Therapeutical effects of vaccine from Trypanosoma cruzi amastigote surface protein 2 by simultaneous inoculation with live parasites

The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 – Control Group (CTRL); Group 2 – Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 – Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 ⁸ plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 ⁸ pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection.     

Oxidative stress parameters in blood, liver, and kidney of diabetic rats treated with curcumin and/or insulin

This study evaluated the effects of curcumin and/or insulin on antioxidant enzyme activity in blood, liver, and kidney, as well as on lipid peroxidation and delta aminolevulinic dehydratase (δ-ALA-D) activity, and a histopathological analysis of streptozotocin-induced diabetic rats. The animals were divided into six groups (n = 6): control/saline (C); control/curcumin (CCur); diabetic/saline (D); diabetic/insulin (DIns); diabetic/curcumin (DCur); and diabetic/insulin/curcumin (DInsCur). After 30 days of treatment with curcumin and/or insulin, the animals were sacrificed and the liver, kidney, and serum were used for experimental determinations. Results of histopathological analysis showed that the treatment with insulin ameliorate renal and hepatic lesions from both DIns and DInsCur groups. TBARS levels were significantly increased in serum, liver, and kidney in D group and the administration of curcumin and insulin prevented this increase in DIns and DCur groups. The activities of catalase (CAT), superoxide dismutase, and δ-ALA-D presented a significant decrease in the liver and kidney D group when compared to C group (P < 0.05). The animals treated with curcumin and insulin presented an increase of CAT activity, revealing a positive interaction between both substances. The treatments with curcumin or insulin prevented oxidative stress in blood, through modulation of enzymatic antioxidant defenses. These findings contributed to the comprehension that antioxidants from medicinal plants could be used as adjuvant in the treatment of this endocrinopathy and not as single therapy.     

Mucopolysaccharidosis I, II, and VI: Brief review and guidelines for treatment

Mucopolysaccharidoses (MPS) are rare genetic diseases caused by the deficiency of one of the lysosomal enzymes involved in the glycosaminoglycan (GAG) breakdown pathway. This metabolic block leads to the accumulation of GAG in various organs and tissues of the affected patients, resulting in a multisystemic clinical picture, sometimes including cognitive impairment. Until the beginning of the XXI century, treatment was mainly supportive. Bone marrow transplantation improved the natural course of the disease in some types of MPS, but the morbidity and mortality restricted its use to selected cases. The identification of the genes involved, the new molecular biology tools and the availability of animal models made it possible to develop specific enzyme replacement therapies (ERT) for these diseases. At present, a great number of Brazilian medical centers from all regions of the country have experience with ERT for MPS I, II, and VI, acquired not only through patient treatment but also in clinical trials. Taking the three types of MPS together, over 200 patients have been treated with ERT in our country. This document summarizes the experience of the professionals involved, along with the data available in the international literature, bringing together and harmonizing the information available on the management of these severe and progressive diseases, thus disclosing new prospects for Brazilian patients affected by these conditions.     

Nitric oxide inhibits neutrophil migration by a mechanism dependent on ICAM-1: role of soluble guanylate cyclase.

In the present study, we addressed the role of intercellular adhesion molecule type 1 (ICAM-1/CD54) in neutrophil migration to inflammatory site and whether the inhibitory effect of nitric oxide (NO) upon the neutrophil rolling, adhesion and migration involves down-modulation of ICAM-1 expression through a cyclic GMP (cGMP) dependent mechanism. It was observed that neutrophil migration induced by intraperitoneal administration of endotoxin (LPS), carrageenan (Cg) or N-formyl peptide (fMLP) in ICAM-1 deficient (ICAM-1-/-) is similar to that observed in wild type (WT) mice. The treatment of mice with NO synthase (NOS) inhibitors, NG-nitro-l-arginine, aminoguanidine or with a soluble guanylate cyclase (sGC) inhibitor, ODQ enhanced LPS- or Cg-induced neutrophil migration, rolling and adhesion on venular endothelium. These parameters induced by LPS were also enhanced by 1400 W, a specific iNOS inhibitor, treatment. On the other hand, the treatment of the mice with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, reduced these parameters induced by LPS or Cg by a mechanism sensitive to ODQ pretreatment. The NOS inhibitors did not enhance LPS-, Cg- or fMLP-induced migration and adhesion in ICAM-1-/- mice. Moreover, genetic (iNOS-/- mice) or pharmacological inhibition of NOS or of sGC enhanced LPS-induced ICAM-1 expression on mesenteric microcirculation vessels of WT mice. By contrast, SNAP reduced the ICAM-1 expression by a mechanism dependent on cGMP. In conclusion, the results suggest that although during inflammation, ICAM-1 does not contribute to neutrophil migration, it is necessary for the down-modulatory effect of inflammation-released NO on the adhesion and transmigration of neutrophils. Moreover, these NO effects are mediated via cGMP.     

An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits.

Aims

We herein report a convenient and affordable method based on in house made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes.

Results

This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41–59%), the hard to transfect murine T cells (mean 38%, 36–42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as in vitro expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis.

Conclusions

We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.     

Frog Virus 3 Open Reading Frame 97R Localizes to the Endoplasmic Reticulum and Induces Nuclear Invaginations

Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.