Immunogenicity and potential protection of DNA vaccine of Leishmania martiniquensis against Leishmania infection in mice.

INTRODUCTION: In Thailand, Leishmania martiniquensis is the predominant species causing cutaneous and visceral leishmaniasis. Its incidence has been increasing among immunocompetent and immunocompromised hosts. We developed a prototype DNA vaccine using a partial consensus sequence of the cysteine protease B (cpb) gene derived from L. martiniquensis from Thai patients. METHODOLOGY: The laboratory inbred strain of albino BALB/c mice were immunized intramuscularly three times at 2-week intervals (weeks 0, 2, and 4) with cpb plasmid DNA (pcDNA_cpb) with or without the adjuvant, monoolein (pcDNA_cpb-MO). Mice were challenged at week 6 with L. martiniquensis promastigotes. Sera were analysed for IgG1, IgG2a, interferon gamma and interleukin 10 (IFN-γ and IL-10, respectively) levels at weeks 0, 4, and 9. Additionally, livers and spleens were also analysed for parasite burden using immunohistochemistry and real-time polymerase chain (qPCR) assays. RESULTS: Three weeks after promastigote challenge, vaccinated mice showed significantly increased levels of IgG2a and IFN-γ while IL-10 level was significantly reduced when compared with those in the control group (p < 0.01). Parasite burden in the livers and spleens of vaccinated mice significantly decreased. In addition, a significant increase in mature granuloma formation in the livers when compared with those of the control group (p < 0.05) was found, indicating increased T-helper cells (Th1)-induced inflammation and destruction of amastigotes. Monoolein produced a booster effect to enhance the mouse Th1 protective immunity. CONCLUSIONS: The prototype DNA vaccine could induce a Th1 immune response that conferred potential protection to the L. martiniquensis promastigote challenge in BALB/c mice.

Microhemodynamic indices to evaluate the effectiveness of herbal medicine in diabetes: A comparison between alpha-mangostin and curcumin in the retina of type 2 diabetic rats.

OBJECTIVE: This study was aimed to develop microhemodynamic indices to evaluate the effectiveness of herbal medicine in diabetic tissues. METHODS: Male Sprague-Dawley rats were divided into four groups: normal control rats (Control), type 2 diabetic rats without (DM2) and with supplementation of alpha mangostin (DM2-MG) or curcumin (DM2-CUR). Alpha-mangostin or curcumin (200 mg/kg BW) were fed followed by i.p. injection of streptozotocin (STZ). Mean arterial pressure (MAP) and retinal blood flow (RBF) were measured and retinal flow resistance (RFR) was calculated. Three indices were developed to evaluate the effectiveness of herbal medicines in RFR-MAP diagram based on experimental data of MAP and RFR in type 2 diabetic rats. These indices are α, ß, and γ where α is a ratio of reduction in MAP, ß is a ratio of reduction in RFR increasing with MAP increase, and γ indicates a ratio of reduction in RFR. RESULTS: The elevated MAP and RFR and decreased RBF were observed in DM2 rats.Interestingly, alpha-mangostin or curcumin supplementation significantly increased RBF while decreased MAP and RFR. Using α, ß and γ indices, it was found that alpha-mangostin is more effective than curcumin in type 2 diabetic retina. CONCLUSIONS: These microhemodynamic indices may be useful to compare various herbal medicines in different tissues.

Alpha-mangostin attenuation of hyperglycemia-induced ocular hypoperfusion and blood retinal barrier leakage in the early stage of type 2 diabetes rats.

The present study examined effects of alpha-mangostin (α-MG) supplementation on the retinal microvasculature, including ocular blood flow (OBF) and blood-retinal barrier (BRB) permeability in a type 2 diabetic animal model. Male Sprague-Dawley rats were divided into four groups: normal control and diabetes with or without α-MG supplementation. Alpha-mangostin (200 mg/Kg/day) was administered by gavage feeding for 8 weeks. The effects of α-MG on biochemical and physiological parameters including mean arterial pressure (MAP), OBF, and BRB leakage were investigated. Additionally, levels of retinal malondialdehyde (MDA), advance glycation end products (AGEs), receptor of advance glycation end products (RAGE), tumour necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF) were evaluated. The elevated blood glucose, HbA1c, cholesterol, triglyceride, serum insulin, and HOMA-IR were observed in DM2 rats. Moreover, DM2 rats had significantly decreased OBF but statistically increased MAP and leakage of the BRB. The α-MG-treated DM2 rats showed significantly lower levels of retinal MDA, AGEs, RAGE, TNF-α, and VEGF than the untreated group. Interestingly, α-MG supplementation significantly increased OBF while it decreased MAP and leakage of BRB. In conclusion, α-MG supplementation could restore OBF and improve the BRB integrity, indicating its properties closely associated with antihyperglycemic, antioxidant, anti-inflammatory, and antiglycation activities.

Effects of Long-Term Alpha-mangostin Supplementation on Hyperglycemia and Insulin Resistance in Type 2 Diabetic Rats Induced by High Fat Diet and Low Dose Streptozotocin.

OBJECTIVE: The present study investigated the effects of long-term supplementation of alpha-mangostin (MG; a xanthone isolated from mangosteen fruit) on hyperglycemia, and insulin resistance in type 2 diabetic rats. MATERIAL AND METHOD: Type 2 diabetes (DM2) was induced in male Sprague-Dawley rats by feeding high fat diet for three weeks followed by an IP injection of low dose streptozotocin. The rats were divided into four groups: control and diabetes without or with alpha-MG supplementation (CON, DM2, CON-MG and DM2-MG group, respectively). Alpha-MG was administered by gavage feeding in the amount of 200 mg/kg BW/day for 8 or 40 weeks. Fasting blood glucose, plasma HbA1c, cholesterol, and triglyceride were determined in all groups of rats. Serum insulin, calculated HOMA-IR and Oral glucose tolerance test were also carried out. RESULTS: The results showed that both 8 and 40 weeks DM2 groups had a significant increase in fasting blood glucose, HbA1c, plasma cholesterol and triglyceride compared with their aged-match control groups. Furthermore, the serum insulin and HOMA-IR were significantly elevated in 8 weeks DM2 whereas these two parameters were significantly decreased in 40 weeks DM2 group compared with their aged-match CON groups (p < 0.001). The OGTT showed impaired glucose tolerance in DM2 groups. Interestingly, alpha-MG supplemented DM2-MG group had significantly decreased levels of fasting blood glucose, HbA1c, plasma cholesterol, triglyceride when compared with the untreated DM2 groups. Supplementation of alpha-MG for 40 weeks in DM2-MG group showed significantly increase serum insulin levels compared with that of DM2 group (p < 0.001). Moreover alpha-MG supplemented DM-MG group demonstrated a better glucose tolerance pattern which was different from that of DM2 group at both 8 weeks and 40 weeks experimental periods. CONCLUSION: Long-term alpha-mangostin supplementation has anti-hyperglycemic, anti-hyperlipidemic effects and increase insulin sensitivity by improving beta-cell functions in type 2 diabetes mellitus.

The correlation of parathyroid hormone and heart rate variability in CAPD patients.

OBJECTIVE: To investigate the correlation of parathyroid hormone (PTH) and cardiac autonomic nervous system (ANS) measured by heart rate variability (HRV) method in continuous ambulatory peritoneal dialysis (CAPD) patients. MATERIAL AND METHOD: Healthy subjects (HS) and two groups of CAPD patients classified by the PTH concentration: high PTH group (H-PTH; PTH = 150-300 pg/ml) and ultra-high PTH group (UH-PTH; PTH > 300 pg/ml) were studied. Time and frequency domains of HRV were analyzed. For the frequency domain, the fast Fourier transform of the total power (TP), low frequency (LF), high frequency (HF), and LF/HF ratio were transformed by natural logarithm (ln). The Pearson's correlation was used to analyze the correlation between lnPTH and the parameters of HRV RESULTS: Time and frequency domains of HS were at highest values whilst LF/HF ratio was the lowest. For UH-PTH CAPD patients, the values of standard deviation of R-R interval (SDNN), root mean square of the difference of R-R interval (RMSSD), lnTP and lnHF were significantly lower whereas lnLF was not significantly different compared to H-PTH. In addition, lnHF was found to have the highest negative correlation value with lnPTH concentration (r = -0.53). CONCLUSION: PTH, a serious uremic toxin, influences the initiation of ANS dysfunction. According to decreased lnHF a decrease in parasympathetic activity was demonstrated in UH-PTH. Consequently, the modality that can stimulate the parasympathetic activity should be considered in CAPD patients who were hyperparathyroidism.

Long-term effects of oral vitamin C supplementation on the endothelial dysfunction in the iris microvessels of diabetic rats.

The current study was aimed to investigate effects of long-term supplementation of vitamin C on the iris microcirculation in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar-Furth rats by intravenous injection of STZ (55 mg/kg b.w.). The rats were divided into three groups: control rats (CON), STZ-induced diabetic rats (STZ), and STZ rats supplemented with vitamin C (STZ-vitC). For supplementation of vitamin C, ascorbic acid (1 g/l) was added into the drinking water. The experiments were performed at different periods (8, 12, 24 and 36 weeks) after injection of STZ. Blood glucose, tissue lipid peroxidation and plasma vitamin C were measured. To examine the endothelial function, leukocyte adhesion to the venular endothelium was evaluated in the iris post-capillaries by means of counting the number of leukocytes labeled with rhodamine 6G. Blood flow perfusion in the iris was monitored using a laser Doppler flow meter. In the STZ rats, hyperglycemia was induced with an increase in HbA(1c) and lipid peroxidation but with a decrease in the plasma vitamin C level which improved by vitamin C supplementation. The number of adherent leukocytes increased significantly, associated with reduction in the iris blood flow perfusion, at 8, 12, 24 and 36 weeks after injection of STZ. In the STZ-vitC rats, the iris blood flow perfusion was significantly increased in comparison with the STZ rats, while the leukocyte adhesion was decreased at 24 and 36 weeks. The statistical analysis shows that the leukocyte adhesion decreased with increase in the iris blood flow perfusion in STZ and STZ-vitC rats. In conclusion, vitamin supplementation suppressed leukocyte adhesion and thus endothelial dysfunction, associated with increase in iris blood flow perfusion in diabetes. The antioxidant vitamin C may be a therapeutic agent for preventing diabetic retinopathy.

Combined effects of curcumin and vitamin C to protect endothelial dysfunction in the iris tissue of STZ-induced diabetic rats.

This study was aimed to evaluate the combined effect of curcumin with vitamin C supplementation on hyperglycemic and dyslipidemia conditions and endothelial cell dysfunction induced in diabetic rats. Wistar Furth rats were used and divided into four groups: control (single injection of 0.9% sterile saline), STZ (streptozotocin, Sigma, 55 mg/kg.BW, i.v.), STZ-vitC (1 g/l ascorbic acid mixed in drinking water), STZ-cur (daily oral treatment of 300 mg/kg.BW curcumin; Cayman Chemical Co., USA), and STZ-cur+vitC (1 g/l ascorbic acid mixed in drinking water and oral treatment of 300 mg/kg.BW curcumin). On 8th week after STZ-injection, the microcirculation in the iris tissue was observed using intravital fluorescence videomicroscopy, and also leukocyte adhesion in the venule was examined for each group. Blood glucose (BG), lipid profiles, glycosylated hemoglobin (HbA1c) were measured in blood samples collected at the end of each experiment. The contents of liver malondialdehyde (MDA) were also quantified for each group. Feeding curcumin (STZ-cur) could decrease BG, HbA1c, dyslipidemia, and MDA significantly, compared to STZ. In cases of feedings curcumin with vitamin C, these results were more effective in all aspects, including leukocyte adhesion. In conclusion, curcumin might increase the effect of vitamin C in protecting the function of endothelial cells through its anti-oxidant with hypoglycemic and hypolipidemic actions.

Effects of Yahom on the regional cerebral blood flow in rat using fluorescence videomicroscopy.

This study was conducted to investigate effects of Yahom on the regional cerebral blood flow (rCBF) in rats using fluorescence videomicroscopy. Male Wistar Furth rats weighing 200-250 g were used, and divided into three groups: experimental I, II and control groups. The experimental I and II groups received single oral administration of Yahom 2 and 4 g/kg.bw, and topical application of Yahom on the pial cerebral cortex, respectively, while the control group received oral administration of 1 ml of 5% Tween. The rCBF was monitored using laser Doppler flowmetry at different periods (5-120 minutes) after the administration of Yahom or Tween. The mean arterial pressure (MAP) was measured through a femoral artery. The cerebral microcirculation was observed and recorded under an intravital fluorescence videomicroscopic system. The arteriolar diameter was measured based on the recorded videomicroscopic images. The MAP and rCBF increased after the oral administration of Yahom, while they did not alter in the control group. The maximum responses of MAP and rCBF were approximately 16% and 33% at 45 min and 30 min after the administration of Yahom 4 g/kg.bw, respectively. The topical application of Yahom induced vasodilation in the pial microvessels. In conclusion, single oral administration of Yahom may increase the regional cerebral blood flow under the effect of cerebral microvascular vasodilation.

Chronic changes of the iris microvasculature of streptozotocin-induced diabetic rats using fluorescence videomicroscopy.

This study was aimed to investigate chronic changes of the iris microvasculature in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar-Furth rats by intravenous injection of STZ (55 mg/kg.bw). The rats were divided into control (CON) and diabetic (STZ) groups. The experiments were performed at 8, 12, 24 and 36 weeks after the injection of STZ. The iris microcirculation was visualized under a fluorescence videomicroscope. Intraluminal diameters of microvessels were measured based on the FITC-dextran images. Leukocyte adhesion to the microvascular endothelium was evaluated by counting leukocytes (labeled with rhodamine 6G). The iris blood perfusion was measured using laser Doppler flowmetry. Tissue lipid peroxidation of the eye was evaluated. The results demonstrated that the lipid peroxidation increased significantly after the injection of STZ. Both the diameters of arterioles (or precapillaries) and the iris blood perfusion decreased significantly in STZ rats, compared to the control levels. Adherent leukocytes increased significantly at 8, 12, 24 and 36 week after the injection of STZ, compared with the control levels. This indicates that the increased in oxygen-derived free radicals may be a major contributor for iris vascular endothelial dysfunction in diabetes mellitus, including leukocyte adhesion and reducing the arteriolar diameter. The present model may be useful for assessing long-term effects of therapeutic agents on diabetic retinopathy.

Cerebral endothelial dysfunction in diabetes: intravital microscopic analysis using streptozotocin-induced diabetic rats.

Endothelial function of cerebral microvessel in diabetes was evaluated using streptozotocin (STZ)-induced diabetic rats (blood glucose of >/=300 mg/dl). At 36 weeks after STZ injection, the rats were anesthetized with sodium pentobarbital. The cerebral microcirculation in control and STZ groups was directly observed, using intravital fluorescence videomicroscopy. To evaluate the endothelial function in vivo, the number of leukocytes adhering to postcapillary venules were counted, and cerebral arteriolar responses to acetylcholine (ACh) and nitroglycerine (NTG) were examined. The results showed that the leukocyte adhesion to cerebral postcapillary venular endothelium increased significantly in STZ-rats, compared with control rats. The vasodilatory responses of cerebral arterioles (20-30 microm) to ACh decreased significantly in STZ-rats, compared with control rats (p<0.01), but the responses to NTG did not alter in diabetes. These results indicate that the impaired responses should occur on the endothelial cell. In conclusion, endothelial dysfunction induced in diabetes are characterized by impaired endothelium-dependent vasodilation and increased leukocyte adhesion to endothelial cells.

Maturity of pericytes in cerebral neocapillaries induced by growth factors: fluorescence immuno-histochemical analysis using confocal laser microscopy.

The maturity of pericytes in cerebral neocapillaries induced by two different growth factors: basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), was examined using an immunohistochemical staining technique. Cerebral angiogenesis was induced in mice by implanting a sandwich system of bFGF/PDGF gel and nylon-mesh over the exposed cortex. On 28th day after incubation, a small volume of cerebral tissue with the nylon-mesh was isolated and stained using tetramethyl rhodamine isothiocyanate (TRITC)-labeled secondary antibody to the primary antibody against NG_2 proteoglycan and fluorescein isothiocyanate (FITC)-labeled Griffonia simplicifolia (GS)-lectin. Using a confocal laser microscopic system, we observed the cerebral neocapillaries on the upper surface of the nylon-mesh and evaluated the maturity of pericytes stained with NG_2 based on the fluorescence immunohistological images. The pericyte appeared rich in neocapillaries induced by PDGF. It was suggested that pericytes might play a key role in the regulation of blood flow in neovessels.

The effect of long-term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium in STZ-induced diabetic rats.

The effect of long-term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium was investigated in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar Furth rats by intravenous injection of STZ. The vitamin C, ascorbic acid, was supplemented with drinking water (1 g/l). The rats were divided into control and diabetic groups without or with supplementation of vitamin C. The cerebral microcirculation was directly observed through a cranial window after different periods (12, 24 and 36 weeks) of vitamin C supplementation, using fluorescence videomicroscopy. Leukocyte adhesion to the venular endothelium was examined by labeling leukocytes with rhodamin 6G. The number density of adherent leukocytes in STZ-diabetic rats was increased significantly, compared with control rats. This increase in leukocyte adhesion was prevented by the long-term supplemented vitamin C. It was suggested that the antioxidant effect of vitamin C might be responsible for the prevention of leukocyte adhesion in diabetes mellitus.