accessed by blood sampling 2 weeks before stenting, within the first 24 h after procedure and additional samples after four weeks and 6 months. Minimal luminal diameter (1.76 +/- A 0.56 mm Vs. 1.70 +/- A 0.86 mm; P = 0.7), restenosis rate, diameter stenosis, late loss, and binary restenosis rates were comparable in placebo and azithromycin group in the 6 months follow-up. Serum levels of C-reactive protein presented a three fold significant increase in the control group one day after stenting but did not change in the azithromycin group (8.5 [3.0;16.4] Vs. 2.9 [1.7;6.6]-median [25;75 percentile] P < 0.01). Azithromycin does not improve late angiographic outcomes but attenuates the elevation of C-reactive protein levels after stenting, indicating an anti-inflammatory effect.

Molluscum contagiosum: immunomorphological aspects of keratinocytes markers of differentiation and adhesion

Background Molluscum contagiosum (MC) is a Molluscipox virus infection of keratinocytes with hyperplasia and intracytoplasmic inclusions-the molluscum bodies (MBs). Few papers address cytokeratins (K) profile in MC, mainly focusing terminal keratinization process. Methods Forty-one MC lesions were subjected to immunohistochemical technique to verify K1, K10, K14, K16, involucrin, filaggrin, E-cadherin and p63 expression. MC immunolabeling pattern was compared to adjacent normal appearing epidermis (ANAE). Results In MC and ANAE, K1/K10 were expressed in suprabasal layers, K14 was expressed in basal and suprabasal layers and K16 was expressed through all spinous layer. Involucrin and filaggrin were observed in granular, spinous and in basal layer of ANAE and MC. E-cadherin was present up to the first layers of MC while ANAE exhibited E-cadherin labeling at basal and spinous layers. Basal and spinous layers keratinocytes nuclei, in both MC and ANAE, express p63. Conclusion Infection by Molluscipox virus alters keratinocyte differentiation status. The presence of K14 and p63 in spinous layer, as well as early expression of involucrin and filaggrin, associated to a hyperproliferative state disclosed by K16 expression, may be a result of disruption in keratinocytes maturation process. The changes observed at ANAE may represent early events in keratinization disturbance. Callegaro CF, Sotto MN. Molluscum contagiosum: immunomorphological aspects of keratinocytes markers of differentiation and adhesion.

Inflammatory epidermolysis bullosa acquisita - Case report

We report a case of an inflammatory variant of epidermolysis bullosa acquisita in a 53-year-old male, with itching blistering eruption on the trunk, armpits and limbs for six months. The skin biopsy specimen showed subepidermal blister with neutrophils. Direct immunofluorescence revealed linear depositions of IgG, IgA, IgM and C3 at the basement membrane; indirect immunofluorescence and salt split skin were negative. Antinuclear antibodies were also negative. Improvement of the blisters followed treatment with systemic corticotherapy and some lesions healed with milia. This is a rare presentation of epidermolysis bullosa acquisita, with inflammatory lesions at first.

Effects of phonophoresis with Arnica montana onto acute inflammatory process in rat skeletal muscles: An experimental study

This study aimed at verifying the effects of phonophoresis associated with Arnica montana on the acute phase of an inflammatory muscle lesion. Forty Wistar male rats (300 +/- 50 g), of which the Tibialis Anterior muscle was surgically lesioned, were divided into four groups (n = 10 each): control group received no treatment; the ultrasound group (US) was treated in pulsed mode with 1-MHz frequency, 0.5 W/cm(2) intensity (spatial and temporal average - SATA), duty cycle of 1: 2 (2 ms on, 4 ms off, 50%), time of application 3 min per session, one session per day, for 3 days; the phonophoresis or ultrasound plus arnica (US+A) group was treated with arnica with the same US parameters plus arnica gel; and the arnica group (A) was submitted to massage with arnica gel, also for 3 min, once a day, for 3 days. Treatment started 24 h after the surgical lesion. On the 4th day after lesion creation, animals were sacrificed and sections of the lesioned, inflamed muscle were removed for quantitative (mononuclear and polymorphonuclear cell count) and qualitative histological analysis. Collected data from the 4 groups were statistically analyzed and the significance level set at p < 0.05. Results show higher mononuclear cell density in all three treated groups with no significant difference between them, but values were significantly different (p < 0.0001) when compared to control group`s. As to polymorphonuclear cell density, significant differences were found between control group (p = 0.0134) and US, US+A and A groups; the arnica group presented lesser density of polymorphonuclear cells when compared (p = 0.0134) to the other groups. No significant difference was found between US and US+A groups. While the massage with arnica gel proved to be an effective anti-inflammatory on acute muscle lesion in topic use, these results point to ineffectiveness of Arnica montana phonophoresis, US having seemingly checked or minimized its anti-inflammatory effect. (C) 2008 Elsevier B. V. All rights reserved.

Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

Inflammation and Biochemical Features of Bariatric Candidates: Does Gender Matter?

Background Accumulated fat is an accepted trigger of inflammation and metabolic syndrome but specific biochemical associations in males and females are still debated. In a prospective study, multiple variables were analyzed to search for gender-related correlations. Methods Bariatric candidates (n=94) were consecutively investigated. Age was 34.9 +/- 10.4 years (68.1% females) and body mass index (BMI) was 40.8 +/- 4.6 kg/m(2). Methods included anthropometrics, inflammatory indices (C-reactive protein (CRP), white blood cell count (WBC), ferritin) and general biochemical profile. Results Ferritin, but not CRP or WBC, was substantially more elevated in males. Serum albumin, uric acid, creatinine, and liver enzymes AST and ALT were also higher in men. Even after BMI was adjusted, all differences remained significant, and several, notably ferritin, withstood waist circumference control. Ferritin and CRP correlated with anthropometrics, glucose-related measurements, and liver enzymes, whereas WBC was only associated with triglycerides in females. Conclusions (1) Males displayed more severe inflammation according to ferritin profile, and also more signs of liver derangement; (2) all differences continued after BMI discrepancies were adjusted for, and ferritin was significant also after control of waist girth; (3) in both genders inflammatory markers often correlated with different anthropometrics, liver enzymes, and markers of glucose homeostasis; and (4) inflammatory and biochemical gender-related dissimilarities might have prognostic implications for cardiovascular risk and other comorbidities, and deserve additional studies.

Markers of vascular differentiation, proliferation and tissue remodeling in juvenile nasopharyngeal angiofibromas

Juvenile nasopharingeal angiofibroma (JNA) is a histologically benign locally aggressive tumor characterized by irregular vessels embedded. in a fibrous stroma. Excessive vascularity results in bleeding complications, and the inhibition of angiogenesis is a promising strategy for managing extensive JNA tumors. To better characterize the endothelial components of JNA, we aimed to evaluate markers of vascular differentiation and proliferation, such as friend leukemia integration-1 (FLI-1) and endoglin, lymphatic markers, including podoplanin and vascular endothelial growth factor receptor 3 (VEGFR3) and its cognate ligand VEGFC, GLUT-1, a diagnostic marker that discriminates between hemangiomas and vascular malformations, and two markers of tissue remodeling, stromelysin 3 (ST3) and secreted acid protein rich in cysteine (SPARC). Antigens were assessed immunohistochemically in vessels and stromal cells of JNA archival cases (n=22). JNA endothelial cells were positive for endoglin, VEGFC and FLI-1, whereas podoplanin and VEGFR3 were negative in all cases. Both endothelial cells and fibroblasts stained for ST3 and SPARC. GLUT-1 was investigated in JNA cases, in infantile hemangiomas (n=123) and in vascular malformations (n=135) as controls. JNAs and vascular malformations were GLUT-1-negative, while hemangiomas showed positive staining. The presence of markers of endothelial differentiation and proliferation highlighted the hyper-proliferative state of JNA vessels. The absence of podoplanin and VEGFR3 underscores their blood endothelial cell characteristic. The absence of GLUT-1 discriminates JNAs from hemangiomas. ST3 and SPARC up-regulation in endothelial cells and fibroblasts may contribute to a compensatory signaling for controlling angiogenesis. Some of these markers may eventually serve as therapeutic targets. Our results may aid in the understanding of JNA pathophysiology.

TNF receptor-associated periodic syndrome (TRAPS): Description of a novel TNFRSF1A mutation and response to etanercept

TRAPS is the most common of the autosomal dominant periodic fever syndromes. It is caused by mutations in the TNFRSF1A gene, which encodes for the type 1 TNF-receptor (TNFR1). We describe here a Brazilian patient with TRAPS associated to a novel TNFRSF1A de novo mutation and the response to anti-TNF therapy. The patient is a 9-year-old girl with recurrent fevers since the age of 3 years, usually lasting 3 to 7 days, and recurring every other week. These episodes are associated with mild abdominal pain, nausea, vomiting and generalized myalgia. Recurrent conjunctivitis and erysipela-like skin lesions in the lower limbs also occur. Laboratory studies show persistent normocytic normochromic anemia, thrombocytosis, elevated erythrocyte sedimentation rate and C-reactive protein. IgD levels are normal. Mutational screening of TNFRSF1A revealed the association of a novel C30F mutation with the common R92Q low-penetrance mutation. The R92Q mutation is seen in 5% of the general population and is associated with an atypical inflammatory phenotype. The patient had a very good response to etanercept, with cessation of fever and normalization of inflammatory markers. Our report expands the spectrum of TNFRSF1A mutations associated with TRAPS, adding further evidence for possible additive effects of a low-penetration R92Q and cysteine residue mutations, and confirms etanercept as an efficacious treatment alternative.