Antiganglioside Antibodies and Inflammatory Response in Cutaneous Melanoma

Introduction. Endogenously produced antiganglioside antibodies could affect the evolution of cutaneous melanoma. Epidemiological and experimental evidence suggest “chronic inflammation” to be one of the hallmarks in skin cancers. The aim of the study was to characterize the relation between antiganglioside antibodies and inflammation in cutaneous melanoma focusing on gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, GQ1b. Material and Method. We performed an observational study that included 380 subjects subdivided into three groups: patients with metastatic melanoma (170 cases), patients with primary melanoma (160 cases), and healthy subjects (50 subjects). The assessment of antiganglioside antibodies, IgG, and IgM classes, against -GM1, -GM2, -GM3, -GD1a, -GD1b, -GT1b, -GQ1b was performed using immunoblot technique (EUROLine kit). Results. The presence of IgG and IgM antiganglioside antibodies in primary melanoma was (%), as follows: anti-GM1 (5.0 and 13.1), -GM2 (1.8 and 18.1), -GM3 (0.6 and 5.6), -GD1a (0.6 and 15.0), -GD1b (3.7 and 10.7), -GT1b (0.0 and 13.1), -GQ1b (0.0 and 5.0). In metastatic melanoma, the level of antiganglioside antibodies was significantly lower compared with primary melanoma (p<0.05), while in the control group they were absent. Antiganglioside antibodies anti-GM1 and -GD1a were positively correlated, while anti-GM3, -GD1b, and -GT1b were negatively associated with the inflammatory markers, interleukin 8 (IL-8), and C reactive protein (CRP). Conclusions. Tumour ganglioside antigens generate an immune response in patients with primary melanomas. The host’s ability to elaborate an early antiganglioside response could be considered as a defence mechanism, directed toward eliminating a danger signal from the tumour microenvironment. Antiganglioside antibodies associated with inflammation markers could be used as diagnostic, monitoring, and treatment tools in patients with cutaneous melanoma.

Lyme Disease: Diversity of Borrelia Species in California and Mexico Detected Using a Novel Immunoblot Assay

Background: With more than 300,000 new cases reported each year in the United States of America (USA), Lyme disease is a major public health concern. Borrelia burgdorferi sensu stricto (Bbss) is considered the primary agent of Lyme disease in North America. However, multiple genetically diverse Borrelia species encompassing the Borrelia burgdorferi sensu lato (Bbsl) complex and the Relapsing Fever Borrelia (RFB) group are capable of causing tickborne disease. We report preliminary results of a serological survey of previously undetected species of Bbsl and RFB in California and Mexico using a novel immunoblot technique. Methods: Serum samples were tested for seroreactivity to specific species of Bbsl and RFB using an immunoblot method based on recombinant Borrelia membrane proteins, as previously described. A sample was recorded as seropositive if it showed immunoglobulin M (IgM) and/or IgG reactivity with at least two proteins from a specific Borrelia species. Results: The patient cohort consisted of 90 patients residing in California or Mexico who met the clinical case definition of chronic Lyme disease. Immunoblot testing revealed that 42 patients were seropositive for Bbsl (Group 1), while 56 patients were seropositive for RFB (Group 2). Eight patients were seropositive for both Bbsl and RFB species. Group 1 included patients who were seropositive for Bbss (14), B. californiensis (eight), B. spielmanii (10), B. afzelii/B. garinii (10), and mixed infections that included B. mayonii (three). Group 2 included patients who were seropositive for B. hermsii (nine), B. miyamotoi (seven), B. turicatae (nine), and B. turcica (two). In the remaining Group 1 and Group 2 patients, the exact Borrelia species could not be identified using the immunoblot technique. Conclusions: Lyme disease is associated with a diverse group of Borrelia species in California and Mexico. Current testing for Lyme disease focuses on detection of Bbss, possibly resulting in missed diagnoses and failure to administer appropriate antibiotic therapy in a timely manner. The genetic diversity of Borrelia spirochetes must be considered in future Lyme disease test development.

Perturbation of PTEN-PI3K/AKT Signalling Impaired Autophagy Modulation in Dystrophin-Deficient Myoblasts

Alteration of single protein regulation has given a massive implication in Muscular Dystrophy pathogenesis. Herein, we investigated the contribution of defected dystrophin that has impaired PI3K/Akt signalling and subsequently reduced autophagy in dystrophin-deficient myoblasts. In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen condition for 10 days to induce differentiation. Analyses of protein expression has been done by using immunoblot technique, immunofluorescence and flow cytometry. In our myoblasts differentiation system, the dfd13 myoblasts did not achieved terminal differentiation as fewer myotube formation and fast-myosin heavy chain expression almost not detected. Immunoblot analysis showed that PTEN expression is profoundly increased in dfd13 myoblasts throughout the differentiation day. As a result, the PI3K activity is decreased and has caused serine/threonine kinase Akt inactivation. Both residues; Thr308 and Ser473, on Akt were found not phosphorylated. The mTOR activation by Ser2448 phosphorylation was decreased indicates an impairment for raptor and rictor binding. Unable to form complexes; mTORC1 target protein, p70S6K1 activation was found reduced at the same time explained un-phosphorylated-Akt at Ser473 by rictor-mTORC2. As one of Akt downstream protein, transcription factor FoxO3 regulation was found impaired as it was highly expressed and highly mainly localised in the nucleus in dfd13 towards the end of the differentiation day. This occurrence has caused higher activation of autophagy related genes; Beclin1, Atg5, Atg7, in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features. As a conclusion, destabilisation of plasma membrane owing to dystrophin mutation has caused the alteration of plasma membrane protein regulation particularly PTEN-PI3K, thus impaired autophagy modulation that critical for myoblasts development.

Anti-MDA5 Antibodies in a Large Mediterranean Population of Adults with Dermatomyositis

A new myositis-specific autoantibody directed against melanoma differentiation-associated gene 5 (anti-MDA5) has been described in patients with dermatomyositis (DM). We report the clinical characteristics of patients with anti-MDA5 in a large Mediterranean cohort of DM patients from a single center, and analyze the feasibility of detecting this autoantibody in patient sera using new assays with commercially available recombinant MDA5. The study included 117 white adult patients with DM, 15 (13%) of them classified as clinically amyopathic dermatomyositis (CADM). Clinical manifestations were analyzed, with special focus on interstitial lung disease and its severity. Determination of anti-MDA5 antibodies was performed by a new ELISA and immunoblot technique. In sera, from 14 (12%) DM patients (8 CADM), MDA5 was recognized by ELISA, and confirmed by immunoblot. Eight of the 14 anti-MDA5-positive patients (57.14%) presented rapidly-progressive interstitial lung disease (RP-ILD) versus 3 of 103 anti-MDA5-negative patients (2.91%) (P<0.05; OR: 44.4, 95% CI 9.3–212). The cumulative survival rate was significantly lower in anti-MDA5-positive patients than in the remainder of the series (P<0.05). Patients with anti-MDA5-associated ILD presented significantly lower 70-month cumulative survival than antisynthetase-associated ILD patients. Among the cutaneous manifestations, only panniculitis was significantly associated with the presence of anti-MDA5 antibodies (P<0.05; OR: 3.85, 95% CI 1.11–13.27). These findings support the reliability of using commercially available recombinant MDA5 for detecting anti-MDA5 antibodies and confirm the association of these antibodies with RP-ILD in a large series of Mediterranean patients with DM.

Idiopathic Granulomatous Mastitis: An Autoimmune Disease?

Purpose. This study aimed to investigate the autoimmune basis of idiopathic granulomatous mastitis (IGM) by determining the anti-nuclear antibody (ANA) and extractable nuclear antigen (ENA) levels of patients diagnosed with IGM.Material and Methods. Twenty-six IGM patients were evaluated. Serum samples were analyzed for autoantibodies by indirect immunofluorescence (IIF) using a substrate kit that induced fluorescein-conjugated goat antibodies to human immunoglobulin G (IgG). IIF patterns were read at serum dilutions of 1 : 40 and 1 : 100 for ANA positivity. Using the immunoblot technique, the sera of patients were assayed at dilutions of 1 : 40 and 1 : 100 for human autoantibodies of the IgG class to 15 lines of highly purified ENAs.Results. In the IIF studies for ANA, positivity was identified for four different patterns in the 1 : 40 diluted preparations, for three different patients in the 1 : 100 diluted preparations and only one pattern was identified at the 1 : 320 dilution. In the ENA studies, positivity was identified for four different pattern in the 1 : 40 dilution, and only one pattern was identified at the 1 : 100 dilution.Conclusion. This study was not able to support the eventual existence of an autoimmune basis for IGM.

Serum Helicobacter pylori Specific Antibodies Among Children With Undernourishment: A Case Control Study

Infection with Helicobacter pylori has been implicated in the development of acute and chronic gastritis, peptic ulcer disease, non-ulcer dyspepsia, and gastric MALT (mucosa-associated lymphoid tissue) lymphoma and extragastroduodenal disorders. H. pylori infection may lead to development of hypochlorhydria and subsequent malnourishment. This study examined the affiliation between H. pylori infection and some sociodemographic and nutritional variables including breast feeding practice, poverty, and availability of safe drinking water. Three hundred children with age ranging from zero to twelve years were enrolled in the study. Their nutritional status was assessed following standard protocol. A precoded questionnaire was filled up for obtaining data. Serostatus of anti H. pylori antibody was determined by in-house ELISA using formalin fixed H. pylori whole cell antigen. The cut-off value of the ELISA was further validated by Immunoblot technique. Among the 149 case (malnourished) children, 107 (72%) contained anti-H. pylori antibodies and among the control (nonmalnourished) population (n=151), 91 (60%) possessed anti-H. pylori antibodies, with an overall seroprevalence of 66%. Malnourishment was found to be more widespread among female (56%) than male children (44%). Highly significant association (p = 0.000) was reported between poor socioeconomic status and development of malnourishment. The degree of severity of malnourishment increased inversely with the practice of exclusive breast-feeding up to 5 months of age (p = 0.010). The odds ratio relating breastfeeding to malnourishment reflected 52% reduction in the risk of development of malnourishment among breast-fed children. The use of unsafe drinking water was associated with 5.33 times higher odds of developing malnourishment. Poverty reduction strategies associated with improvement of hygiene condition and promotion of breast feeding can actively contribute to the improvement of the nutritional status of the children of Bangladesh. DOI: http://dx.doi.org/10.3329/bjm.v28i1.11805 Bangladesh J Microbiol, Volume 28, Number 1, June 2011, pp 25-31

Studying the central control of food intake and obesity in rats

The central nervous system regulates energy intake and expenditure through a complex network of neurotransmitters and neuromodulators. It is of great interest to understand the relevance of these systems to the physiological control of energy balance and to the disturbances of obesity. The present paper discusses some of the methods to address this field used at the laboratory of Endocrine Physiology of Universidade Federal de São Paulo. Initially, different experimental models of rat obesity are presented, namely the hypothalamic induced monosodium glutamate model, the Zucker genetic model, and the dietary model. The principles of brain microdialysis are also presented, the technique applied to obtain representative samples of the extracellular fluid of brain sites involved in feeding control. The microdialysate levels of serotonin, an important anorexigenic neurotransmitter, are determined by HPLC with electrochemical detection. The immunoblot technique (Western blot) is used to determine hypothalamic levels of proteins relevant to the anorexigenic effect of serotonin and to analyze the acute activation of the insulin signaling cascade in the hypothalamus. The final section addresses the potential applications of proteomics in the study of the central control of feeding.

Apoprotein(A) Isoforms and Plasma LP(A) Concentration in Members of Four Families

Apoprotein(A) Isoforms and Plasma LP(A) Concentration in Members of Four FamiliesApoprotein(a) is a multikringle protein which shows a genetically inherited size polymorphism. The APO(a) gene is located at the telomeric region of chromosome 6q2.6-q 2.7. Apo(a) size polymorphism is a major determinant of Lp(a) levels. The aim of this study is to describe the influence of apo(a) size polymorphism on the plasma Lp(a) levels in the members of four families. K3EDTA plasma was obtained from every subject after over-night fast. Apo(a) isoforms were determined by 3-15% SDS-PAGE followed by Western immunoblot technique. Plasma Lp(a) level was de - termined with immunonephelometric method. Every child inherited one isoform from its mother and the other from its father. The children from the first family had Lp(a) levels similar to those measured in their parents. The daughters from the second and fourth family inherited the dominant S3 apo(a) isoform from their mothers and also mother's high Lp(a) levels (0.365 g/L - daughter from the second, and 0.465 g/L and 0.446 g/L - daughter from the fourth family respectively). The elder daughter from the third family, carrier of double banded S4S1 apo(a) isoform, had the highest Lp(a) level among the children from all four families. We found out a generation decrease of the Lp(a) level in two families. On the basis of our findings we concluded that the inheritance of the apo(a) isoforms in the members of all four families is in accordance with the simple Mendelian's model and that the apo(a) size polymorphism influences the Lp(a) level in the blood of the examined subjects.

Protein extraction from activated sludge

Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer.