Serum 1,3-beta-D-glucan as a noninvasive test to predict histologic activity in patients with inflammatory bowel disease

BACKGROUND 1,3-beta-D-glucan(BG)is a ubiquitous cell wall component of gut microorganisms.We hypothesized that the serum levels of BG could reflect active intestinal inflammation in patients with inflammatory bowel disease.AIM To determine whether the serum BG concentrations correlate with intestinal inflammation.METHODS A prospective observational study was performed in a tertiary referral center,from 2016 to 2019,in which serum BG was determined in 115 patients with Crohn’s disease(CD),51 with ulcerative colitis(UC),and 82 controls using a photometric detection kit.Inflammatory activity was determined by ileocolonoscopy,histopathology,magnetic resonance enterography,and biomarkers,including fecal calprotectin(FC),C-reactive protein,and a panel of cytokines.The ability of BG to detect active vs inactive disease was assessed using the area under the receiver operating characteristic curve.In subgroup analysis,serial BG was used to assess the response to therapeutic interventions.RESULTS The serum BG levels were higher in CD patients than in controls(P=0.0001).The BG levels paralleled the endoscopic activity in CD patients and histologic activity and combined endoscopic and histologic activity in both CD and UC patients.The area under the curve(AUC)in receiver operating characteristic analysis to predict endoscopic activity was 0.694[95%confidence interval(CI):0.60-0.79;P=0.001]in CD,and 0.662(95%CI:0.51-0.81;P=0.066)in UC patients.The AUC in receiver operating characteristic analysis to predict histologic activity was 0.860(95%CI:0.77-0.95;P<0.001)in CD,and 0.786(95%CI:0.57-0.99;P=0.015)in UC patients.The cut-off values of BG for both endoscopic and histologic activity were 60μg/mL in CD,and 40μg/mL in UC patients.Performance analysis showed that the results based on BG of 40 and 60μg/mL were more specific for predicting endoscopic activity(71.8%and 87.2%for CD;and 87.5%and 87.5%for UC,respectively)than FC(53.3%and 66.7%for CD;and 20%and 80%for UC,respectively);and also histologic activity(60.5%and 76.3%for CD;and 90.0%and 95.0%for UC,respectively)than FC(41.7%and 50.0%for CD;and 25%and 50%for UC,respectively).Regarding the clinical,endoscopic,and histologic activities,the BG levels were reduced following therapeutic intervention in patients with CD(P<0.0001)and UC(P=0.003).Compared with endoscopic(AUC:0.693;P=0.002)and histologic(AUC:0.868;P<0.001)activity,no significant correlation was found between serum BG and transmural healing based on magnetic resonance enterography(AUC:0.576;P=0.192).Positive correlations were detected between BG and IL-17 in the CD(r:0.737;P=0.001)and the UC group(r:0.574;P=0.005),and between BG and interferon-gamma in the CD group(r:0.597;P=0.015).CONCLUSION Serum BG may represent an important novel noninvasive approach for detecting mucosal inflammation and therapeutically monitoring inflammatory bowel diseases,particularly in CD.

Changes in cellular proliferation and plasma products are associated with liver failure

AIM To study the differences in immune response and cytokine profile between acute liver failure and selflimited acute hepatitis.METHODS Forty-six patients with self-limited acute hepatitis(AH), sixteen patients with acute liver failure(ALF), and twenty-two healthy subjects were involved in this study. The inflammatory and anti-inflammatory products in plasma samples were quantified using commercial enzyme-linked immunoassays and quantitative real-time PCR. The cellular immune responses were measured by proliferation assay using flow cytometry. The groups were divided into viral- and non-viral-induced selflimited AH and ALF. Thus, we worked with five groups: Hepatitis A virus(HAV)-induced self-limited acute hepatitis(HAV-AH), HAV-induced ALF(HAV-ALF), nonviral-induced self-limited acute hepatitis(non-viral AH), non-viral-induced acute liver failure(non-viral ALF), and healthy subjects(HC). Comparisons among HAV and non-viral-induced AH and ALF were performed.RESULTS The levels of mitochondrial DNA(mt DNA) and the cytokines investigated [interleukin(IL)-6, IL-8, IL-10, interferon gamma, and tumor necrosis factor] were significantly increased in ALF patients, independently of etiology(P < 0.05). High plasma mt DNA and IL-10 were the best markers associated with ALF [mt DNA: OR = 320.5(95%CI: 14.42-7123.33), P < 0.0001; and IL-10: OR = 18.8(95%CI: 1.38-257.94), P = 0.028] and death [mt DNA: OR = 12.1(95%CI: 2.57-57.07), P = 0.002; and IL-10: OR = 8.01(95%CI: 1.26-50.97), P = 0.027]. In the cellular proliferation assay, NK^(bright), NKT and regulatory T cells(TReg) predominated in virusspecific stimulation in HAV-induced ALF patients with an anergic behavior in the cellular response to mitotic stimulation. Therefore, in non-viral-induced ALF, anergic behavior of activated T cells was not observed after mitotic stimulation, as expected and as described by the literature. CONCLUSION mt DNA and IL-10 may be predictors of ALF and death. TReg cells are involved in immunological disturbance in HAV-induced ALF.

B-1 cells modulate the murine macrophage response to Leishmania major infection

AIM To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major(L. major) in vitro.METHODS Peritoneal macrophages obtained from BALB/c andBALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10(IL-10) production was quantified in the cellular supernatants using an enzymelinked immunosorbent assay. The levels of the lipid mediator prostaglandin E2(PGE2) were determined using a PGE2 enzyme immunoassay kit(Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major.RESULTS We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. CONCLUSION Our results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell cultures.

Towards a Comprehensive Search of Putative Chitinases Sequences in Environmental Metagenomic Databases

Chitinases catalyze the hydrolysis of chitin, a linear homopolymer of β-(1,4)-linked N-acetylglucosamine. The broad range of applications of chitinolytic enzymes makes their identification and study very promising. Metagenomic approaches offer access to functional genes in uncultured representatives of the microbiota and hold great potential in the discovery of novel enzymes, but tools to extensively explore these data are still scarce. In this study, we develop a chitinase mining pipeline to facilitate the comprehensive search of these enzymes in environmental metagenomic databases and also to explore phylogenetic relationships among the retrieved sequences. In order to perform the analyses, UniprotKB fungal and bacterial chitinases sequences belonging to the glycoside hydrolases (GH) family-18, 19 and 20 were used to generate 15 reference datasets, which were then used to generate high quality seed alignments with the MAFFT program. Profile Hidden Markov Models (pHMMs) were built from each seed alignment using the hmmbuild program of HMMER v3.0 package. The best-hit sequences returned by hmmsearch against two environmental metagenomic databases (Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis—CAMERA and Integrated Microbial Genomes—IMG/M) were retrieved and further analyzed. The NJ trees generated for each chitinase dataset showed some variability in the catalytic domain region of the metagenomic sequences and revealed common sequence patterns among all the trees. The scanning of the retrieved metagenomic sequences for chitinase conserved domains/signatures using both the InterPro and the RPS-BLAST tools confirmed the efficacy and sensitivity of our pHMM-based approach in detecting putative chitinases sequences. These analyses provide insight into the potential reservoir of novel molecules in metagenomic databases while supporting the chitinase mining pipeline developed in this work. By using our chitinase mining pipeline, a larger number of previously unannotated metagenomic chitinase sequences can be classified, enabling further studies on these enzymes.

Microbe/Bug-Busters Visit the Interactive House: An Itinerant Scenographic Device for Health Education

The term Ecology is derived from οἶκος (Greek “house” or environment), but our habitations usually overlooked in ecology or environmental studies. The expression “at home” usually means safety and comfort, but at home we are under risk of innumerous parasitic/microbial infections and contaminations/ envenomation. During the COVID-19 pandemic we were forced to stay at home, but the virus and other pathogens were also home-delivered. Education for health is highly effective in health promotion, particularly in poor areas. Thus, prophylactic interventions approaching household environment are required. The present activity aims community empowerment and engagement in controlling parasitic diseases and other infections such as Chagas disease, leishmaniasis, malaria, arboviruses etc. Inspired on the use of a house maket by Dr. Virgínia Schall to demonstrate Aedes mosquitoes breeding sites. We also employ house makets displaying pathogen vectors breeding/hiding sites. Although some makets can be opened, revealing intradomiciliary milieu, we intended to offer the public a literally “insider” view of this largely overlooked scenery. The “Interactive House”, also known as “House with no viruses and other bugs” is an educative, interactive, ludic device elaborated on an inflatable igloo, with furniture and utensils crafted using reused/recycled or low-cost materials. Live mosquito larvae were placed at peridomicile in water-accumulating plant pot saucer, leaves-clogged gutter, used tire, dog water bowl as well as within the domicile, in the shower drain trap. Evidengue®, crafted in cloth to block mosquito oviposition developed is presented. Participants enjoy taking part in the activity, seem amused, play, laugh and smile while enthusiastically take pictures. This educative activity permits health communication in a ludic interactive way, which may contribute to health promotion in areas with public health problems.

An unfolding monkeypox outbreak in Europe and beyond

Dear Editor,Monkeypox is a zoonotic disease caused by the monkeypox virus(MPXV),a double-stranded DNA virus in the genus Orthopoxvirus,family Poxviridae.MPXV is known to be transmitted between animals such as rats and squirrels,but animal-to-human and human-to-human transmission has been known in Africa since 1970.Transmission is linked to direct contact with body fluids,skin lesions,and patient items as well as respiratory droplets in case of prolonged face-to-face contact[1].Until recently,only a few cases were associated with travel to endemic countries or contact with infected animals imported from endemic countries.

Zea mays L. Pollen： An Approach to Its Quality Control

Zea mays L. is one of the biggest cropping systems among the sustainable development agronomy. Pollen from this crop source is unexplored and apiculture can be a good partner adding value to the product and creating new jobs helping to solve some social issues as unemployment. However, food safety is crucial, thus the aim of this study was to explore the flavonoid/phenolic profiles from Z. mays L. pollen as a fingerprint for this plant identification and also to demonstrate how the method of bee pollen samples （honeybee collected pollen） is applied. For this purpose, several sources ofZ. mays L. pollen were analyzed, including corn hybrids and genetic modified samples collected at the breeding fields. For this work, samples were taken at several years from 2000 to 2012 and collected from different countries and locations, such as Portugal, Mexico and Brazil. Results showed, for the first time, that the fingerprint （flavonoid/phenolic profile） for Z. mays L. pollen does not change over the time of sampling neither with the region of harvesting. The high performance liquid chromatography-diode array detector （HPLC/DAD） fingerprints of phenolic/flavonoid extract from Z. mays remain unchanged for all samples analyzed from different countries, hybrids and/or genetic modified plants. This is also the first study reporting these phenolic compounds not only in pollen collected directly from hybrid plants, but also in Z. mays bee pollen. The described fingerprinting method is easy, fast and accurate for the characterization of Z. mays L. pollen samples and complete microscopic analysis because it is species-specific.

<i>Schistosoma mansoni</i>: Phytochemical Effect on Aquatic Life Cycle

Background: Two aspects need to be considered for schistosomiasis control: morbidity and transmission. In this context, many soluble substances have been tested and Euphorbia milii latex is one of the most promising Brazilian molluscicides. Phytochemical studies involving simulation of the applicability of this latex on all aquatic forms of the S. mansoni life cycle are rare in the literature. The aim of this work was to evaluate the effect of E. milii latex on S. mansoni in the egg, miracidium and different developmental stages in Biomphalaria glabrata. Methods: The laboratory study was designed to simulate the different forms of exposure of the life cycle stages of S. mansoni to the LC50 of E. milii latex;we tested the exposition from four situations of S. mansoni contact with the latex and observed the exposure on different snails’ infection stage too. All snails were analyzed weekly for cercarial shedding and reproductive biology. Results: The results showed that contact of S. mansoni eggs and miracidia with the LC50 of E. milii negatively influenced the development of the parasite life cycle in the intermediate host, with consequent reduction of cercarial shedding. The exposure of infected snails affected the reproductive biology and cercarial shedding in all intra-mollusk development stages of S. mansoni, but the reduction was greater in the first, fourth, fifth, sixth, seventh and eighth weeks of infection. The LC50 of E. milii latex had toxic action on eggs and miracidia, and the number of cercariae shed by snails during the study period declined by about 80%. Conclusions: We can conclude that the use of natural biodegradable compounds containing low concentrations of substances already characterized as having eco-toxico- logical potential can be an important tool to reduce the transmission of Schistosomiasis.

Serological Evidence of Human Coinfection by Brazilian Spotted Fever and Bartonellosis

Brazilian spotted fever and bartonellosis are zoonotic, emerging and under diagnosed diseases. Pets may be co-infected by multiple pathogens and become transmissions sources to humans. The study reports the first case of active co-infection by Brazilian spotted fever and bartonellosis based on serological evidence. The authors aim to demonstrate the importance of performing systematic syndromic investigations on nonspecific febrile syndromes, guided by the epidemiological history and considering the possibility of co-infection by zoonosis sharing the same ecological niche.

Ultrastructural and enzymatic alterations in the ovary of Rhodnius prolixus infected with Trypanosoma rangeli

Objective:To investigate the morphological structure of ovarian follicular cells and biochemical parameters of both ovaries and fat bodies(sites of vitellogenesis)from Rhodnius(R.)prolixus infected with Trypanosoma(T.)rangeli.Methods:Adult virgin females of R.prolixus were fed upon a membrane apparatus containing heat-inactivated citrated rabbit blood and a suspension of T.rangeli epimastigotes(Macias strain).Females from the control group and all the males received parasite-free blood.Transmission electron microscopy was used to reveal the morphological aspects of ovarian follicle cells in both control and parasite-infected groups.Protein profile,proteolytic activities and Western blotting analyses were performed in either ovary or fat body samples of control and parasite-infected groups.Results:According to the ultrastructural data,T.rangeli infection elicited a degeneration process in the ovarian follicular cells of R.prolixus.Proteolytic assays indicated a reduction in the activity of aspartic peptidases in the ovary and fat body from parasite-infected group,while a significant increase in the cysteine peptidase activity was measured in both insect organs.Additionally,immunoblotting revealed that vitellogenin was overexpressed in the ovary of parasite-infected insects.Conclusions:T.rangeli infection seems to elicit an early programmed cell death in the ovarian follicle cells as well as induces the modulation on the activities of different peptidase classes in either ovaries or fat bodies and the overexpression of the vitellogenin in the ovary of R.prolixus.

Experience of more than 3000 cycles of Brazilian women using the contraceptive vaginal ring (nuvaring^&reg;)

Objective: To evaluate the profile of the Brazilianvaginal ring user, reasons for choosing it, satisfaction, menstrual pattern, tolerability, efficacy, side effects and reasons for discontinuation. Methods: We reviewed the files of 148 users who attended the Hospital de Jacarepaguáand private clinics, between 2005 and 2011, in a total of 3107 cycles. Kaplan-Meier survival curves and regression models were used in statistical analysis. Results: The profile was: single, nulligravida, 30 years (mean) and with university education. The previous contraception used was mostly the oral hormonal contraception (75.7%). Women chose the ring for: practicality (32.4%), forgetting the pill (32.4%) and irregular vaginal bleeding (13.5%). The satisfaction degree was 80.5% (119). There were good cycle control and no pregnancies. Thirty-eight patients discontinued the use (25.7%). The main reasons were wish for pregnancy (6%), spotting (3.4%) and increased vaginal discharge (2.7%). At the end of the first year, use of the greatest reduction of continuity probability was observed (19.3%). Women with side effects were 68% less likely to be satisfied with the method than those without (p-value Conclusion: Nuvaring&reg;is an excellent contraceptiveoption due to its sefficacy, cycle control, tolerability, practicality and safety.

Additional Records of the American Elephantfish <i>Callorhinchus callorynchus</i>(Chondrichthyes, Holocephali, Chimaeriformes) in Southeastern Brazil

Chimaeras, fish in the order Chimaeriformes, are among the rarest and least studied fish in the class Chondrichthyes (cartilaginous fish). Previous records have indicated that a species of chimaera known as the elephantfish, or cockfish, Callorhinchus callorynchus, may be found in marine waters off southeastern Brazil, as far north as the state of São Paulo. Here we report that C. callorynchus, caught with bottom gillnet, was found in an urban fish market in the city of Rio de Janeiro (~23˚S). This and other records from a variety of places call into question the distribution limits of the American elephantfish in the southwestern Atlantic Ocean and whether, at least seasonally, this species may often be found as far north as the state of Rio de Janeiro. Because of scientific and conservation interests in the poorly known Chimaeriformes, we recommend long-term monitoring of the fish catch in ports along the southern Brazilian coast, to better understand the natural history of the intriguing chimeras.

Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin

Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.

Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree

Endophytic fungi were identified from different parts of the medicinal parasitic mistletoe Cladocolea micrantha and from its host Anacardium occidentale, suggesting a strict host-parasite relationship. Eight fungal endophytes were isolated and morphologically characterized. The ascomycete Guignardia mangiferae and strains of Mycelia sterilia were prevalent in the isolations. The unequivocal identification of Guignardia mangiferae at a probabilistic degree close to 100% was carried out by DNA extraction followed by PCR analyses of the ITS-1 and ITS-2 regions and comparison of the genetic sequence with the NCBI database.

Functional biomarker signatures of circulating T-cells and its association with distinct clinical status of leprosy patients and their respective household contacts

Background: Leprosy is a chronic infectious disease classified into two subgroups for therapeutic purposes:paucibacillary(PB)and multibacillary(MB),closely related to the host immune responses.In this context it is noteworthy looking for immunological biomarkers applicable as complementary diagnostic tools as well as a laboratorial strategy to follow-up leprosy household contacts.Methods:: The cross-sectional study enrolled 49 participants,including 19 patients and 30 healthy controls.Peripheral blood mononuclear cells(PBMC)were isolated and incubated in the presence of Mycobacterium leprae bacilli.The cells were prepared for surface(CD4+and CD8+)and intracytoplasmic cytokine staining(IFN-γ,IL-4 and IL-10).Multiple comparisons amongst groups were carried out by ANOVA,Kruskal–Wallis,Student T or Mann–Whitney test.Comparative analysis of categorical variables was performed by Chi-square.Functional biomarker signature analysis was conducted using the global median values for each biomarker index as the cut-off edge to identify the proportion of subjects with high biomarker levels.Results: The cytokine signature analysis demonstrated that leprosy patients presented a polyfunctional profile of T-cells subsets,with increased frequency of IFN-γ+T-cell subsets along with IL-10+and IL-4+from CD4+T-cells,as compared to health Controls(Venn diagram report).Moreover,statistical analysis was carried out using parametric or non-parametric variance analysis followed by pairwise multiple comparisons,according to the data normality distribution.L(PB)displayed a polyfunctional profile characterized by enhanced percentage of IFN-γ+,IL-10+and IL-4+produced by most T-cell subsets,as compared to L(MB)that presented a more restricted cytokine functional profile mediated by IL-10+and IL-4+T-cells with minor contribution of IFN-γproduced by CD4+T-cells.Noteworthy was that HHC(MB)exhibited enhanced frequency of IFN-γ+T-cells,contrasting with HHC(PB)that presented a cytokine profile limited to IL-10 and IL-4.Conclusions: Our data demonstrated that L(PB)displayed enhanced percentage of IFN-γ+,IL-10+and IL-4+as compared to L(MB)that presented functional profile mediated by IL-10+and IL-4+T-cells and HHC(MB)exhibited enhanced frequency of IFN-γ+T-cells,contrasting with HHC(PB).Together,our findings provide additional immunological features associated with leprosy and household contacts.These data provide evidence that biomarkers of immune response can be useful complementary diagnostic/prognostic tools as well as insights that household contacts should be monitored to access putative subclinical infection.