Familial hypercholesterolemia: Is there a role for PCSK9 and thrombin generation?

INTRODUCTION: Familial hypercholesterolemia (FH) is an autosomal dominant genetic disease. The prevalence of FH has previously been reported as 1 in 500 in the general population. This study aimed to evaluate the proprotein convertase subtilisin/kexin 9 (PCSK9) levels, lipid profile and thrombin generation in FH patients undergoing treatment or not. METHODS: Eighty individuals with FH were selected and distributed in 2 groups: individuals treated with statins alone or conjugate therapy (statin + ezetimibe) (T = 53) and those non treated (NT = 27). PCSK9 levels were determined by ELISA, the lipid profile by colorimetric enzyme method and thrombin generation assay (TGA) by CAT method. RESULTS: Individuals treated with conjugate therapy (statin + ezetimibe) showed a significant reduction in the levels of total cholesterol (TC) low density lipoprotein cholesterol (LDLc) and in the potential for thrombin generation (ETP with low and high concentration of tissue factor), compared to the treated individuals with monotherapy (statins). PCSK9 was positively correlated with increased levels of TC, LDLc and triglycerides, while TGA parameters were positively correlated with PCSK9 and lipid profile. CONCLUSION: PCSK9 levels appear to be associated with components of the lipid and hemostatic profiles, in addition to being influenced by age. In general, our findings suggest that combined therapy for the treatment of FH is associated with a significant improvement in both lipid and hemostatic profiles assessed by TGA, suggesting a reduction in atherogenic and thrombogenic risks and, therefore, more promising compared to the use of statin monotherapy.

Evaluation of potential biomarkers for the diagnosis and monitoring of Systemic Lupus Erythematosus using the Cytometric Beads Array (CBA).

BACKGROUND: Systemic Lupus Erythematosus (SLE) is an autoimmune, multisystemic disease. Currently diagnosis depends on complex criteria developed by the American College of Rheumatology. Moreover, the lack of specific biomarkers also challenges the diagnosis. METHODS: Inflammatory biomarkers such as IL-8, IP-10, MIG, MIP-1α and RANTES were measured in serum samples from SLE patients and subjects in control groups (patients with other autoimmune diseases and healthy individuals). Forty-six SLE patients (22 patients with low activity, SLEDAI-2 K ≤ 4, 24 patients with moderate/high activity, SLEDAI-2 K > 4), 42 patients with other autoimmune diseases (OAD group), and 8 healthy volunteers participated in this study. RESULTS: MIG (p < .001) and RANTES (p < .001) concentrations in SLE patients and healthy controls, and IP-10 concentrations in SLE patients with different disease activities (low activity, p < .01, moderate/high activity, p < .05) differed significantly. IL-8 (p < .001) and MIP-1α (p < .001) concentrations in SLE patients differed from those in patients from the OAD group. IL-8 (p < .05), IP-10 (p < .01), MIG (p < .05), MIP-1α (p < .001), and RANTES (p < .05) were correlated with SLE activity; their concentrations in SLE patients with low and moderate/high activity differed significantly. CONCLUSIONS: Given the findings of this study, one can envision the possibility of future use of some of these cytokines to assist in the screening of SLE patients, or even in monitoring disease activity.

Slow-release nutrient capsules for microorganism stimulation in oil remediation.

As the concern towards environmental deterioration grows worldwide, new technological achievements become essential for all countries. Among the technologies with great potential of bioremediation is microencapsulation of active material. Several studies have investigated the use of controlled release of active materials as a way of biostimulation and supplying the nutrients necessary for the bioremediation process. In fact, as the use of microorganisms has a great potential in degrading crude oils, this work aims to use that technology and to associate it to produce controlled-release capsules of nitrogen, phosphorus, and potassium (N, P, and K) for bioremediation purposes. For the capsule formulation, polymers of sodium alginate, Capsul®, and the commercial fertilizer NPK from Sempre Verde Inc. were used. Crude oil was the only carbon source and mineral medium for microorganism growth. Controlled-release nutrient capsules, with 4 mm in diameter, made of 3.0 % alginate (w/v) and 4.0 % Capsul® (w/v) were produced. Those capsules were used in association with a microbial consortium, in a liquid phase bioremediation process, having degraded 43.6 % of the total hydrocarbon within 240 h, evidencing thus as a promising tool for hydrocarbon bioremediation.

Plasma lipoproteins in visceral leishmaniasis and their effect on Leishmania-infected macrophages.

This work aimed at investigating the lipid profile of zoonotic visceral leishmaniasis (VL) patients' sera and the effect of lipoproteins on the in vitro production of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 and IL-12 by Leishmania infantum-infected and uninfected macrophages. Lipids were quantified in 26 VL patients' sera and 26 healthy controls from a VL endemic area. The patients' sera had higher triglyceride and very low density lipoprotein (VLDL) levels, and much lower apolipoprotein A1, total cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL) levels than the control sera. Lipoprotein fractions were obtained by ultracentrifugation of sera. The addition of LDL and HDL to Leishmania-infected and uninfected macrophages, in physiological concentrations, enhanced the production of IL-6 and IL-10, but not of IL-12. LDL stimulated the production of TNF-alpha only in infected macrophages, whereas HDL stimulated the production of lower amounts of TNF-alpha in both infected and uninfected macrophages. VLDL stimulated only the production of IL-10. It is proposed herein that LDL may influence the development of VL by promoting the production of TNF-alpha by infected macrophages. A decrease in plasma LDL in some VL patients (to 20 mg/mL or less); however, would tend to reduce the production of TNF-alpha and therefore to limit the development of immune-mediated pathology, not withstanding the fact that it would perhaps increase the permissiveness of macrophages to Leishmania growth.

Schistosoma mansoni triose phosphate isomerase peptide MAP4 is able to trigger naïve donor immune response towards a type-1 cytokine profile.

We evaluated the ability of naïve monocyte-derived dendritic cells (DC) to sensitize autologous peripheral blood mononuclear cells (PBMC) to the schistosome vaccine candidate MAP4 using a priming in vitro (PIV) assay. MAP4 is a multiple antigen peptide containing B- and T-cell epitopes derived from the glycolytic enzyme triose phosphate isomerase. PBMC primed and restimulated with MAP4 first and secondary recalls (MAP4 PIV cells) were examined for cell phenotype and cytokine production. We found that after the first recall stimulation with MAP4, the major cell population was predominantly CD4(+) T-cell subsets (68.5%), CD8(+high) (16%) and CD19(+) (10%). Additionally, MAP4 PIV cells significantly expressed CD4(+)-HLA-DR(+), -CD54(+), -CD45RO(+) (P < 0.0001) and -CD25(+) (P < 0.0004) together with significant expression of CD80(+) on CD19(+) B cells (P < 0.007). Cytokine production from activated MAP4 PIV cells was predominantly Th1-like, consisting mainly of IFN-gamma. Interestingly, IFN-gamma production was suppressed when Schistosoma mansoni-soluble egg antigen (SEA) was added to a MAP4 PIV cell culture. Furthermore, addition of MAP4 to a SEA PIV cell culture significantly reduced secretion of IL-10. The present findings add to the knowledge gained from studies in the mouse model, and our results show that naïve donor DC, sensitized with MAP4, were able to prime and clonally expand MAP4-specific T cells towards a Th1-type response.

Naïve donor responses to Schistosoma mansoni soluble egg antigens.

Schistosome infection induces profound Th-biasing and immune suppression. Although much has been examined in mice, few studies have examined responses of naïve humans to schistosome antigens. In this study, we examined the response of naïve human peripheral blood mononuclear cells (nPBMC) to stimulation with Schistosoma mansoni soluble egg antigen (SEA) using a priming in vitro (PIV) assay. We found that SEA induced a pronounced CD4+ T-helper cell response based on cytokine secretion and phenotyping markers. SEA-stimulated nPBMC (SEA cells) at day 7 post-priming and after the first recall consisted predominantly of Th0-like CD4+ T cells. Following the second recall, the majority of donor (10/12) responses were Th2-like. The cell population consisted of approximately 64% CD4+, 17% CD8(+high), 12% CD19+, and 7% CD23+ cells. The CD4+ population also expressed HLA-DR+, CD54+, CD45RO+ and CD25+ whereas the CD19+ cells expressed CD80 and CD86. Following priming, we detected high levels of IL-6, IFN-gamma, IL-12p40, IL-10 and IL-5. Upon restimulation, SEA cells secreted IL-5 and high levels of IL-10, typical of a Th2-like response. The data presented herein shows that the majority of naïve donor dendritic cells, following stimulation with SEA, prime and clonally expand SEA-specific T cells towards a Th2-type response. However, two donors responded with an atypical response, producing IFN-gamma coincident with low levels of IL-10. Whether this differential response was due to HLA or other genes was not determined but is currently under investigation.

Participation of hippocampal nicotinic receptors in acquisition, consolidation and retrieval of memory for one trial inhibitory avoidance in rats.

One-trial step-down inhibitory avoidance in rats involves the activation of two separate memory types, a short-term system (STM) that lasts 3-6 h, and a long-term system (LTM) that takes 3-6 h to be formed and lasts for many days or even months. Here we investigate the effect of nicotinic receptor (nAChR) ligands infused bilaterally in the hippocampus on STM and LTM formation and on LTM retrieval of this task. Rats were implanted with chronic cannulae in the CA1 region of the dorsal hippocampus, trained using a 0.5 mA foot shock, and tested twice, first 1.5 h after training to measure STM, and again at 24 h to measure LTM. The drugs used were the nAChR antagonists, mecamylamine (1, 3 and 10 microg/side) and dihydro-beta-erythroidine (DHbetaE; 2, 6 and 18 microg/side) and the agonist, nicotine (0.6, 1 and 3 microg/side). They were given either 15 min before training, immediately after training or 15 min prior to LTM retrieval. Mecamylamine and DHbetaE impaired and nicotine enhanced STM, LTM and retrieval similarly. The results indicate that nAChRs in CA1 participate in the regulation of both STM and LTM formation, and on the retrieval of LTM.

Anti-beta2 glycoprotein I and anticardiolipin antibodies in leptospirosis, syphilis and Kala-azar.

OBJECTIVE: Reports have shown that anticardiolipin (aCL) antibodies present in patients with autoimmune diseases are dependent on the cofactor,beta2 glycoprotein I (beta2 GPI), as opposed to aCL antibodies seen in infectious diseases such as syphilis, HIV hepatitis C, etc. The assay for anti-beta2GPI antibodies has been reported to be more specific for antiphospholipid syndrome (APS). However, the prevalence of these antibodies in diseases such as leishmaniasis and leptospirosis remains unknown. The aim of the present study was determine the prevalence of antibodies to cardiolipin and to beta2GPI in patients with different infectious diseases, including leptospirosis, syphilis and leishmaniasis. METHODS: Samples from patients with Kala-azar (visceral leishmaniasis), syphilis or leptospirosis were tested for IgG and IgM anticardiolipin and IgG anti-beta2GPI antibodies by ELISA. RESULTS: In patients with Kala-azar the prevalence of IgG aCL, IgM aCL and anti-beta2GPI was 6% (2/30), 3% (1/30) and 53% (16/30), respectively. In syphilis the prevalence was 18% (14/74), 13% (10/74) and 10% (8/70), respectively. In leptospirosis the frequency of these antibodies was 23% (9/39), 10% (4/39) and 17% (6/34), respectively. There was no statistical correlation between aCL and anti-beta2GPI antibodies in these diseases. DISCUSSION: This study clearly shows a significant prevalence of anti-beta2GPI antibodies in leptospirosis and leishmaniasis and syphilis. This indicates that the assay for antibeta2GPI antibodies should be thoroughly validated before it is introduced as a definitive tool for the diagnosis of APS, testing a larger number of sera from patients with a wider range of clinical conditions.

Lewis(x)-containing oligosaccharide attenuates schistosome egg antigen-induced immune depression in human schistosomiasis.

The proliferative and interleukin (IL)-10 responses to Lacto-n-fucopentaose III (LNFPIII) that contains Lewis(x)(Le(x))-trisaccharide was assessed in PBMC from humans infected with Schistosoma mansoni. All patient groups with low, medium, and high egg counts in their feces responded to polyvalent LNFPIII-HSA (where HSA = human serum albumin) conjugate. PBMC of all subjects showed a significant proliferative response to this sugar conjugate. However, the levels of interleukin (IL)-10 induced by LNFPIII-HSA were higher in groups with low and medium egg counts than those with high egg. Soluble egg antigens (SEA) also induced IL-10 production by PBMC from infected patients. Interestingly, the SEA-induced IL-10 production was remarkably inhibited by pretreatment of PBMC with free ligands of LNFPIII (monovalent form). These LNFPIII-pretreated PBMC displayed appreciable increase in the level of proliferation to SEA stimulation. We propose that the observed bystander immune potentiation rendered by free LNFPIII is due to the reduced IL-10 level which, presumably, up-regulate expression of co-stimulatory molecules on APC. The ensemble of results indicates that the Le(x)-containing LNFPIII is a potent immunoreactive epitope in SEA that negatively influences PBMC response against this parasite antigens via IL-10.

A new proposal to adjust Moran's I for population density.

We analyse the effect of using prevalence rates based on populations with different sizes in the power of spatial independence tests. We compare the well known spatial correlation Moran's index to three indexes obtained after adjusting for population density, one proposed by Oden, another proposed by Waldhör, and a third proposed by us in this paper. We find an effect of spatially correlated populations in the type I error probability on the test based on Moran's and Waldhör's indexes. We conclude also that the test proposed by Oden is powerful to test risk heterogeneity, but it has disadvantages when the interest is solely on the spatial correlation of morbidity risks. In this latter case, we recommend using our proposed test which is more powerful than the usual Moran's index applied directly to the rates.

Comparative analysis by polymerase chain reaction amplified minicircles of kinetoplast DNA of a stable strain of Trypanosoma cruzi from São Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area.

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment length polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

Association of hepatosplenic schistosomiasis with HLA-DQB1*0201.

Major histocompatibility class II alleles of 351 persons living in an area endemic for Schistosoma mansoni in northeastern Brazil were characterized at three loci (DRB1, DQA1, and DQB1). Contingency analyses were used to compare allele frequencies with high egg excretion, proliferative response to schistosome soluble egg antigens (SEA), and occurrence of severe, biopsy-confirmed hepatosplenic disease. There were no associations of HLA-DR or DQ with egg excretion. Patients positive for DRB1*01, DQA1*0101, or DQB1*0501 were less likely to respond to SEA than was the overall study population. However, using stringent Bonferroni correction (multiplying P values by the number of alleles tested; P x 35), none of these associations with SEA responsiveness remained significant. Hepatosplenic disease was less likely in patients positive for DRB1*11 and was more likely in patients positive for DRB1*07 or DQB1*0201. However, only the DQB1*0201 association remained significant (odds ratio = 3.72; P < .005) following Bonferroni correction.