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Oleuropein (OP) is an appreciated compound present not only in fruits but also in leaves of olive trees, which can be transformed into hydroxytyrosol (HT), a substance with high antioxidant activity. In this work, the transformation of an agricultural residue containing OP (olive leaves or wastewater from mills) to the high added value compound HT is accomplished through different enzymatic strategies. Different enzymes were used, immobilized on various supports by diverse binding forces: beta-glucosidase encapsulated in siliceous material, esterases and lipases immobilized on hydrophobic supports (octyl-functionalized amorphous silica and periodic mesoporous organosilica), and esterase immobilized on amine-functionalized ordered mesoporous silica. All these biocatalysts were tested for oleuropein hydrolysis through two different reaction approaches: a) split of glucosidic bond catalyzed by beta-glucosidase (β-glu), followed by hydrolysis of the aglycon and further ester hydrolysis. 5 mg·mL -1 of β-glu fully hydrolyzed 5 mM OP at pH 7 and 50°C in 7 days, and further enzymatic hydrolysis of the aglycon yielded near to 0.5 mM HT in the best conditions tested. b) via direct hydrolysis of the ester bond to produce hydroxytyrosol in a one-step reaction using esterases or lipases. The latter reaction pathway catalyzed by lipase from Penicillium camemberti immobilized on octyl-silica (4 mg·mL -1 ) at 35°C and pH 6 directly produced 6.8 mM HT (1 mg·mL -1 ), transforming in 12 days near to 30% of the initial 25 mM OP from a commercial olive leaves extract.

Introduction: Leptospirosis is a global zoonosis that affects more than one million people per year, with a lethality rate of approximately 15%. Chemokines are crucial in the immune response against Leptospira , recruiting leukocytes to the site of infection and regulating immune activity. In previous studies, we have shown that CCL2, CXCL5, and CCL8 are involved in the leptospirosis process, although the mechanisms are not understood., Methods: In this study, we present the frequency of Leptospira serovars in human samples. We then evaluated the profile of various chemokines in sera from patients diagnosed with leptospirosis, assessing the possible correlation between them. Moreover, we evaluated the changes in the chemokine profile on different days after the first symptoms. The frequency of the Leptospira serovars in human samples is presented., Results and Discussion: The main findings were that CCL5, CXCL5, and CXCL9 are highly expressed during leptospirosis, indicating a special role of these molecules in the immunity and pathogenesis of the disease. The correlation analysis of detected chemokines CXCL11, CXCL9, CCL3, and CCL2 helps to clarify the role of each cytokine in leptospirosis. The possible use of CCL5 as a biomarker for complementary diagnosis of the disease is suggested., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mariano, Blanco, de Souza, de Freitas, Ho, Martins, Romero and da Silva.)

Zeolitic imidazolate frameworks (ZIFs) are widely used MOFs because of certain characteristics, but also because they can be prepared at room temperature using water as the unique solvent. However, these a priori sustainable conditions inevitably entail a huge and somehow unusable excess of linker. Here, we present the formation of ZIFs at room temperature in water, starting from mixtures with a linker/metal ratio of two, that is, coinciding with the stoichiometry found in the final MOFs, in the presence of amines. ZIF-8 can be prepared with triethylamine (TEA), giving a yield of Zn of 96.6%. Other bases, like NaOH, tetraethylammonium hydroxide or ammonium hydroxide, do not lead to ZIF-8 under the same conditions. The so-obtained ZIF-8 contains TEA inside its cavities, making it less porous than its conventionally prepared counterparts. Amine can be removed by mild thermal treatments (200-250 °C). Such thermal treatments induce the generation of g-C 3 N 4 -like species which could give added value to these materials as potential photocatalysts, increasing their affinity to CO 2 , as proved in this work. This methodology can be successfully extended to other amines, like N,N-dicyclohexylmethylamine, as well as to other prepared ZIFs, like Co-based ZIF-67, isostructural to ZIF-8.

The diagnosis of human leptospirosis is mainly based on serological assays. Since the extraction by N-butanol has only been studied as an antigen for the diagnosis of cattle leptospirosis, this study aimed to investigate the feasibility of the N-butanol preparation for the diagnosis of human leptospirosis and compare it with sonicated and thermo-resistant antigens in IgM dot-blot test. Paired serum samples from 147 laboratory-confirmed leptospirosis cases were tested. The control group consisted of 148 serum samples from healthy individuals and nonleptospirosis cases. N-butanol antigens from serovar Copenhageni (ButC3) and serovar Patoc (ButP3) showed reactivity with antileptospiral antibodies from patients with confirmed leptospirosis. In the acute phase, sensitivities of IgM dot-blot assay with ButC3 and ButP3 antigens were 47.6% and 51.0%, respectively. In the convalescent phase, sensitivities were 95.9% (ButC3) and 93.2% (ButP3), and no significant differences were observed among the IgM dot-blot tests with other antigens. The specificity of the IgM dot-blot test with ButC3 antigen was good (92.6%), but with ButP3 (83.1%), it was significantly lower than with the other tests. The IgM dot-blot test described in this study is simple to perform and presents reliable visual results. Antigens prepared by N-butanol proved to be valuable diagnostic markers of leptospirosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Laccases are natural catalysts with remarkable catalytic activity. However, their application is limited by their lack of stability. Metal-organic frameworks (MOFs) have emerged as a promising alternative for enzyme immobilization. Enzymes can be immobilized in MOFs via two approaches: postsynthetic immobilization and in situ immobilization. In postsynthetic immobilization, an enzyme is embedded after MOF formation by covalent interactions or adsorption. In contrast, in in situ immobilization, a MOF is formed in the presence of an enzyme. Additionally, MOFs have exhibited intrinsic enzyme-like activity. These materials, known as nanozymes when they have the ability to replace enzymes in certain catalytic processes, have multiple key advantages, such as low cost, easy preparation, and large surface areas. This review presents a general overview of the most recent advances in both enzyme@MOF biocatalysts and MOF-based nanozymes in different applications, with a focus on laccase, which is one of the most widely investigated enzymes with excellent industrial potential., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2023. Published by Elsevier Inc.)

Introduction. Brazil was one of the most affected countries by the COVID-19 pandemic. Instituto Adolfo Lutz (IAL) is the reference laboratory for COVID-19 in São Paulo, the most populous state in Brazil. In April 2020, a secondary diagnostic pole named IAL-2 was created to enhance IAL's capacity for COVID-19 diagnosis. Hypothesis/Gap Statement. Public health laboratories must be prepared to rapidly respond to emerging epidemics or pandemics. Aim. To describe the design of IAL-2 and correlate the results of RT-qPCR tests for COVID-19 with secondary data on suspected cases of SARS-CoV-2 infection in the São Paulo state. Methodology. This is a retrospective study based on the analysis of secondary data from patients suspected of infection by SARS-CoV-2 whose clinical samples were submitted to real-time PCR after reverse transcription (RT-qPCR) at IAL-2, between 1 April 2020 and 8 March 2022. RT-qPCR Ct results of the different kits used were also analysed. Results. IAL-2 was implemented in April 2020, just over a month after the detection of the first COVID-19 case in Brazil. The laboratory performed 304,250 RT-qPCR tests during the study period, of which 98 319 (32.3 %) were positive, 205827 (67.7 %) negative, and 104 (0.03 %) inconclusive for SARS-CoV-2. RT-qPCR Ct values≤30 for E/N genes of SARS-CoV-2 were presented by 79.7 % of all the samples included in the study. Conclusion. IAL was able to rapidly implement a new laboratory structure to support the processing of an enormous number of samples for diagnosis of COVID-19, outlining strategies to carry out work with quality, using different RT-qPCR protocols.

We describe 5 cases of yellow fever vaccine-associated viscerotropic disease (YEL-AVD) in 2 familial clusters during the 2017-2018 yellow fever (YF) vaccination campaign in São Paulo state, Brazil. The first case was that of a 40-year-old white man who died of icterohemorrhagic syndrome, which was confirmed to be YEL-AVD by using real-time reverse transcription PCR to detect 17DD YF vaccine in the liver. Ten years previously, his brother died of a clinically similar disease without a confirmed diagnosis 9 days after YF vaccination. The second cluster included 3 of 9 siblings in whom hepatitis developed in the first week after receiving fractionated doses of YF vaccine. Two of them died of hemorrhagic diathesis and renal and respiratory failure, and 17DD-YF vaccine was detected in serum samples from all patients and in the liver in 1 case. Genetic factors might play a substantial role in the incidence of YEL-AVD.

Leptospirosis is a public health concern with lethality around 15% of the total cases. The current vaccines against Leptospira infection based on bacterins have several limitations, which require urgent development of new ones. In this context, groundbreaking approaches such as peptide-vaccines could be used to come around with promising results. Our goal was to identify conserved and immunogenic epitopes from the lipoprotein LruC that could interact with Major Histocompatibility Complex (MHC) I and II. LruC is a conserved lipoprotein expressed during leptospirosis that is considered among vaccine candidates and can be used as source for development of peptide-based vaccines. We searched for peptides that would be recognized by antibodies from either serum of hamsters previously immunized with low-LPS bacterin vaccines or from serum of patients diagnosed with leptospirosis. Immuno properties of seven peptides from LruC protein were evaluated in silico and by Dot Blot assay, and validate by ELISA. Preliminary results pointed one promising peptide that was recognized by the sera. In conclusion, the immunoinformatic approach helps the search and screening of peptides, while the Dot Blot assay, a simple and effective tool, helps to test and validate them. Thus, these prospective techniques together were validated to identify and validate potential peptides for further investigation as peptide-based vaccines or diagnostic methods., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mariano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cholesterol mediates its proliferative and metastatic effects via the metabolite 27-hydroxycholesterol (27-HC), at least in breast and endometrial cancer. We determined the serum lipoprotein profile, intratumoral cholesterol and 27-HC levels in a cohort of patients with well-differentiated papillary thyroid carcinoma (PTC; low/intermediate and high risk), advanced thyroid cancers (poorly differentiated, PDTC and anaplastic thyroid carcinoma, ATC) and benign thyroid tumors, as well as the expression of genes involved in cholesterol metabolism. We investigated the gene expression profile, cellular proliferation, and migration in Nthy-ori 3.1 and CAL-62 cell lines loaded with human low-density lipoprotein (LDL). Patients with more aggressive tumors (high-risk PTC and PDTC/ATC) showed a decrease in blood LDL cholesterol and apolipoprotein B. These changes were associated with an increase in the expression of the thyroid's LDL receptor, whereas 3-hydroxy-3-methylglutaryl-CoA reductase and 25-hydroxycholesterol 7-alpha-hydroxylase were downregulated, with an intratumoral increase of the 27-HC metabolite. Furthermore, LDL promoted proliferation in both the Nthy-ori 3.1 and CAL-62 thyroid cellular models, but only in ATC cells was its cellular migration increased significantly. We conclude that cholesterol and intratumoral accumulation of 27-HC promote the aggressive behavior process of PTC. Targeting cholesterol metabolism could be a new therapeutic strategy in thyroid tumors with poor prognosis.

This study was conducted to develop a selective medium for the detection of Leptospira spp. in clinical samples. Serovars of Leptospira spp., environmental bacteria and the fungus from contaminated cultures of patients with suspected leptospirosis were inoculated into EMJH medium containing amphotericin B, 5-fluorouracil (5-FU), furazolidone and neomycin used singly or combined. Medium with 5-FU at the concentration of 200 μg ml -1 did not show any inhibitory effect against the fungus, Gram-negative bacilli and any of the leptospira strains except serovar Pyrogenes. The highest concentration of neomycin and furazolidone that did not inhibit the growth of leptospires was 4 μg ml -1 . All strains of Leptospira spp. grew on 5-FU (100 μg ml -1 ) in combination with neomycin (4 μg ml -1 ) and on 5-FU (100 μg ml -1 ) in combination with furazolidone (4 μg ml -1 ). The highest concentration of amphotericin B (500 μg ml -1 ) that inhibited the growth of the fungus also inhibited the bacteria and most of serovars of Leptospira spp. The most effective antibiotic combinations that inhibited the majority of environmental bacteria growth without affecting leptospiral growth were EMJH with 5-FU (100 μg ml -1 ) in combination with neomycin (4 μg ml -1 ). In conclusion, these findings will help the development of new selective media to isolate leptospires., Significance and Impact of the Study: Leptospirosis is one of the most widespread zoonotic diseases in the world. Since certain serovars are often associated with the symptoms and severity of the disease, the isolation and identification of the leptospires usually permits the prediction of sources of infection. Attempts to isolate Leptospira spp. from clinical specimens are often frustrated by overgrowth of the slow-growing bacteria by more rapidly growing contaminants. In this study, we evaluated selective agents to develop a new selective medium to isolate leptospires. The results demonstrated that the association of drugs in concentrations that allowed the growth of leptospires is to be more effective in inhibiting bacterial contaminants., (© 2018 The Society for Applied Microbiology.)

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O 2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Z basic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 μmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O 2 -dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.

Leptospirosis is a zoonotic infection caused by pathogenic members of the genus Leptospira spp. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the disease. The aim of this study was to evaluate the ability of serology by the microscopic agglutination test (MAT) to predict the serogroups compared with results of identification of leptospires in São Paulo, Brazil. MAT correctly assigned the serogroup of the infecting isolate in 49/52 cases (94.23%). The serogroup Icterohaemorrhagiae was the predominant serogroup (88.46%). This study showed the usefulness of the MAT to correctly identify the infecting serogroup with a good overall agreement between the serologically-identified infecting serogroup and by identification of the isolate and can be used in epidemiological surveys in São Paulo. However, it should be complemented by the identification of Leptospira isolates., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The use of biomass fuel for cooking in traditional cookstove designs negatively affects respiratory health of communities in developing countries. Indoor pollution affects particularly women and children, who are participating in food preparation. The effects of smokeless cookstove designs on indoor pollution are well documented, but few studies exist to assess the effects of improved stove designs on the respiratory health of community members., Methods: This study uses peak expiratory flow rate (PEFR) measurements in a before-and-after format to assess respiratory function of inhabitants of all 30 houses of Buenas Noches in central Honduras. PEFRs are measured before and 6 months after the installation of Justa stoves in people's homes. Health behaviors, respiratory symptoms and fire wood use are evaluated in a door-to-door survey format., Results: A total of 137 eligible women and children between 6 and 14 years participated in the study. PEFR improved by 9.9-18.5% (P < 0.001) depending on the participants' exposure to indoor pollution. Health complaints like cough and behaviors like clinic visits did not change with the introduction of smokeless cookstove technology., Conclusions: Smokeless stoves improve respiratory health in an environment of high levels of indoor pollution., (© The Author 2014. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Household contacts are important sources of Bordetella pertussis in infants. A total of 353 household contacts of 97 index cases were evaluated for pertussis by culture and polymerase chain reaction. Twenty eight contacts were positive (8.0%). The presence of symptoms did not influence the rate of diagnosed bacteriologic pertussis in communicants. We conclude that contacts with an index case can be positive for B. pertussis independently of the presence of symptoms.

Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit a regular 2D hexagonal packing of cylindrical mesopores of uniform size, for non-covalent immobilization of laccase. Synthesis conditions were adjusted in order to obtain supports with different particle shape, where those with shorter channels had higher loading capacity. Despite the similar isoelectric points of silica and laccase and the close match between the size of laccase and the pore dimensions of these SBA-15 materials, immobilization was achieved with very low leaching. Surface modification of macro-/mesoporous amorphous silica by grafting of amine moieties was proved to significantly increase the isoelectric point of this support and improve the immobilization yield.

The aim of this study was to analyse the nested polymerase chain reaction (nested PCR) in human serum samples of patients with clinical manifestations of leptospirosis. The cases of leptospirosis were defined by the microagglutination test (MAT). The samples were collected in 2010. Of 1042 serum samples collected from 521 patients, 28 (5.4%) were considered positive cases of leptospirosis, and 493 (94.6%) were negative. Twenty-three confirmed cases had no MAT-detectable antibodies in the acute sample (mean of 5.6 days after onset). Nested PCR was positive in 22/23 (95.7%) patients during the acute phase of the disease, with negative results by MAT. Nested PCR was negative in all convalescent serum samples with positive results by MAT. All negative cases of leptospirosis were negative by nested PCR. The nested PCR is an alternative diagnostic tool for early detection of leptospires in sera during the first 7 days of the disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the microagglutination test (MAT) results in serum samples dried on filter paper and stored at different temperatures during 1day, 7days, 30days and 1year to determine the stability of sera antibody against leptospires. Serum samples collected onto filter paper for the detection of leptospires antibody was compared with MAT in a study of 300 serum samples from patients with suspected leptospirosis. Among 300 fresh serum samples analyzed by MAT 156 (52%) were positive and 144 (48%) negative. All the negative fresh serum samples were negative when dried on filter paper (specificity 100%). The sensitivity of MAT performed on dried serum samples was 100%. Storage on filter paper at room temperature and at 4°C for 1 and 7days did not affect the MAT titers. For up to 7days, 98.72% of dried serum samples had titers identical to those of the corresponding serum samples, and 1.18% of dried serum samples showed 1 dilution of difference. After a storage period of one month a prozone phenomenon was observed. After a storage period of one year all serum samples were negative. Serum samples collected onto filter paper are a convenient source of antibodies for serological diagnosis and epidemiological surveys., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Leptospirotic renal lesions frequently produce a polyuric form of acute kidney injury with a urinary concentration defect. Our study investigated a possible effect of the glycolipoprotein, (GLPc) extracted from L. interrogans, on vasopressin (Vp) action in the guinea pig inner medullary collecting duct (IMCD)., Methods: The osmotic water permeability (Pf µm/s) was measured by the microperfusion in vitro technique. AQP2 protein abundance was determined by Western Blot. Three groups were established for study as follows: Group I, IMCD from normal (ngp, n = 5) and from leptospirotic guinea-pigs (lgp-infected with L. interrogans serovar Copenhageni, GLPc, n = 5); Group II, IMCD from normal guinea-pigs in the presence of GLPc (GLPc group, n = 54); Group III, IMCD from injected animals with GLPc ip (n = 8)., Results: In Group I, PFS were: ngp--61.8±22.1 and lgp--8.8±12.4, p<0.01 and the urinary osmolalities were: lgp--735±64 mOsm/Kg and ngp--1,632±120 mOsm/Kg. The lgp BUN was higher (176±36 mg%) than the ngp (56±9 mg%). In Group II, the Pf was measured under GLPc (250 µg/ml) applied directly to the bath solution of the microperfused normal guinea-pig IMCDs. GLPc blocked Vp (200 pg/ml, n = 5) action, did not block cAMP (10(-4) M), and Forskolin (Fors--10(-9) M) action, but partially blocked Cholera Toxin (ChT--10(-9) M) action. GLP from L.biflexa serovar patoc (GLPp, non pathogenic, 250 µg) did not alter Vp action. In Group III, GLPc (250 µg) injected intraperitoneally produced a decrease of about 20% in IMCD Aquaporin 2 expression., Conclusion: The IMCD Pf decrease caused by GLP is evidence, at least in part, towards explaining the urinary concentrating incapacity observed in infected guinea-pigs.

A collection of 101 Leptospira isolates was tested by multilocus sequence typing (MLST) and by traditional serotyping. MLST divided the isolates into 4 sequence types (STs), while serotyping classified them into 6 serogroups. Two isolates failed to generate products for some genes by MLST. MLST was less discriminatory than serotyping for uncommonly occurring isolates from humans in Brazil.

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.

Aims: Development of a simple, specific, rapid and inexpensive Dot-ELISA test for early diagnosis of human leptospirosis., Methods and Results: Serum samples from 90 patients diagnosed with leptospirosis were analysed by Dot-ELISA test incorporating Glycolipoprotein (GLP) antigen from serovars Copenhageni and Patoc. Results were compared with those obtained with microscopic agglutination test, currently, the gold standard reference serological method. Serum samples from healthy blood bank donors and patients diagnosed with diseases other than leptospirosis were used as negative controls. The specificities of both GLP-based assays were 97.1% and 100% with serum samples from patients with other diseases and with serum samples from healthy control group, respectively. With serum samples from patients with acute leptospirosis, sensitivity was 76.6% with Dot-ELISA Copenhageni and 90.0% with Dot-ELISA Patoc. With serum samples from patients in convalescence, sensitivity was 100% with both GLP-based assays., Conclusions: This Dot-ELISA provides a candidate antigen for serodiagnosis of leptospirosis during all phases of illness and could be a good alternative method for the early diagnosis of leptospirosis., Significance and Impact of the Study: The Dot-ELISA test is simple, specific, rapid and inexpensive. It is suitable for identifying a large number of samples and, hence, reducing the death rate of patients with leptospirosis.

Aims: Leptospirosis is a public health problem worldwide. Traditionally, microscopic agglutination test (MAT) and cross-agglutinin absorption test (CAAT) are used to identify leptospires. However, these techniques are laborious and time-consuming, requiring the maintenance of a collection of more than 200 reference strains and correspondent rabbit antisera. The purpose of this study was to evaluate the pulsed-field gel electrophoresis (PFGE) method for discrimination of Leptospira serovars., Methods and Results: Fourteen clinical isolates of Leptospira spp. were analysed by MAT before being characterized by PFGE. The isolates were compared with a library of 206 different reference Leptospira serovars. All the isolates gave clear profiles with high resolution. PFGE and MAT results were in agreement for all clinical isolates evaluated. Twelve isolates were classified as serovar Icterohaemorrhagiae/Copenhageni by PFGE. By MAT, these isolates were classified as serogroup Icterohaemorrhagiae with titres ranging from 3200 to 25 600. Two isolates were classified as serovar Canicola by PFGE, and as serogroup Canicola by MAT with titres higher than 3200., Conclusions: PFGE offers the advantages of simple, reliable and reproducible results., Significance and Impact of the Study: PFGE provides a convenient tool for the identification of clinical isolates.

Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira. In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni(2+)-charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n = 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.

Using an indirect immunofluorescence method on human umbilical vein endothelial cells (HUVEC), we investigated the presence of antiendothelial cell antibodies (AECA) in 136 pre- and posttransplant serum samples sequentially collected from 31 patients during the first year after cardiac transplantation. A healthy control group was also included (n = 87). Colocalization studies demonstrated a positive staining pattern of different cytoskeletal components (cytoskeletal-antiendothelial cell antibodies, CSK-AECA) including antivimentin, antiactin, antitubulin, and anticytokeratin among heart transplanted patients. Frequency of CSK-AECA in the control group and at day 0 in the transplant group was 18.3 and 22.5%, respectively (p = NS). A progressive increase in the frequency of CSK-AECA was observed after cardiac transplantation: 13.3% at day 15; 22.2% at day 30; 53.8% at day 90, and 58% at day 360. Interestingly, rejection episodes within the first year after transplantation occurred in 83.3% of CSK-AECA-positive and in 30.7% of CSK-AECA-negative patients (p = 0.0045). The presence of antibodies was detected prior to the rejection event and was associated with a poor clinical outcome: rejection episodes occurred at a mean of 36.14 +/- 17 days after transplantation in patients with preexisting AECA and 174.25 +/- 51.9 days after de novo antibody appearance in patients with no antibodies at day 0 (p = 0.029). In conclusion, a progressive increase in the frequency of CSK-AECA was observed following cardiac transplantation; the presence of these antibodies is strongly associated and precedes the rejection episodes. Thus, CSK-AECA could be a good marker for acute graft rejection.

Immobilized enzyme derivatives, in organic media at neutral pH and moderate temperatures, should be mainly and perhaps uniquely inactivated by promotion of conformational changes on their 3D structure. Subsequent irreversible inactivation mechanisms (intermolecular aggregations, chemical modifications, thiol-disulfide exchanges) are thus impossible. However, simple reincubation in aqueous medium of enzymes previously inactivated by solvents usually yields significant but slow and incomplete reactivations. Disruption of incorrect protein structures by denaturing agents (urea, guanidine) is proposed as a new strategy to get rapid, complete and technologically feasible reactivations. By using multipoint immobilized chymotrypsin derivatives, we have evaluated the possibility of unfolding and further refolding of native (non-inactivated) derivatives by different denaturing conditions. After unfolding in 8 M guanidine, derivatives were quickly and completely refolded up to 100% of catalytic activity in 10 minutes. Besides, successive cycles of unfolding and refolding could be exactly reproduced. Finally we checked the possibility to reactivate chymotrypsin derivatives inactivated by dioxane. Simple reincubations in aqueous media yielded a poor reactivation even after 24 hours. However, unfolding in 8 M guanidine enabled complete reactivation in less than 2 hours. From this point of view, by working under 'chemically inert conditions' (moderate pH and temperatures), fully dispersed covalently immobilized enzyme derivatives seem to behave as almost everlasting catalysts despite the very deleterious effect of organic media.

Chymotrypsin linked to agarose beads by multi-point covalent attachment catalyzes synthesis of Ac-Trp-OEt in 3-pentanone even when the thermodynamic water activity (aw) of the system is reduced to as low as 0.4. If fully hydrated catalyst is added to the reaction mixture before removal of water, product is formed linearly once aw has stabilized. The initial rate is reduced from that if aw is kept close to 1 (0.47 mmol s-1 (kg enzyme)-1), to 50% (aw 0.9), 25% (aw 0.4) and < 1% (aw 0.25). The large drop between aw of 1 and 0.9 probably reflects the effects of water removal on the agarose gel structure. Catalyst partly dried (even only to aw 0.86) before adding to the organic phase is inactive. At reduced aw, the equilibrium (when reached) is shifted in favor of the ester, as expected.

The synthesis of N-acetyl tryptophan phenylethyl ester in organic media is catalyzed by suspended agarose beads with multipoint covalently attached chymotrypsin. A dilute aqueous phase is trapped within the gel beads and may be manipulated separately from the organic phase. The equilibrium position becomes more favorable as the solvent polarity decreases, with K(eq) increasing 38 times between 2-butanone and 1,1,1-trichloroethane. The more apolar solvents also give faster kinetics. Addition of cosolvents (up to 10% dimethylformamide or 20% acetonitrile) does not affect the rate but does substantially reduce the equilibrium yield, presumably also by making the organic phase more polar. With trichloroethane as solvent the enzyme appears to be kinetically saturated with 1M phenylethanol. Doubling this concentration also does not cause the expected increase in equilibrium conversion, probably again because K(eq) is reduced in the more polar organic phase. Increased temperature raises the reaction rate as expected but has little effect on the equilibrium., ((c) 1992 John Wiley & Sons, Inc.)

We have developed different activity/stability tests to evaluate the possibilities of fully dispersed chymotrypsin derivatives as industrial catalysts in biphasic systems. We have tested different immiscible organic solvents (log P ranged from 0.65 to 2.8) and used different enzyme derivatives (soluble chymotrypsin and one-point and multipoint covalent attached derivatives). Special emphasis has been given to the role of the "exact composition of the aqueous phase."High phosphate concentrations largely protect every hymotrypsin derivative from the distorting effects of dissolved solvent molecules. The effects on the activity and stability of soluble chymotrypsin due to saturating solvent concentrations in an aqueous solution, and the much more severe effects of contact with the phase interface in a stirred biphasic system, all show the opposite trend for the influence of solvent polarity to that generally observed for biocatalysts. For example, deleterious effects decline in the order chloroform, dichloromethane, ethyl acetate. On the contrary, with or without stirring, our stabilized chymotrypsin-agarose derivatives are much more stable against these water-immiscible solvents, and their relative effects follow the normal trend. From these integrated activity and stability tests we can conclude that fully dispersed immobilized-stabilized derivatives seem to be an interesting alternative to develop industrial biphasic processes catalyzed by chymotrypsin.

By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 M ammonium sulfate (KCS) or 90% organic cosolvents (TCS]. In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of 180 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst.

We have found that penicillin G sulfoxide (pen G SO) behaves as a general stabilizing agent of two bacterial penicillin G acylases (PGAs) from E. coli and from K. citrophila), and this role is related to a strong inhibitory effect on the enzymes. The stabilizing effect has been observed during two different inactivation processes: (i) thermal inactivation of soluble enzymes at alkaline pH, and (ii) inactivation of immobilized enzymes as a consequence of covalent multiinteraction with highly activated agarose aldehyde gels. At the same time, pen G SO behaves as a strong competitive inhibitor of these two enzymes. The inhibition constant is more than 10-fold lower than the one corresponding to another smaller competitive inhibitor, phenylacetic acid (PAA), the structure of which is exactly the acyl donor moiety corresponding to pen G SO. In turn, PAA hardly exerts any stabilizing effect on PGAs. The stabilizing effect of pen G SO allowed the preparation of derivatives of these PGAs preserving full catalytic activity in spite of being 1,400- and 650-fold more stable than the corresponding soluble or one-point attached immobilized enzymes.

Synthesis of dipeptides benzoyl Arginine leucinamide and kyotorphin catalyzed by highly stabilized derivatives of trypsin and chymotrypsin have been performed. Extreme experimental conditions could be tested and parameters of industrial interest could be improved provided the high activity and stability of the derivatives in these unfavourable environments. Thermodynamically controlled synthesis catalyzed by trypsin could be optimized and 97% conversion was obtained in 90% organic cosolvents. 100% yields were achieved in kinetically controlled synthesis catalyzed by trypsin in aqueous medium in the presence of IM Ammonium Sulphate. Higher starting concentrations of poorly soluble substrates of chymotrypsin could be used in a reaction medium containing 50% DMF and 95% yield were obtained.

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.