Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation.

The development of therapeutic vaccines against chronic hepatitis B requires the capacity of the formulation to subvert a tolerated immune response as well as the evaluation of histopathological damage resulting from the treatment. In the present study, the dynamicity of induced immune response to hepatitis B surface antigen (HBsAg) was evaluated in transgenic mice that constitutively express the HBsAg gene (HBsAg-tg mice). After immunization with a vaccine candidate containing both surface (HBsAg) and core (HBcAg) antigens of hepatitis B virus (HBV), the effect of vaccination on clearance of circulating HBsAg and the potential histological alterations were examined. Transgenic (tg) and non-transgenic (Ntg) mice were immunized by intranasal (IN) and subcutaneous (SC) routes simultaneously. A control group received phosphate-buffered saline (PBS) by IN route and aluminum by SC route. Positive responses, at both humoral and cellular levels, were obtained after five immunizations in HBsAg-tg mice. Such responses were delayed and of lower intensity in tg mice, compared to vaccinated Ntg mice. Serum IgG response was characterized by a similar IgG subclass pattern. Even when HBsAg-specific CD8+ T cell responses were clearly detectable by gamma-interferon ELISPOT assay, histopathological alterations were not detected in any organ, including the liver and kidneys. Our study demonstrated, that it is possible to subvert the immune tolerance against HBsAg in tg mice, opening a window for new studies to optimize the schedule, dose, and formulation to improve the immune response to the therapeutic vaccine candidate. These results can be considered a safety proof to support clinical developments for the formulation under study. HOW TO CITE THIS ARTICLE: Freyre FM, Blanco A, Trujillo H, Hernández D, García D, Alba JS, Lopez M, Merino N, Lobaina Y, Aguilar JC. Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation. Euroasian J Hepato-Gastroenterol 2016;6(1):25-30.

Growth-hormone-releasing peptide 6 (GHRP6) prevents oxidant cytotoxicity and reduces myocardial necrosis in a model of acute myocardial infarction.

Therapies aimed at enhancing cardiomyocyte survival following myocardial injury are urgently required. As GHRP6 [GH (growth hormone)-releasing peptide 6] has been shown to stimulate GH secretion and has beneficial cardiovascular effects, the aim of the present study was to determine whether GHRP6 administration reduces myocardial infarct size following acute coronary occlusion in vivo. Female Cuban Creole pigs were anaesthetized, monitored and instrumented to ensure a complete sudden left circumflex artery occlusion for 1 h, followed by a 72 h reperfusion/survival period. Animals were screened clinically before surgery and assigned randomly to receive either GHRP6 (400 microg/kg of body weight) or normal saline. Hearts were processed, and the area at risk and the infarct size were determined. CK-MB (creatine kinase MB) and CRP (C-reactive protein) levels and pathological Q-wave-affected leads were analysed and compared. Evaluation of the myocardial effect of GHRP6 also included quantitative histopathology, local IGF-I (insulin-growth factor-I) expression and oxidative stress markers. GHRP6 treatment did not have any influence on mortality during surgery associated with rhythm and conductance disturbances during ischaemia. Infarct mass and thickness were reduced by 78% and 50% respectively, by GHRP6 compared with saline (P<0.01). More than 50% of the GHRP6-treated pigs did not exhibit pathogological Q waves in any of the ECG leads. Quantitative histopathology and CK-MB and CRP serum levels confirmed the reduction in GHRP6-mediated necrosis (all P<0.05). Levels of oxidative stress markers suggested that GHRP6 prevented myocardial injury via a decrease in reactive oxygen species and by the preservation of antioxidant defence systems (all P<0.05). Myocardial IGF-I transcription was not amplified by GHRP6 treatment compared with the increase induced by the ischaemic episode in relation to expression in intact hearts (P<0.01). In conclusion, GHRP6 exhibits antioxidant effects which may partially contribute to reduce myocardial ischaemic damage.

Epidermal growth factor intralesional infiltrations can prevent amputation in patients with advanced diabetic foot wounds.

This study examined if a series of epidermal growth factor (EGF) local infiltrations can enhance the healing process of complicated diabetic wounds. Twenty-nine in-hospital patients with diabetic neuropathic or ischaemic lesions with high risk of amputation were treated in a non controlled pilot study conducted at the National Institute of Angiology, Havana. Lesions, classified as Wagner's grade 3 or 4, included ulcers > or = 20 cm2 for > or = 25 days or amputation residual bases > or = 30 cm2 for > or = 15 days, healing refractory despite comprehensive wound care. EGF (25 microg) intralesional infiltrations (approximately 250 microl of a 25 microg/ml solution/injection point) were performed thrice weekly up to the eighth week. Wound closure was monitored during the treatment and recurrence examined for a year following discharge from hospital. Eighty-six per cent of the patients treated showed a productive granulation at infiltration session 8. Histological examination at this point indicated a substantial wound matrix transformation, granulation tissue cell repopulation and angiogenesis. Of the 29 patients treated, amputation was prevented in 17 (58.6%) of them who completed 24 infiltration sessions. They averaged 71.1 +/- 18.3% of reepithelisation during a mean in-hospital period of 66.5 +/- 4.9 days. Wound recurrence after 1 year of follow-up appeared in only one patient. Preliminary evidences suggest that EGF intralesional infiltrations may be effective in reducing diabetic lower limb amputation.

Use of growth-hormone-releasing peptide-6 (GHRP-6) for the prevention of multiple organ failure.

Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer (HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given alone and in combination with the potent protective factor EGF (epidermal growth factor) in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion). In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P<0.01), but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that, compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage (histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 microg/kg of body weight, intraperitoneally) alone truncated these effects by 50-85% (all P<0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments. In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.

Methylglyoxal administration induces diabetes-like microvascular changes and perturbs the healing process of cutaneous wounds.

Increased formation of MG (methylglyoxal) and related protein glycation in diabetes has been linked to the development of diabetic vascular complications. Diabetes is also associated with impaired wound healing. In the present study, we investigated if prolonged exposure of rats to MG (50-75 mg/kg of body weight) induced impairment of wound healing and diabetes-like vascular damage. MG treatment arrested growth, increased serum creatinine, induced hypercholesterolaemia (all P < 0.05) and impaired vasodilation (P < 0.01) compared with saline controls. Degenerative changes in cutaneous microvessels with loss of endothelial cells, basement membrane thickening and luminal occlusion were also detected. Acute granulation appeared immature (P < 0.01) and was associated with an impaired infiltration of regenerative cells with reduced proliferative rates (P < 0.01). Immunohistochemical staining indicated the presence of AGEs (advanced glycation end-products) in vascular structures, cutaneous tissue and peripheral nerve fibres. Expression of RAGE (receptor for AGEs) appeared to be increased in the cutaneous vasculature. There were also pro-inflammatory and profibrotic responses, including increased IL-1beta (interleukin-1beta) expression in intact epidermis, TNF-alpha (tumour necrosis factor-alpha) in regions of angiogenesis, CTGF (connective tissue growth factor) in medial layers of arteries, and TGF-beta (transforming growth factor-beta) in glomerular tufts, tubular epithelial cells and interstitial endothelial cells. We conclude that exposure to increased MG in vivo is associated with the onset of microvascular damage and other diabetes-like complications within a normoglycaemic context.