Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than CKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with CKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity. (C) 2009 Elsevier B.V. All rights reserved.

Treatment of obstructive sleep apnea syndrome in patients from a teaching hospital in Brazil: is it possible?

The aim of this study was to evaluate the efficacy of a cost-effective intra-oral appliance for obstructive sleep apnea syndrome built into a large teaching hospital. Out of 20 evaluated and treated patients, 14 concluded the study: eight men and six women, with a mean age of 42-46 (mean + SD) years and mean body mass index of 27.66. Inclusion criteria were mild or moderate apnea-hypopnea index (AHI) according to a polysomnographic study. All patients were treated with the monobloco intra-oral appliance. They were then submitted to a follow-up polysomnographic study after 60 days using the appliance. An orofacial clinical evaluation was carried out with the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) questionnaire and with clinical evaluation questionnaire devised by the Orofacial Pain Team before and 60 days after fitting the intra-oral appliance. The AHI showed a statistically meaningful (p = 0.002) reduction from 15.53 to 7.82 events per hour, a non-statistically significant oxygen saturation increase from 83.36 to 84.86 (p = 0.09), and Epworth`s sleepiness scale reduction from 9.14 to 6.36 (p = 0.001). Three patients did not show any improvement. The most common side effect during the use of the appliance/device was pain and facial discomfort (28.57%), without myofascial or temporomandibular joint pain as evaluated by the RDC/TMD questionnaire. The intra-oral device produced a significant reduction of the apnea-hypopnea index during the study period with the use of the monobloco intra-oral appliance. Patients did not show prior myofascial pain or 60 days after use of the intra-oral appliance.

Current management practices for acromegaly: an international survey

To determine whether peer-reviewed consensus statements have changed clinical practice, we surveyed acromegaly care in specialist centers across the globe, and determined the degree of adherence to published consensus guidelines on acromegaly management. Sixty-five acromegaly experts who participated in the 7th Acromegaly Consensus Workshop in March 2009 responded. Results indicated that the most common referring sources for acromegaly patients were other endocrinologists (in 26% of centers), neurosurgeons (25%) and primary care physicians (21%). In sixty-nine percent of patients, biochemical diagnoses were made by evaluating results of a combination of growth hormone (GH) nadir/basal GH and elevated insulin like growth factor-I (IGF-I) levels. In both Europe and the USA, neurosurgery was the treatment of choice for GH-secreting microadenomas and for macroadenomas with compromised visual function. The most widely used criteria for neurosurgical outcome assessment were combined measurements of IGF-I and GH levels after oral glucose tolerance test (OGTT) 3 months after surgery. Ninety-eight percent of respondents stated that primary treatment with somatostatin receptor ligands (SRLs) was indicated at least sometime during the management of acromegaly patients. In nearly all centers (96%), the use of pegvisomant monotherapy was restricted to patients who had failed to achieve biochemical control with SRL therapy. The observation that most centers followed consensus statement recommendations encourages the future utility of these workshops aimed to create uniform management standards for acromegaly.

Intranasal Corticosteroid Administration Reduces Nonspecific Bronchial Hyperresponsiveness and Improves Asthma Symptoms

Introduction. Rhinitis and asthma are currently recognized as manifestations of a single syndrome, the chronic allergic respiratory syndrome. Nearly all individuals with asthma have rhinitis, and severe rhinitis has been associated with worse outcomes in asthma patients. Intranasal treatment has been reported to be beneficial for the lower airways. Methods. This was a randomized, double-blind, placebo-controlled study. The objective was to evaluate the effects that treatment with intranasal beclomethasone dipropionate (BDP; 400 g/d) has on nasal and bronchial symptoms, as well as on lung function test results and bronchial responsiveness to histamine in patients with allergic rhinitis and asthma. We evaluated 33 patients, divided into two groups: treatment (n = 17); and placebo (n = 16). Over the course of the 125-day study period, each patient reported daily rhinitis and asthma symptoms, as well as the need for additional medication. All patients were submitted to spirometry and histamine challenge at baseline and at each subsequent evaluation (on days 50 and 75). Results. In comparison with the patients in the placebo group, those in the BDP treatment group presented significantly fewer nasal symptoms on day 50 and fewer asthma symptoms on day 75 (p 0.01 for both); required rescue medications less often; and presented a significantly lower degree of bronchial responsiveness to histamine on day 75 (p 0.01). Conclusion. In this study, intranasal BDP was effective in treating rhinitis as well as asthma. The benefits for the lower airways were observed only after prolonged treatment and might be better evaluated through nonspecific bronchial challenge.

ICAM-1 (Lys469Glu) and PECAM-1 (Leu125Val) polymorphisms in diffuse astrocytomas

Cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) play an important role in glioma invasion and angiogenesis. The aim of this study was to investigate whether specific genetic polymorphisms of ICAM-1 and PECAM-1 could be associated with glioma development and progression. Single-nucleotide polymorphism in codon 469 of ICAM-1 and codon 125 of PECAM-1 were examined in 158 patients with astrocytomas and 162 controls using polymerase chain reaction and restriction enzyme analysis. The distribution of PECAM-1 polymorphic genotypes in astrocytomas did not show any significant difference. However, a specific ICAM-1 genotype (G/G, corresponding to Lys469Glu) exhibited higher frequency in grade II astrocytomas compared to controls, grade III, and grade IV astrocytomas; suggesting that this polymorphism could be involved in the development of grade II astrocytomas.

HLA-G polymorphisms in women with squamous intraepithelial lesions harboring human papillomavirus

Human papillomavirus (HPV) infection is etiologically associated with low-(LSIL) and high-grade squamous intraepithelial lesions (HSIL) and with cervical cancer. The progression or regression of the lesions may depend, among other factors, on the host heritable immune response. Because human leukocyte antigen (HLA)-G molecules are involved in the modulation of innate and adaptive immune responses, and because no previous studies have evaluated HLA-G polymorphism in patients with SIL, we conducted a study to assess the association between HLA-G polymorphisms and cervical lesions harboring HPV infection. Cervico-vaginal scrapings and blood samples were collected from 125 women with SIL (68 LSIL and 57 HSIL) and from 94 healthy women without HPV infection and cytological abnormalities. HPV type and HLA-G polymorphisms in exons 2, 3 and 8 (14 bp insertion/deletion) were evaluated by PCR methodology, and digested with restriction endonucleases. The Genepop software and the EM and PHASE algorithms were used for statistical analysis. A significant protective association was observed between the presence of the G*0103 allele and SIL and between the G0101/G0104 genotype and HSIL in the group of patients compared to control. The presence of the G0104/+14 bp and G0104/-14 bp haplotypes conferred susceptibility to SIL compared to control. In addition, patients possessing the G0104/+14 bp haplotype and harboring HPV-16 and -18 co-infections were particularly associated with HSIL. These findings suggest that HLA-G polymorphisms may be associated with HPV infection and SIL, consequently representing a profile of predisposition to cervical cancer. Modern Pathology (2009) 22, 1075-1082; doi: 10.1038/modpathol.2009.67; published online 1 May 2009

Efficiency of the DNA repair and polymorphisms of the XRCC1, XRCC3 and XRCC4 DNA repair genes in systemic lupus erythematosus

Impaired DNA repair efficiency in systematic lupus erythematosus (SLE) patients has been reported ill some studies, mainly regarding the repair of oxidative damage, but little is known about repair kinetics towards primarily single-stranded DNA breaks. In the present study, we aimed to investigate: (a) the efficiency of SLE peripheral blood leucocytes in repairing DNA damage induced by ionizing radiation and (b) the association of DNA repair gene (XRCC1 Arg399Gln, XRCC3 Thr241Met and XRCC4 Ile401Thr) polymorphisms in SLE patients, considering the whole group, or stratified sub-groups according to clinical and laboratory features. A total of 163 SLE patients and 125 healthy control were studied. The kinetics of DNA strand break repair was evaluated by the comet assay, and genotyping for DNA repair genes was performed by PCR-RFLP. Compared with controls. SLE leucocytes exhibited decreased efficiency of DNA repair evaluated at 30 min following irradiation. A significant association with DNA repair gene polymorphisms was not observed for the whole group of SLE patients; however, the XRCC1Arg399Gln polymorphism was associated with the presence of anti-dsDNA antibody. The concomitance of two DNA repair polymorphic sites was associated with the presence of neuropsychiatric manifestations and antiphospholipid antibody syndrome. Taken together, these results indicated that SLE leucocytes repair less efficiently the radiation-induced DNA damage, and DNA repair polymorphic sites may predispose to the development of particular clinical and laboratory features. Lupus (2008) 17, 988-995.

The Association of Hemoglobin A1c With Incident Heart Failure Among People Without Diabetes: The Atherosclerosis Risk in Communities Study

OBJECTIVE-This study sought to investigate an association of HbA1c (A1C) with incident heart failure among individuals without diabetes and compare it to fasting glucose. RESEARCH DESIGN AND METHODS-We studied 11,057 participants of the Atherosclerosis Risk in Communities (ARIC) Study without heart failure or diabetes at baseline and estimated hazard ratios of incident heart failure by categories of A1C (<5.0, 5.0-5.4 [reference], 5 5-59, and 6.0-6.4%) and fasting glucose (<90, 90-99 [reference], 100-109, and 110-125 mg/dl) using Cox proportional hazards models. RESULTS-A total of 841 cases of incident heart failure hospitalization or deaths (International Classification of Disease, 9th/10th Revision, 428/150) occurred during a median follow-up of 14.1 years (incidence rate 5.7 per 1,000 person-years). After the adjustment for covariates including fasting glucose, the hazard ratio of incident heart failure was higher in individuals with A1C 6.0-6.4% (1.40 [95% CI, 1 09-1.79]) and 5.5-6.0% (1.16 [0.98-1 37]) as compared with the reference group. Similar results were observed when adjusting for insulin level or limiting to heart failure cases without preceding coronary events or developed diabetes during follow-up. In contrast, elevated fasting glucose was not associated with heart failure after adjustment for covariates and A1C. Similar findings were observed when the top quartile (A1C, 5.7-6.4%, and fasting glucose, 108-125 mg/dl) was compared with the lowest quartile (<5 2% and <95 mg/dl, respectively). CONCLUSIONS-Elevated A1C (>= 5.5-6 0%) was associated with incident heart failure in a middle-aged population without diabetes, suggesting that chronic hyperglycemia prior to the development of diabetes contributes to development of heart failure. Diabetes 59:2020-2026, 2010

Measurement of IgE antibodies to shrimp tropomyosin is superior to skin prick testing with commercial extract and measurement of IgE to shrimp for predicting clinically relevant allergic reactions after shrimp ingestion

Background: Shrimp is a frequent cause of food allergy. Tropomyosin is the major allergen in shrimp, and it shares homology to tropomyosins from other crustaceans, dust mites, cockroach, and parasites. Objective: The aim of this study was to determine the value of detection of IgE to shrimp tropomyosin in the diagnosis of shrimp allergy. Methods: We have studied 35 patients with asthma, rhinitis, or both who were sensitized to Dermatophagoides pteronyssinus. All subjects underwent skin prick testing in addition to double-blind, placebo-controlled food challenges (DBPCFC); oral open challenges; or both with shrimp. Measurements of IgE to shrimp and shrimp tropomyosin were carried out by means of CAP and chimeric ELISA, respectively. Results: Oral challenges confirmed the diagnosis of shrimp allergy in 7 patients. IgE measurement to shrimp tropomyosin was positive in 71.4% of the patients with shrimp allergy. Of the 28 patients without shrimp allergy, only 7.1% (2/28) had IgE to shrimp tropomyosin compared with 25% (7/28) who had IgE to shrimp and 35.7% (10/28) who had positive skin prick test responses to shrimp. Sensitivity was similar for all 3 methods (71.4%); in contrast, specificity of IgE to shrimp tropomyosin (92.8%) was greater than that of IgE to shrimp (75%) and skin prick testing (64.2%). With regard to diagnostic efficiency, measurement of IgE to shrimp tropomyosin was superior to measurement of IgE to shrimp and skin prick testing (88.5%, 74.2%, and 65.7%, respectively). Conclusion: Use of measurements of IgE to shrimp tropomyosin provided added value to the diagnosis of shrimp allergy. (J Allergy Clin Immunol 2010;125:872-8.)

Increased endothelin-1 reactivity and endothelial dysfunction in carotid arteries from rats with hyperhomocysteinemia

There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries. Vascular reactivity to ET-1 and ET(A) and ET(B) receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ET(A) and ET(B) receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [(125)I]-ET-1. HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ET(A) or ET(B) receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ET(A) but not of ET(B) receptors abolished enhancement in HHcy tissues. ET(A) and ET(B) receptor gene expressions were not up-regulated. ET(A) receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A(2) receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO. Increased ET(A) receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ET(A) receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility. British Journal of Pharmacology (2009) 157, 568-580; doi:10.1111/j.1476-5381.2009.00165.x; published online 9 April 2009 This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009.

TNF-alpha Knockout Mice Have Increased Corpora Cavernosa Relaxation

Erectile dysfunction is considered an early clinical manifestation of vascular disease and an independent risk factor for cardiovascular events associated with endothelial dysfunction and increased levels of pro-inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine, suppresses endothelial nitric oxide synthase (eNOS) expression. Considering that nitric oxide (NO) is of critical importance in penile erection, we hypothesized that blockade of TNF-alpha actions would increase cavernosal smooth muscle relaxation. In vitro organ bath studies were used to measure cavernosal reactivity in wild type and TNF-alpha knockout (TNF-alpha KO) mice and NOS expression was evaluated by western blot. In addition, spontaneous erections (in vivo) were evaluated by videomonitoring the animals (30 minutes). Collagen and elastin expression were evaluated by Masson trichrome and Verhoff-van Gieson stain reaction, respectively. Corpora cavernosa from TNF-alpha KO mice exhibited increased NO-dependent relaxation, which was associated with increased eNOS and neuronal NOS (nNOS) cavernosal expression. Cavernosal strips from TNF-alpha KO mice displayed increased endothelium-dependent (97.4 +/- 5.3 vs. Control: 76.3 +/- 6.3, %) and nonadrenergic-noncholinergic (93.3 +/- 3.0 vs. Control: 67.5 +/- 16.0; 16 Hz) relaxation compared to control animals. These responses were associated with increased protein expression of eNOS and nNOS (P < 0.05). Sympathetic-mediated (0.69 +/- 0.16 vs. Control: 1.22 +/- 0.22; 16 Hz) as well as phenylephrine-induced contractile responses (1.6 +/- 0.1 vs. Control: 2.5 +/- 0.1, mN) were attenuated in cavernosal strips from TNF-alpha KO mice. Additionally, corpora cavernosa from TNF-alpha KO mice displayed increased collagen and elastin expression. In vivo experiments demonstrated that TNF-alpha KO mice display increased number of spontaneous erections. Corpora cavernosa from TNF-alpha KO mice display alterations that favor penile tumescence, indicating that TNF-alpha plays a detrimental role in erectile function. A key role for TNF-alpha in mediating endothelial dysfunction in ED is markedly relevant since we now have access to anti-TNF-alpha therapies. Carneiro FS, Sturgis LC, Giachini FRC, Carneiro ZN, Lima VV, Wynne BM, Martin SS, Brands MW, Tostes RC, and Webb RC. TNF-alpha knockout mice have increased corpora cavernosa relaxation. J Sex Med 2009;6:115-125.

Increases in circulating matrix metalloproteinase-9 levels following fibrinolysis for acute pulmonary embolism

Introduction: Fibrinolyis is one of the first line therapies in high risk pulmonary embolism (PE) according to current guidelines. Previous studies showed that brinolytic therapy with tPA (tissue plasminogen activator, or alteplase) upregulates the concentrations of matrix metalloproteinases (MMPs) and contributes to hemorrhagic transformation after cardioembolic stroke. However, no previous study has described the circulating MMPs levels following fibrinolysis for acute PE. Materials and Methods: We serially measured the circulating levels of MMPs (MMP-9 and MMP-2) and their endogenous inhibitors, the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in alteplase and in streptokinase-treated patients with acute PE by gelatin zymography and by enzyme-linked immunosorbent assays, respectively. Results: We found that therapy of PE streptokinase or with alteplase is associated increased pro-MMP-9, but not MMP-2, concentrations for up to 24 hours, whereas no significant changes were found in TIMP-1 or TIMP-2 concentrations. This alteration returned to normal 3 to 5 days after thrombolysis. This is the first study reporting on MMPs alterations following fibrinolysis for acute PE. Conclusions: We found transient increases in circulating pro-MMP-9 levels following fibrinolysis for acute PE. Our findings support the hypothesis that increased MMP-9 levels may underlie the risk of intracerebral hemorrhage or other bleeding complication of thrombolysis for acute PE, and the use of MMP inhibitors may decrease such risk. (C) 2010 Elsevier Ltd. All rights reserved.

Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: Involvement of cytokines and nitric oxide

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125-1000 mu g/cavity), and at 2-96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 mu g/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-alpha, IL-1 beta and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 mu g/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1 beta, TNF-alpha and CINC-1. (C) 2009 Elsevier Ltd. All rights reserved.

Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model

Aim of the study: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. it comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). Material and methods: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII), and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. Results: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. Conclusions: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Antimicrobial Activities of Ethanol Extract and Coumestans from Eclipta alba (L.) Hassk (Asteraceae)

Ethanol extract and fractions from aerial parts of Eclipta alba (L.) Hassk (Asteraceae) were screened for the antibacterial and antifungal activities against different species of human pathogenic bacterial ATCC, antibiotic-resistant clinical isolates and strains of the dermatophyte Trichophyton rubrum (wild and mutant for TruMDR2 gene) using a microdilution method. Demethylwedelolactone/wedelolactone (DWL/WL) and only wedelolactone (WL), both in a high homogeneity degree, were efficient to inhibit the ATCC strains of Staphylococus aureus (Minimal Inhibitory Concentration MIC = 75 mu g/mL), Staphylococcus epidemidis (MIC = 125 mu g/mL) and Escherichia coli (MIC = 125 mu g/mL) as well as antibiotic-resistant clinical isolates of Enterococcus spp (MIC = 250 mu g/mL) and S. aureus (MIC = 125 mu g/mL). Ethanol extract was more effective than the purified fractions against Trichophyton rubrum strains (MIC = 125 mu g/mL), suggesting that anti-fungal activity is not only related to demethylwedelolactone and wedelolactone, but also to a synergistic action between these coumestans and other compounds found in that extract. Thus, this work suggests that E. alba possesses a significant antimicrobial activity, including that against multi-drug resistant microorganisms, which could be of relevance for the treatment of infectious diseases.