Monitoring Leishmania infantum Infections in Female Lutzomyia longipalpis by Using DNA Extraction on Cation Exchange Paper and PCR Pool Testing

Visceral leishmaniasis remains a serious public health issue, and Brazil was among the seven countries with the highest prevalence of this disease worldwide. The measures to control this disease are not easily developed, and the improvement of its diagnosis, surveillance, and control is still needed. This study aimed to carry out the polymerase chain reaction (PCR) diagnosis of Leishmania infantum in vector samples in some municipalities of the State of São Paulo, which included two municipalities with human disease transmission and two with dog transmission only. Vectors were collected in traps with luminous bait. Next, they were killed at -4 °C and kept in 70% alcohol. Groups of ten female insects (pools) were mashed on cation exchange paper (fine cellulose phosphate with 18 µEq/cm² ionic exchange capacity) for DNA extraction. The PCR was carried out to identify the natural infection of the Leishmania genus in female Lutzomyia longipalpis (Lu. Longipalpis). Out of the 3,880 Lu. longipalpis phlebotomines, 1060 were female and 2820 were male (3:1). The method used to extract the DNA in pools of ten phlebotomines and the PCR resulted in sensitivity, specificity, practicality, and faster analyses when compared to the individual analysis method. The procedure described can be used on a large scale in the leishmaniasis epidemiological surveillance, enabling a higher number of analyses and the optimization of human resources because the traditional diagnostic method is carried out via desiccation of the insect digestive system and microscopic examination, which is time-demanding and there is the need of manual skills.

Endocytosis of tight junctions caveolin nitrosylation dependent is improved by cocoa via opioid receptor on RPE cells in diabetic conditions.

PURPOSE: Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. METHODS: Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). RESULTS: Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. CONCLUSIONS: The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

The contribution of hypertension to diabetic nephropathy and retinopathy: the role of inflammation and oxidative stress.

Diabetes and hypertension frequently coexist and constitute the most notorious combination for the pathogenesis of diabetic nephropathy and retinopathy. Large clinical trials have clearly demonstrated that tight control of glycemia and/or blood pressure significantly reduces the incidence and progression of diabetic retinopathy (DR) and nephropathy. However, the mechanism by which hypertension interacts with diabetes to induce and/or exacerbate nephropathy and retinopathy is very unclear. Substantial evidence implicates the involvement of chronic inflammation and oxidative stress in the pathogenesis of DR and nephropathy. In addition, hypertension causes oxidative stress and inflammation in the kidney and retina. In the present review, we summarized data obtained from our research along with those from other groups to better understand the role of hypertension in the pathogenesis of diabetic nephropathy and retinopathy. It is suggested that oxidative stress and inflammation may be common denominators of kidney and retinal damage in the concomitant presence of diabetes and hypertension.

A decrease in retinal progenitor cells is associated with early features of diabetic retinopathy in a model that combines diabetes and hypertension.

PURPOSE: Hyperglycemia and hypertension contribute to the development of diabetic retinopathy, and this may involve alterations in the normal retinal cell cycle. In this work, we examined the influence of diabetes and hypertension on retinal cell replication in vivo and the relationship between these changes and several early markers of diabetic retinopathy. METHODS: Diabetes was induced with streptozotocin in 4- and 12-week-old spontaneously hypertensive rats (SHR) and their Wistar Kyoto (WKY) controls. The rats were killed 15 days later. Retinal cells stained with bromodeoxyuridine (BrdU) were seen in rats of both ages. RESULTS: In 12-week-old rats, the number of BrdU-positive retinal cells was higher in SHR than in WKY rats. After 15 days of diabetes mellitus, there was a marked reduction in cell replication only in diabetic SHR (p=0.007). The BrdU-positive cells expressed neural, glial, or vascular progenitor markers. There was greater expression of p27(Kip1) in the ganglion cell layer of both diabetic groups (p=0.05), whereas in the inner nuclear layer there was enhanced expression only in diabetic SHR (p=0.02). There was a marked increase in the retinal expression of fibronectin (p=0.04) and vascular endothelial growth factor (p=0.02) in diabetic SHR that was accompanied by blood-retinal barrier breakdown (p=0.01). DISCUSSION: Concomitant diabetes and hypertension attenuated the proliferation of retinal cells, and it is associated with an increase in p27(Kip1) expression, fibronectin accumulation, and blood-retinal barrier breakdown. The replicative retinal cells displayed characteristics of progenitor cells.