Effect of brain-derived neurotrophic factor Val66Met polymorphism and serum levels on the progression of mild cognitive impairment

Objectives. Abnormalities in neurotrophic systems have been reported in Alzheimer`s disease (AD), as shown by decreased serum brain-derived neurotrophic factor (BDNF) levels and association with BDNF genetic polymorphisms. In this study, we investigate whether these findings can be detected in patients with mild cognitive impairment (MCI), which is recognized as a high risk condition for AD. We also address the impact of these variables on the progression of cognitive deficits within the MCI-AD continuum. Methods. One hundred and sixty older adults with varying degrees of cognitive impairment (30 patients with AD, 71 with MCI, and 59 healthy controls) were longitudinally assessed for up to 60 months. Baseline serum BDNF levels were determined by sandwich ELISA, and the presence of polymorphisms of BDNF and apolipoprotein E (Val66Met and APOE*E4, respectively) was determined by allelic discrimination analysis on real time PCR. Modifications of cognitive state were ascertained for non-demented subjects. Results. Mean serum BDNF levels were reduced in patients with MCI and AD, as compared to controls (509.2 +/- 210.5; 581.9 +/- 379.4; and 777.5 +/- 467.8 pg/l respectively; P < 0.001). Baseline serum BDNF levels were not associated with the progression of cognitive impairment upon follow-up in patients with MCI (progressive MCI, 750.8 +/- 463.0; stable MCI, 724.0 +/- 343.4; P = 0.8), nor with the conversion to AD. Although Val66Met polymorphisms were not associated with the cross-sectional diagnoses of MCI or AD, the presence of Met-BDNF allele was associated with a higher risk of disease-progression in patients with MCI (OR = 3.0 CI(95%) [1.2-7.8], P = 0.02). We also found a significant interaction between the APOE*E4 and Met-BDNF allele increasing the risk of progression of cognitive impairment in MCI patients (OR = 4.4 CI(95%) [1.6-12.1], P = 0.004). Conclusion. Decreased neurotrophic support, as indicated by a reduced systemic availability of BDNF, may play role in the neurodegenerative processes that underlie the continuum from MCI to AD. The presence of Met-BDNF allele, particularly in association with APOE*E4, may predict a worse cognitive outcome in patients with MCI.

Cytokine Profile in Pleural Fluid and Serum After Lung Transplantation

Background. Lung transplantation is the procedure of choice in several end-stage lung diseases. Despite improvements in surgical techniques and immunosuppression, early postoperative complications occur frequently. Objective. To evaluate the pleural inflammatory response after surgery. Patients and Methods. Twenty patients aged 18 to 63 years underwent unilateral or bilateral lung transplantation between August 2006 and March 2008. Proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and IL-8 and vascular endothelial growth factor in pleural fluid and serum were analyzed. For cytokine evaluation, 20-mL samples of pleural fluid and blood (right, left, or both chest cavities) were obtained at 6 hours after surgery and daily until removal of the chest tube or for a maximum of 10 days. Data were analyzed using analysis of variance followed by the Holm-Sidak test. Results. All effusions were exudates according to Light`s criteria. Pleural fluid cytokine concentrations were highest at 6 hours after surgery. Serum concentrations were lower than those in pleural fluid, and IL-1 beta, IL-6, and IL-8 were undetectable at all time points. Conclusions. There is a peak concentration of inflammatory cytokines in the first 6 hours after transplantation, probably reflecting the effects of surgical manipulation. The decrease observed from postoperative day 1 and thereafter suggests the action of the immunosuppression agents and a temporal reduction in pleural inflammation.

Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

Evaluation of the prognostic significance of serum galectin-3 in American Joint Committee on Cancer stage III and stage IV melanoma patients

Galectin-3 (Gal-3) is a member of the P-galactoside-binding lectins family and has been implicated in angiogenesis, tumor invasion, and metastatic process in vitro and in vivo. As we showed recently that advanced melanoma patients presented high serum level of Gal-3, we investigated the association of this protein with the outcome of melanoma patients. Whether this protein could be a biomarker has riot been assessed, and we compared the prognostic value of serum Gal-3 in multivariate analysis with lactate dehydrogenase, C-reactive protein and S100B. We conclude that Gal-3 could be of prognostic value in melanoma patients; more precisely, this protein has a strong independent prognostic signification with a cut-off value of 10 ng/ml. After these data, we believe that serum Gal-3 measurement can have an important role in the follow-up and management of advanced American Joint Commission on Cancer stage III and stage IV melanoma patients. Further studies will uncover whether Gal-3 will be able to open new therapeutic perspectives. Melanoma Res 19:316-320 (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Early onset of primary hypogonadism revealed by serum anti-Mullerian hormone determination during infancy and childhood in trisomy 21

Male patients with an extra sex chromosome or autosome are expected to present primary hypogonadism at puberty owing to meiotic germ-cell failure. Scarce information is available on trisomy 21, a frequent autosomal aneuploidy. Our objective was to assess whether trisomy 21 presents with pubertal-onset, germ-cell specific, primary hypogonadism in males, or whether the hypogonadism is established earlier and affects other testicular cell populations. We assessed the functional status of the pituitary-testicular axis, especially Sertoli cell function, in 117 boys with trisomy 21 (ages: 2 months-20 year). To compare with an adequate control population, we established reference levels for serum anti-Mullerian hormone (AMH) in 421 normal males, from birth to adulthood, using a recently developed ultrasensitive assay. In trisomy 21, AMH was lower than normal, indicating Sertoli cell dysfunction, from early infancy, independently of the existence of cryptorchidism. The overall prevalence rate of AMH below the 3rd percentile was 64.3% in infants with trisomy 21. Follicle-stimulating hormone was elevated in patients <6 months and after pubertal onset. Testosterone was within the normal range, but luteinizing hormone was elevated in most patients <6 months and after pubertal onset, indicating a mild Leydig cell dysfunction. We conclude that in trisomy 21, primary hypogonadism involves a combined dysfunction of Sertoli and Leydig cells, which can be observed independently of cryptorchidism soon after birth, thus prompting the search for new hypotheses to explain the pathophysiology of gonadal dysfunction in autosomal trisomy.

Toll-like receptor activity in Recurrent Aphthous Ulceration

Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, virus, fungal or host tissues. Activation of TLRs causes the production of cytokines that mediate inflammatory responses and drive T helper (Th) 1 and 2 cell development. As an exaggerated Th1 immune response is supposed to be involved in pathogenesis of Recurrent Aphthous Ulceration (RAU), we suggest that RAU patients may have an imbalance in TLR pathways. To study the function of TLR activation ex vivo, peripheral blood mononuclear cells (PBMCs) from RAU patients (n = 17) and controls (n = 17) were exposed to TLR2 [lipoteichoic acid (LTA), heat-killed Listeria monocytogenes (HKLM) and PamC3CSK4], TLR3 [Poly(I:C)], TLR4 [lipopolysaccharide (LPS)], TLR5 (flagellin) and TLR7 (imiquimod) ligands, and the time course of supernatant tumor necrosis factor-alpha (TNF-alpha) levels was quantified by enzyme-linked immunosorbent assay. In addition, serological and salivary TNF-alpha and soluble CD14 levels were quantified. The TNF-alpha produced by PBMCs in contact with each TLR ligand and autologous serum or saliva at the same time was also investigated. The data were analyzed by statistical multivariate tests. The control group had a higher response to LTA, whereas RAU had a higher response to HKLM. LTA and LPS interfered with the salivary stimulation of the RAU PBMC and HKLM with the stimulation of the control. Autologous serum was capable of inhibiting TLR2 responsiveness to LTA and enhancing LPS stimulation. Salivary and serological levels of sCD14 and TNF-alpha were not significantly different. Recurrent Aphthous Ulceration patients have an anomalous activity of the TLR2 pathway that probably influences the stimulation of an abnormal Th1 immune response.

Increased Serum IL-1 beta Level in Alzheimer`s Disease and Mild Cognitive Impairment

Background/Aims: Abnormal inflammatory response has been associated to the pathogenesis of Alzheimer`s disease (AD) and may be a marker of an ongoing neurodegenerative process. The aim of this study was to evaluate the serum levels of interleukin-1 beta (IL-1 beta) in patients with mild cognitive impairment (MCI) and AD. Methods: One hundred and sixty-three older adults ( 58 with mild to moderate AD, 74 with MCI and 31 healthy controls) were recruited for this study. Serum IL-1 beta levels were measured by ELISA. Patients with MCI were subcategorized in single-domain amnestic (aMCI), nonamnestic (naMCI), and multiple-domain (mdMCI) subtypes. Results: Patients with AD and MCI ( all subtypes) had a significant increase in serum IL-1 beta levels as compared to controls (p = 0.03). Patients with mdMCI had serum IL-1 beta levels comparable to those with AD, and significantly higher than those observed in aMCI and naMCI ( p = 0.02). Discussion: The present study provides evidence that inflammatory mechanisms, represented by elevated IL-1 beta, are observed in patients with MCI, specifically in those with impairment in multiple cognitive domains. As these patients are at higher risk of conversion to dementia, we propose that an increased serum IL-1 beta level is a stage marker of the ongoing brain neurodegeneration in the continuum between normal ageing and AD. Copyright (C) 2009 S. Karger AG, Basel

Characterization of acid-base status in maintenance hemodialysis: physicochemical approach

Acidosis is a common and deleterious aspect of maintenance dialysis. Traditionally, it is considered to be an elevated anion gap acidosis caused by the inability to excrete nonvolatile anions. Stewart`s approach made it possible to identify real determinants of the acid-base status and allowed quantification of the components of these disturbances, especially the unmeasured anions. We performed a cross-sectional study to identify and quantify each component of acidosis in hemodialysis maintenance patients. Sixty-four maintenance hemodialysis patients and 14 controls were enrolled in this study. Gasometrical and biochemical analysis were performed before the midweek dialysis session. Quantitative physicochemical analysis was carried out using the Stewart methodology. Hemodialysis patients were found to have mild acidemia (mean pH: 7.33 +/- 0.06 versus 7.41 +/- 0.05) secondary to metabolic acidosis (serum bicarbonate: 18.8 +/- 0.26 versus 25.2 +/- 0.48 mEq/l). The metabolic acidosis was due to retention of unmeasured anions (6.5 +/- 0.29 versus 3.1 +/- 0.62 mEq/l), hyperchloremia (105.1 +/- 0.5 versus 101.8 +/- 0.7 mEq/l), and hyperphosphatemia (5.90 +/- 0.19 versus 3.66 +/- 0.14 mg/dl). Compared with control values, the unmeasured anions and hyperchloremia had a similar acidifying effect (3.4 and 3.3 mEq/l), corresponding to almost 90% of the metabolic acidosis. Unmeasured anions and hyperchloremia are important components of acidosis in maintenance hemodialysis, in addition to phosphorus. Future studies to determine the etiology and consequences of hyperchloremic acidosis are warranted.

Neurosyphilis in HIV-infected patients: Clinical manifestations, serum venereal disease research laboratory titers, and associated factors to symptomatic neurosyphilis

Goal: To describe clinical and laboratory features of human immunodeficiency infection (HIV)-infected patients with neurosyphilis. Study Design: Retrospective study of 27 consecutive cases of HIV-infected patients with a positive Venereal Disease Research Laboratory (VDRL) in cerebrospinal fluid (CSF). Results: Median of age was 36 years and 89% were men. Ten (37%) patients had previous nonneurologic syphilis treatment. At the time of neurosyphilis diagnosis, 10 (37%) patients had early syphilis, and 6 of them were neurologically asymptomatic. Nine (33%) patients had symptomatic neurosyphilis. Twenty-six (96%) patients were classified with early neurosyphilis. The medians of serum VDRL and CD4(+) T cell counts were 1:128 and 182 cell/mu L, respectively. Twenty five (93%) patients presented serum VDRL titers >= 1:16. Five of 6 patients with early syphilis and asymptomatic neurosyphilis, presented serum VDRL >= 1:16. Symptomatic patients showed lower CD4(+) T cell counts (59 cell/mu L vs. 208 cell/mu L, P = 0.03) and higher protein concentration on CSF (118 mg/dL vs. 39 mg/dL, P <0.001) than asymptomatic patients. Conclusions: Most patients had early and asymptomatic neurosyphilis, and more than one third had early syphilis. Patients with symptomatic neurosyphilis showed lower CD4(+) T cell counts and higher protein concentration on CSF than those asymptomatic. Most patients had serum VDRL titers >= 1:16, regardless of syphilis stage.

Evaluation of rhBMP-2 and Natural Latex as Potential Osteogenic Proteins in Critical Size Defects by Histomorphometric Methods

This in vivo study evaluated the osteogenic potential of two proteins, recombinant human bone morphogenetic protein-2 (rhBMP-2) and a protein extracted from natural latex (Hevea brasiliensis, P-1), and compared their effects on bone defects when combined with a carrier or a collagen gelatin. Eighty-four (84) Wistar rats were divided into two groups, with and without the use of collagen gelatin, and each of these were divided into six treatment groups of seven animals each. The treatment groups were: (1) 5 mu g of pure rhBMP-2; (2) 5 mu g of rhBMP-2/monoolein gel; (3) pure monoolein gel; (4) 5 mu g of pure P-1; (5) 5 mu g of P-1/monoolein gel; (6) critical bone defect control. The animals were anesthetized and a 6 mm diameter critical bone defect was made in the left posterior region of the parietal bone. Animals were submitted to intracardiac perfusion after 4 weeks and the calvaria tissue was removed for histomorphometric analysis. In this experimental study, it was concluded that rhBMP-2 allowed greater new bone formation than P-1 protein and this process was more effective when the bone defect was covered with collagen gelatin (P < 0.05). Anat Rec, 293:794-801, 2010. (C) 2010 Wiley-Liss, Inc.

Increased Vascular Permeability, Angiogenesis and Wound Healing Induced by the Serum of Natural Latex of the Rubber Tree Hevea brasiliensis

Increases in vascular permeability and angiogenesis are crucial events to wound repair, tumoral growth and revascularization of tissues submitted to ischemia. An increased vascular permeability allows a variety of cytokines and growth factors to reach the damaged tissue. Nevertheless, the angiogenesis supply tissues with a wide variety of nutrients and is also important to metabolites clearance. It has been suggested that the natural latex from Hevea brasiliensis showed wound healing properties and angiogenic activity. Thus, the purpose of this work was to characterize its angiogenic activity and its effects on vascular permeability and wound healing. The serum fraction of the latex was separated from the rubber with reduction of the pH. The activity of the dialyzed serum fraction on the vascular permeability injected in subcutaneous tissue was assayed according Mile`s method. The angiogenic activity was determined using a chick embryo chorioallantoic membrane assay and its effects on the wound-healing process was determined by the rabbit ear dermal ulcer model. The serum fraction showed evident angiogenic effect and it was effective in enhancing vascular permeability. In dermal ulcers, this material significantly accelerated wound healing. Moreover, the serum fraction boiled and treated with proteases lost these activities. These results are in accordance with the enhancement of wound healing observed in clinical trials carried out with a biomembrane prepared with the same natural latex. Copyright (C) 2009 John Wiley & Sons, Ltd.

Neonatal BCG Immunization Followed by DNAhsp65 Boosters: Highly Immunogenic but not Protective Against Tuberculosis - a Paradoxical Effect of the Vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

Schistosoma mansoni encodes SMT3B and SMT3C molecules responsible for post-translational modification of cellular proteins

The sumoylation pathway is a post-translational modification of nuclear proteins widespread among several organisms. SMT3C is the main protein involved in this process and it is covalently conjugated to a diverse assortment of nuclear protein targets. To date, 3 SUMO paralogues (SMT3C, A/B) have been characterized in mammals and plants. In this work we characterized two SUMO related genes, named SMT3B and SMT3C throughout Schistosoma mansoni life cycle. The SmSMTB/C encodes for proteins sharing significant amino acid homology with SMT3. Phylogenetical analyses revealed that both SmSMT3B/C are distinct proteins. Additionally, SmSMT3B and C are expressed in cercariae, adult worms, eggs and schistosomula however SinSMT3C gene showed an expression level 7 to 9 fold higher than SmSMT3B in eggs, schistosomula and adult worms. The comparison between the SmSMT3C genomic and cDNA sequences established that the encoding sequence is interrupted by 3 introns of 70, 37 and 36 bp. Western Blot has shown SMT3 conjugates are present in nuclear and total protein fractions of adults and cercariae. Therefore our results suggest a functional sumoylation pathway, and the presence of two paralogues also suggests the specificity of substrates for SMT3 in S. mansoni. (c) 2008 Elsevier Ireland Ltd. All rights reserved.