Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column. Results: There were 12 hybridoma strains selected into the polyol-responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA. Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

Trypanosoma Cruzi Strains From Triatomine Collected In Bahia And Rio Grande Do Sul, Brazil.

Collection of triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Northeastern and Southern Brazil respectively, and isolation of Trypanosoma cruzi strains. First, the captured triatomines were identified using insect identification keys, then their intestinal content was examined by abdominal compression, and the samples containing trypanosomatid forms were inoculated in LIT medium and Swiss mice. Six triatomine species were collected in cities in Bahia, namely Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) and T. sordida (460), and two in cities in Rio Grande do Sul, namely T. circummaculata (11) and T. rubrovaria (115). Out of the specimens examined, T. cruzi was isolated from 28 triatomine divided into four different species: T. melanocephala (one), T. lenti (one), T. rubrovaria (16) and T. sordida (10). Their index of natural infection by T. cruzi was 6.4%. The isolation of T. cruzi strains from triatomines found in domestic and peridomestic areas shows the potential risk of transmission of Chagas disease in the studied cities. The maintenance of those T. cruzi strains in laboratory is intended to promote studies that facilitate the understanding of the parasite-vector-host relationship.

Guidelines On The Treatment Of Anemia Of Chronic Renal Failure Using Recombinant Human Erythropoietin: Associação Brasileira De Hematologia, Hemoterapia E Terapia Celular Guidelines Project: Associação Médica Brasileira - 2014.

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Enzymatic Biodiesel Synthesis From Yeast Oil Using Immobilized Recombinant Rhizopus Oryzae Lipase.

The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

Isolation of Pseudomonas aeruginosa strains from dental office environments and units in Barretos, state of São Paulo, Brazil, and analysis of their susceptibility to antimicrobial drugs

A wide variety of opportunistic pathogens has been detected in the tubing supplying water to odontological equipment, in special in the biofilm lining of these tubes. Among these pathogens, Pseudomonas aeruginosa, one of the leading causes of nosocomial infections, is frequently found in water lines supplying dental units. In the present work, 160 samples of water, and 200 fomite samples from forty dental units were collected in the city of Barretos, State of São Paulo, Brazil and evaluated between January and July, 2005. Seventy-six P. aeruginosa strains, isolated from the dental environment (5 strains) and water system (71 strains), were tested for susceptibility to six antimicrobial drugs most frequently used against P. aeruginosa infections. Susceptibility to ciprofloxacin, followed by meropenem was the predominant profile. The need for effective means of reducing the microbial burden within dental unit water lines is emphasized, and the risk of exposure and cross-infection in dental practice, in special when caused by opportunistic pathogens like P. aeruginosa, are highlighted.

Effects of nitrate and phosphate availabilities on growth, photosynthesis and pigment and protein contents in colour strains of Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. (Gigartinales, Rhodophyta)

In Brazil, Hypnea musciformis is the main raw material for carrageenan production and the knowledge of nitrogen and phosphorus metabolism in algae is critical for the success of cultivation because these elements can limit seaweed productivity. Thus, the objective of this study was to evaluate the effects of nitrate (zero to 100 μM) and nitrate plus phosphate (zero to 25 μM) availabilities on the growth, the contents of photosynthetic pigments (phycobiliproteins and chlorophyll a) and proteins, and the photosynthesis and respiration of the brown (BR) and light green (LG) strains of H. musciformis. The results revealed metabolic differences between the colour strains of H. musciformis for nitrogen metabolism: upon nitrate addition, the LG strain stored nitrogen mainly as proteins, while the BR strain stored it as proteins and pigments. Moreover, the respiration of the LG strain and the photosynthesis of the BR strain increased with nitrate concentrations, indicating that the BR strain fixed more photosynthetic carbon than the LG strain.

Evaluation of a recombinant p24 antigen for the detection of Feline Immunodeficiency Virus-specific antibodies

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

Clonal relationship among atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Antifungal activity of bacterial strains from the rhizosphere of Stachytarpheta crassifolia

This study evaluated the antifungal action of biomolecules produced from the secondary metabolism of bacterial strains found in the rhizosphere of semi arid plants against human pathogenic Candida albicans. Crude extracts were obtained using ethyl acetate as an organic solvent and the bioactivity was assessed with a bioautography technique. The results showed that bacterial strains, Alcaligenes faecalis MRbS12 and Bacillus cereus MRbS26, had compounds with antifungal bioactivity. The largest inhibition zones for both compounds were located on spots with Rf values below 0.500, indicating that the molecules possibly had polar characteristics. These results suggested that microorganisms found in the environment could have bioprospecting potential.

Immunomodulatory effects of recombinant BCG expressing pertussis toxin on TNF-alpha and IL-10 in a bladder cancer model

Background: Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a rBCG-S1PT strain that induced a stronger cellular immune response than BCG. This preclinical study was designed to compare the modulatory effects of BCG and rBCG-S1PT on bladder TNF-alpha and IL-10 expression and to evaluate antitumour activity. Methods: For Experiment I, the MB49 bladder cancer cell line was used in C57BL/6 mice. Chemical cauterization of the bladder was performed to promote intravesical tumor implantation. Mice were treated by intravesical instillation with BCG, rBCG-S1PT or PBS once a week for four weeks. After 35 days the bladders were removed and weighed. TNF-

High Prevalence of Side Effects After Recombinant Human Thyrotropin-Stimulated Radioiodine Treatment with 30 mCi in Patients with Multinodular Goiter and Subclinical/Clinical Hyperthyroidism

Background: Treatment of multinodular goiters (MNGs) is highly controversial. Radioiodine (RAI) therapy is a nonsurgical alternative for the elderly who decline surgery. Recently, recombinant human thyrotropin (rhTSH) has been used to augment RAI uptake and distribution. In this study, we determined the outcome of 30 mCi RAI preceded by rhTSH (0.1 mg) in euthyroid (EU) and hyperthyroid (subclinical/clinical) patients with large MNGs. Methods: This was a prospective cohort study. Forty-two patients (age, 43-80 years) with MNGs were treated with 30 mCi RAI after stimulation with 0.1 mg of rhTSH. Patients were divided into three groups, according to thyroid function: EU (n = 18), subclinically hyperthyroid (SC-H, n = 18), and clinically hyperthyroid (C-H, n = 6). All patients underwent a 90-day low-iodine diet before treatment, and those with clinical hyperthyroidism received methimazole 10 mg daily for 30 days. Serum TSH, free thyroxine (FT4), total triiodothyronine (TT3), and thyroglobulin were measured at baseline and at 24, 48, 72, 168 hours, and 1, 3, 6, 9, 12, 18, 24, and 36 months after therapy. Thyroid volume was assessed by computed tomography at baseline and every 6 months. Results: Patients had high iodine urinary excretion (308 +/- 108 mu g I/L) at baseline. TSH levels at baseline were within the normal range (1.5 +/- 0.7 mu U/mL) in the EU group and suppressed (< 0.3 mu U/mL) in the SC-H and C-H groups. After rhTSH, serum TSH peaked at 24 hours reaching 12.4 +/- 5.85 mu U/mL. After RAI administration, patients in both hyperthyroid groups had a higher increase in FT4 and TT3 compared with those in the EU group (p < 0.001). Thyroglobulin levels increased equally in all three groups until day 7. Thyroid volume decreased significantly in all patients. Side effects were more common in the SC-H and C-H groups (31.4% and 60.4%, respectively) compared with EU patients (17.8%). Permanent hypothyroidism was more prevalent in the EU group (50%) compared with the SC-H (11%) and C-H (16.6%) groups. Conclusions: Patients with MNG may have subclinical and clinical nonautoimmune iodine-induced hyperthyroidism. Despite a low-iodine diet and therapy with methimazole, hyperthyroid patients have a significantly higher increase in FT4 and TT3 levels after RAI ablation. This can lead to important side effects related mostly to the cardiac system. We strongly advise that patients with SC-H and C-H be adequately treated with methimazole and low-iodine diet aiming to normalize their hyperthyroid condition before rhTSH-stimulated treatment with RAI.

Near full-length genome analysis of low prevalent human immunodeficiency virus type 1 subclade F1 in Sao Paulo, Brazil

Background: The genetic diversity of the human immunodeficiency virus type 1 (HIV-1) is critical to lay the groundwork for the design of successful drugs or vaccine. In this study we aimed to characterize and define the molecular prevalence of HIV-1 subclade F1 currently circulating in Sao Paulo, Brazil. Methods: A total of 36 samples were selected from 888 adult patients residing in Sao Paulo who had previously been diagnosed in two independent studies in our laboratory as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA was amplified from the purified genomic DNA of all 36 blood samples by 5 fragments overlapping PCR followed by direct sequencing. Sequence data were obtained from the 5 fragments of pure subclade F1 and phylogenetic trees were constructed and compared with previously published sequences. Subclades F1 that exhibited mosaic structure with other subtypes were omitted from any further analysis Results: Our methods of fragment amplification and sequencing confirmed that only 5 sequences inferred from pol region as subclade F1 also holds true for the genome as a whole and, thus, estimated the true prevalence at 0.56%. The results also showed a single phylogenetic cluster of the Brazilian subclade F1 along with non-Brazilian South American isolates in both subgenomic and the full-length genomes analysis with an overall intrasubtype nucleotide divergence of 6.9%. The nucleotide differences within the South American and Central African F1 strains, in the C2-C3 env, were 8.5% and 12.3%, respectively. Conclusion: All together, our findings showed a surprisingly low prevalence rate of subclade F1 in Brazil and suggest that these isolates originated in Central Africa and subsequently introduced to South America.

Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

Background: Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods: In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results: The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions: Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.

Allopatric speciation in ticks: genetic and reproductive divergence between geographic strains of Rhipicephalus (Boophilus) microplus

Background: The cattle tick, Rhipicephalus (Boophilus) microplus, economically impact cattle industry in tropical and subtropical regions of the world. The morphological and genetic differences among R. microplus strains have been documented in the literature, suggesting that biogeographical and ecological separation may have resulted in boophilid ticks from America/Africa and those from Australia being different species. To test the hypothesis of the presence of different boophilid species, herein we performed a series of experiments to characterize the reproductive performance of crosses between R. microplus from Australia, Africa and America and the genetic diversity of strains from Australia, Asia, Africa and America. Results: The results showed that the crosses between Australian and Argentinean or Mozambican strains of boophilid ticks are infertile while crosses between Argentinean and Mozambican strains are fertile. These results showed that tick strains from Africa (Mozambique) and America (Argentina) are the same species, while ticks from Australia may actually represent a separate species. The genetic analysis of mitochondrial 12S and 16S rDNA and microsatellite loci were not conclusive when taken separately, but provided evidence that Australian tick strains were genetically different from Asian, African and American strains. Conclusion: The results reported herein support the hypothesis that at least two different species share the name R. microplus. These species could be redefined as R. microplus (Canestrini, 1887) (for American and African strains) and probably the old R. australis Fuller, 1899 (for Australian strains), which needs to be redescribed. However, experiments with a larger number of tick strains from different geographic locations are needed to corroborate these results.