Loss of end-differentiated ß-cell phenotype following pancreatic islet transplantation.

Replacement of pancreatic ß-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional ß-cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress-induced ß-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated ß-cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin+ /urocortin-3- cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate ß-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative ß-cell sources, graft sites, and ultimately fully vascularized bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature ß-cell phenotype has been maintained.

Correction of diabetes mellitus by transplanting minimal mass of syngeneic islets into vascularized small intestinal segment.

Transplantation of mature islets into portal vein has been most effective thus far, although attrition of transplanted islets constitutes a major limitation, and alternative approaches are required. We analyzed the mechanisms by which islets engrafted, vascularized and functioned over the long term in the small intestinal submucosa. To determine engraftment, survival and function, 350 syngenic islets were transplanted into either intestinal segments or portal vein of diabetic rats. Islet reorganization, vascularization and function were analyzed by histological analysis, RT-PCR analysis as well as glycemic control over up to 1 year. Transplantation of syngeneic islets in marginal numbers successfully restored normoglycemia in diabetic rats. Transplantation of semi-pure islet preparation did not impair their engraftment, vascularization and function. Islets were morphologically intact and expressed insulin as well as glucagon over the year. Expression of angiogenic genes permitted revascularization of transplanted islets. We identified the expression of transcription factors required for maintenance of beta cells. These studies demonstrated that marginal mass of transplanted islets was sufficient to restore euglycemia in streptozotocin-treated rats. These superior results were obtained despite use of an impure preparation of islets in animals with small intestinal segment. Our findings will help advance new horizons for cell therapy in patients with diabetes.

[Antidiabetic activity of berberin and extract, obtained from the bark of Phellodendron lavalei, introduced in subtropic regions of Georgia, in streptozotocin induced diabetic rats].

The goal of the study was to identify antidiabetic impact of berberin and extract, obtained from the bark of Рhеllodendron lavalei, introduced in subtropic regions of Georgia. The study was conducted on Streptozotocin induced diabetic rats. Diabetes in animals was induced by single intraperitoneal injection of streptozotocin in 1M citrate buffer, pH 4.5, dose 40 mg/kg per body weight, 1 ml volume. Treatment was conducted using berberin hydrochloride dose 150 mg/kg and extract of the bark of Рhеllodendron lavalei dose 400 mg/kg per orally, on an everyday basis, once a day during 3 weeks. The following indicators were measured: weight gain during the observation period, amount of food intake g/day, food efficiency ratio (%), relative mass of internal organs and blood glucose level. Blood glucose level was measured after 3 weeks from the day experiment started, after fasting all night. It was determined that per oral administration of berberin hydrochloride dose 150 mg/kg and extract of the bark of Рhеllodendron lavalei dose 400 mg/kg, during 3 weeks caused decrease in blood glucose level in streptozotocin induced diabetic rats. The marker of this efficacy was improvement of symptoms: decrease in hyperglycemic values, decrease in polyuria and polyphagia and relative weight of kidneys and heart, improvement of food efficiency ratio and mark decrease of weight loss. It should be noted that berberins antidiabetic activity was more marked. It is suggested that medicinal plants containing studied active components can be used as a crude drug for obtaining medicinal agent of promising antidiabetic phytotherapy and along with standard treatment can be used against metabolic syndrome and prevention of diabetes type 2.

Bone marrow-derived mesenchymal stem cell plasticity and their application perspectives.

The aim of this review is to summarize recent developments in research on the characteristics of bone marrow-derived mesenchymal stem cell plasticity. Stem cells are uncommitted entities capable of both self-renewal and differentiation into multiple cell lineages. In general, there are certain types of stem cell populations that are identified from embryonic and postnatal tissues. Embryonic stem cells are derived from mammalian blastocytes and theoretically have the ability to generate differentiated cell types arising from the three germ layers: mesoderm, ectoderm and endoderm. In contrast, postnatal stem cells are thought to be tissue specific, committed precursors capable of developing into a restricted number of cell lineages. Bone marrow stromal stem cells (BMSSCs), also known as mesenchymal stem cells, have been identified as a population of organized hierarchical postnatal stem cells with the potential to differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, myoblasts and neural cells. Recently, studies on the plasticity of BMSSCs challenge the traditional dogma that the differentiation and commitment of postnatal stem cells are limited to cell populations resident in their local environment. Current boneregenerative techniques, such as autologous bone grafting, allografts and alloplastic materials, have limitations that hinder their use in a wider range of clinical conditions. Hence, the development of improved methods, such as BMSSC-mediated bone regeneration, is necessary for achieving future viable therapeutic alternatives.

The method of melanocytes isolation.

To judge the therapeutic value and evaluate the mechanism of action of this specific transplantation procedure we have performed an isolation of the melanocytes in experiment on the hens. The black hens (n=5), 3-5 kg of body weight were used in experimental study. Hens were housed in standard laboratory conditions under 12-hr day-night cycles with provision of pelleted diet and water ad libitum. All hens received care according to institutional guidelines. Using a hand dermatome, a shave biopsy specimen of approximately 2 cm2 was taken from the hens' normally pigmented region under local anesthesia (lidocaine hydrochloride, 20 mg/mL). The wound was covered with a transparent occlusive bandage (4 cm x 4 cm) and secured with gauze and adhesive tape. The melanocytes suspension have been obtained by fermentative digestion.

New method for treatment of high hepaticocholedoch strictures (experimental research).

The purpose of the investigation was to achieve hepaticocholedoch recovery with preserved sphincter apparatus. The experiments were carried out on 25 mongrel dogs of both sexes with body weight 12-25 kg. The animals were divided into 3 groups. The hepatocholedoch strictures were induced in the animals of group I (n=10) and II (n=10). On the 4th day following modeling the animals of group II were subjected to relaparatomy, stricture removal and hepatocholedoch distraction by means of distraction device developed by prof. Kakabadze (patent request submitted for approval). On the 18th day after the surgical procedure the animals have undergone the surgery, the distraction device was removed and the anatomical integrity of biliary tract was restored by bilio-biliary anastomosis. Performed investigations have shown that all of the animals of group I have died on the several time points after the surgery. The cause of death was cholestasis. As to animals of group II, 2 dogs have died on 24 and 35 days after the surgery because of technical shortages of the bilio-biliary anastomosis. The rest 8 experimental animals undergo surgery and postoperative period normally and they were under observation in vivarium standard conditions correspondingly. The i.v. cholangiography, performed in survived animals during postoperative period, showed compete contrast liquid flow into the duodenum. The histological test performed during distraction showed mucosal layer sclerosis. Distraction and afford made evident influence on the muscular fibers through the duct axis. Their elongation and attenuation was mentioned after 3 days from distraction. The fibroblasts local accumulation was seen. The hepaticocholedoch mucosal complete recovery was seen after apparatus was taken out and the bilio-biliary anastomosis was done. Thus, new method of hepaticocholedoch strictures surgical correction elaborated by us allows us to restore anatomical integrity of biliary tract with preserved Oddi sphincter.

[Alternative to donor organs--the method of cultivation from fragments of tissues of "new organs" in an organism of the recipient].

A method of "new liver" growing inside of the recipients' organism as an alternative of the donor organs is presented. Experiments were carried out on the 90 Wistar rats. Animals were divided into 3 equal groups. All of the animals were subjected to 40% hepatectomy. The liver fragments were harvested from resected lobe. The animals of the I group received liver fragments transplantation into the greater omentum pocket, animals of the II group--into the peritoneal cavity and animals of the III group--into the "Kakabadze Biocontainer". All animals were under the standard vivarium conditions and were sacrificed in the different time points. The maximal follow up period was 6 months. Performed investigations have shown that vascularized, denuded segment of small intestine is uncial site for the engraftment and future development of transplanted liver fragments.

[Hepaticocholedoch reconstruction by explants and autotransplants].

Authors have elaborated new method of surgical correction of hepaticocholedoch strictures. The experiments were carried out on 25 mongrel dogs of both sexes with body weight 15-25 kg. The surgical procedures were done under endotracheal ether narcosis. The hepaticocholedoch stricture was induced preliminarily in all animals. After the modelling, stricture was cut out, the anatomical integrity of the common bile duct was restored by autovein (n=10) and "GORE-TEX" vascular prosthesis (n=10). The performed investigations have shown that autovenous transplant was subjected to incrustation by bile salts, later on it became necrotic. Therefore the autovenous transplant could not be used in biliary reconstruction. The results of biliary reconstruction by "GORE-TEX" vascular prosthesis were promising. The "GORE-TEX" vascular prosthesis is well adapting with bile ducts tissue and do not incrust by bile salts.

The experimental distention of dissected bile duct for the restoration of its continuity in dogs using a device of own construction.

PURPOSE: The segmental resection of constricted bile duct and end-to-end biliary anastomosis could be an attractive alternative in the treatment of benign biliary tract stricture. The aim of this study was to restore the anatomical integrity of the hepatic-common bile duct after an artificially produced defect while maintaining the large duodenal papilla, using microsurgical technique. MATERIAL AND METHODS: The experiments were carried out on 25 mongrel dogs. The common bile duct was ligated in all of the animals during laparotomy, as a model of bile duct obstruction in humans. Relaparotomy was performed 3 days after the initial operation. The segment of bile duct, 4 cm in length was resected together with the ligature. The continuous bile flow into the duodenum was assured by a polyvinyl catheter introduced into both ends of dissected bile duct. The proximal end of the hepatic-common bile duct was fixed to a device constructed by us for the distention of the bile duct (DDBD). The anterior part of the device was exteriorized through a separate fistula and fixed to the abdominal wall. The hepatic-common bile duct distention was gradually continued during 18 days, by pulling out the mobile part of the device. After 18 days the device was removed and the distended proximal end of the hepatic-common bile duct was anastomosed end-to-end with its distal end. The sequels of this procedure were observed for up to 6 months. RESULTS: The hepatic-common bile duct was distended 4 cm within 18 days. The histopathological examination has shown partial damage of the duct framework due to the distention and tension. However the patency of the duct was preserved and the recovery of normal structures were observed after the device was removed and anastomosis fashioned. CONCLUSION: This method, developed by us, offers the possibility of restoring the integrity of injured extrahepatic bile ducts, allowing effective treatment of benign biliary strictures.

The intracorporeal autoorganic biofilter of portal blood in portal hypertension surgery.

BACKGROUND/AIMS: The authors present the new surgical treatment method for portal hypertension by means of intracorporeal autoorganic biofilter of portal blood at the portosystemic shunt pathway. The hepatized spleen is used as the organic biofilter of portal blood. METHODOLOGY: The experiments were carried out on 20 mongrel dogs, both sexes, 18-20 kg of body weight. The animals were divided into the four equal groups. The comparative evaluation of portal hypertension treatment efficiency by means of several vascular portosystemic shunts were performed after the portal hypertension modeling. On the animals of groups I and II, the mesenterico-caval and splenorenal shunts were performed, respectively. The hepatized autospleen was involved in the portosystemic shunt pathway in the animals of group III. The animals of group IV served as controls. RESULTS: The reduction in perfusional pressure of the blood which flows to the liver led to damage of the organ's functional possibilities, significant disturbances to the hemodynamics and reduction of the liver detoxification possibilities, respectively, for several portocaval shunt creations during the portal hypertension model. On the other hand, the undetoxificated portal blood entrance into the system blood flow via the created shunt over the liver increased. CONCLUSIONS: The hepatized autospleen insertion into the portosystemic bloodflow gave us the opportunity to perform the dosage passing of portal blood into the systemic bloodflow and to maintain the pressure of the portal blood, which flows to the liver. Moreover, the hepatized autospleen has shown such specific liver functions as glucogenesis, albumin synthesis and others.