CAR T Cells Generated Using Sleeping Beauty Transposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor Activity In Vitro and In Vivo.

Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBζ CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBζ CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.

Índice triglicéridos/colesterol de alta densidad y perfil lipídico en adolescentes escolares de una región andina del Perú / Triglycerides/high density cholesterol ratio and lipid profile in school adolescents from an Andean region of Peru

Introducción. El exceso de peso es un fenómeno creciente en población adolescente, sin embargo, los estudios sobre el perfil lipídico en adolescentes peruanos son escasos. Objetivo. Identificar los patrones del índice triglicéridos/colesterol HDL y perfil lipídico en escolares sin síndrome metabólico residentes en zonas urbanas de una región andina del Perú. Métodos. Estudio de fuente secundaria, los datos provienen del estudio «Frecuencia de Síndrome Metabólico en residentes de una región andina del Perú¼. De 586 estudiantes del estudio original, fueron excluidos 19 por tener diagnóstico de síndrome metabólico. Se analizaron los niveles de glucosa, colesterol total, colesterol de alta densidad (HLD), colesterol de baja densidad (LDL) y triglicéridos. El colesterol no HDL fue calculado mediante la diferencia entre el colesterol total y HDL. El índice triglicérido/colesterol de alta densidad (Tg/HDL-C) es el cociente entre la concentración sérica de triglicéridos y colesterol HDL-C. Resultados. Analizamos el perfil lipídico de 567 adolescentes entre 11 y 16 años. La media del índice Tg/HDL-C fue de 2,9 (desviación estándar - DE: 2,35), la mediana fue de 2,3 con un intervalo intercuartil entre 1,62 y 3,51. El índice Tg/HDL-C ≥3 estuvo asociado con mayores niveles de colesterol no HDL, puntaje Z de IMC y perímetro de cintura. Conclusiones. La mitad de adolescentes escolares de una región andina del Perú tuvo un índice Tg/HDL-C entre 1,62 y 3,51. Un corte ≥3 en el índice Tg/HDL-C estuvo asociado a una mayor concentración de colesterol no HDL, puntaje Z de IMC, y perímetro de cintura, estos dos últimos son indicadores de exceso de peso.

Introduction. Excess weight is a growing phenomenon in the adolescent population, however, studies on the lipid profile in Peruvian adolescents are scarce. Objective. To identify the patterns of triglycerides/HDL cholesterol ratio and lipid profile in schoolchildren without metabolic syndrome in urban areas of an Andean region of Peru. Methods. Secondary analysis database from the study «Frequency of Metabolic Syndrome in residents of an Andean region of Peru¼. Of 586 students from the original study, 19 were excluded because they had a diagnosis of Metabolic Syndrome. The levels of glucose, total cholesterol, high density cholesterol (HLD), low density cholesterol (LDL) and triglycerides were analyzed. Non-HDL cholesterol was calculated by the difference between total cholesterol and HDL. The triglyceride / HDL cholesterol index (Tg / HDL-C) is the ratio between the serum concentration of triglycerides and HDL cholesterol. Results. We analyzed the lipid profile of 567 adolescents between 11 and 16 years old. The mean of the Tg/HDL-C ratio was 2.9 (standard deviation - SD: 2,35), the median was 2.3 with an interquartile range between 1,62 and 3,51. The Tg/HDL-C ratio ≥3 was associated with higher levels of non-HDL cholesterol, Z-score of BMI and waist circumference. Conclusions. Half of student adolescents in an Andean region of Peru had a Tg/HDL-C index between 1,62 and 3,51. A cut-point ≥3 in the Tg/HDL-C index was associated with a higher concentration of non HDL cholesterol, Z score of BMI, and waist circumference, these last two are indicators of excess weight.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated Leishmania major or Leishmania braziliensis skin lesions.

OBJECTIVES: This study was designed to verify the cytotoxic activity of S-nitrosoglutathione (GSNO) against intracellular Leishmania amastigotes and to test its efficacy as a topical treatment of localized cutaneous leishmaniasis (LCL) in Leishmania major- or Leishmania braziliensis-infected mice. METHODS: Cytotoxic activity of GSNO was verified in L. major-infected THP-1 macrophages. S-nitrosated proteins were detected by immunofluorescence. Topical treatment was done by daily application of a solution of GSNO in PBS to the skin ulcer of Leishmania-infected mice. BALB/c and interferon-γ-knockout (IFN-γ-KO) C57BL/6 mice were infected with L. major and L. braziliensis, respectively. Ulcer size was measured weekly and the parasite loads were determined in the lesion and lymph nodes. Controls received PBS topically or amphotericin B (AMB) intravenously. RESULTS: The number of intracellular L. major amastigotes was markedly reduced in GSNO-treated cultures; in these, staining for S-nitrosated proteins was present in the cytoplasm and colocalized with intracellular amastigotes. Topical treatment with GSNO of L. major ulcers in BALB/c mice suppressed lesion growth, reduced the parasite load and induced healing comparable to the effect of intravenously administered AMB. Topical GSNO treatment was also efficient at suppressing lesion growth in IFN-γ-KO mice infected with L. braziliensis. CONCLUSIONS: GSNO is cytotoxic to intracellular L. major amastigotes in vitro and had a healing effect on LCL caused by L. major and L. braziliensis in mice. These positive results on the topical therapeutic effect of GSNO in mouse leishmaniasis infections provide the experimental basis for a possible future trial in the treatment of human LCL.