Intrasplenic transplanted adult rat isolated hepatocyte fraction but not cholangiocytes forms bile canaliculi.

BACKGROUND: Hepatocyte transplantation (HT) has been performed in patients with liver-based metabolic disease and acute liver failure as a potential alternative to liver transplantation in countries in which ethical regulations do not allow organ transplantation. One of the problems remains that substances normally secreted by the surviving hepatocytes to bile cannot be removed because of lack of bile canaliculi. We found that ligation of the recipient's common bile duct in hepatocyte transplantation recipients is followed by formation of bile canaliculi. The question arose as to whether the signal released from the obstructed bile vasculature activated the transplanted hepatocytes (HC) or cholangiocyte (CH) to form bile canaliculi. METHODS: We transplanted separately isolated autologous HC and CH to spleens and observed the structural organization of the grafted cells. RESULTS: HC formed glycogen-rich clusters but not cords usually not attached to the CH of the new bile canaliculi. Separate clusters of bile canaliculi with keratin 7 and 19-positive and gamma-glutamyl transpeptidase-positive cells were observed. Transplanted CH remained keratin 7 and 19-positive and gamma-glutamyl transpeptidase positive but did not form canaliculi. CONCLUSIONS: The transplanted HC fraction may contain hepatic progenitor cells for cholangiocytes, but they become activated only under the condition of bile stasis by an as-yet undefined factor.

Lack of healing reaction in the cancer wound.

Surgical wounds in cancer patients have a relatively high dehiscence rate. Although cancer resections are performed so as to include macroscopically non-involved tissues, some cancer cells can be present in the line of transection or surrounding tissues (R1 and R2 resections). The local healing process may facilitate proliferation of these localized cancer cells, and the high cytokine concentration within the healing wound may also attract cancer cells from distant sites to migrate into the wound area. The question arises how the tumor environment influences the wound healing process. The aim of the study was to monitor and compare, using immunohistochemical methods, the healing process of an incision wound performed through a metastatic liver tumor of colon cancer with the healing of a normal liver incision wound. The experiments were carried out on a CC531 colon cancer rat model. We observed impaired healing of cancer wounds at all stages of wound healing. Significantly fewer mononuclear cells infiltrated the cancer than the normal liver wounds. There were no significant differences in the phenotypes of infiltrating mononuclear cells. BrdU incorporation showed rapid proliferation of cancer but not infiltrating cells or fibroblasts in the cancer wounds. We observed no connective tissue formation and poor collagen deposition in cancer wounds. Additionally, cancer wounds were significantly deprived of newly formed vessels. We confirmed that the impaired migration and proliferation of inflammatory cells in cancer wounds and poor scar tissue formation contribute to impaired healing of cancer 'contaminated' wounds.

Lack of functioning lymphatics and accumulation of tissue fluid/lymph in interstitial "lakes" in colon cancer tissue.

There is controversy as to whether intratumoral or peritumoral lymphatics play a dominant role in the metastatic process. The knowledge of how and where exactly tumor cells enter lymphatics is important for therapeutic targeting either the tumor core or peritumoral tissue with drugs or radiation. The basic questions remain: what is the morphological structure of intra- and peritumoral interstitium and lymphatics; what is their hydraulic conductivity?; and do these local physical conditions allow detached tumor cells to migrate to lymphatics? Identification of lymphatics has been based on immunohistochemical staining of lymphatic endothelial cells. This method does not, however, show the tissue fluid filled interstitial space and the shape of minute lymphatic vessels in tumors. We visualized the interstitial space and lymphatics in the central and peripheral regions of tumors using our original method of color stereoscopic lymphography in translucent tissue fragments and simultaneously with immunohistochemical staining of lymphatic and blood endothelial cells. The density of open and compressed lymphatic and blood vessels was measured in the intratumoral "hot spots" and at tumor edge. Moreover, the intratumoral tissue hydraulic conductivity was measured to define force necessary for propelling tissue fluid to peritumoral lymphatics. We found very few rudimentary minor blind lymphatics in the tumor core and numerous minor fluid "lakes" in the interstitium with no visible connection to the peritumoral lymphatics. Lining of "lakes" did not express molecular markers specific for lymphatic endothelial cells. Ninety-five percent of structures of what looked like lymphatics had compressed lumen and the hydraulic conductivity was 3 powers of magnitude lower than in the adjacent non-tumoral tissue. It can be concluded that lack of functioning lymphatics in tumor foci manifested by accumulation of tissue fluid in "lakes," low fluid conductivity and compression of lymphatics by tumor cells, and proliferating connective tissue may hamper escape of tumor cells. The most favorable site of entry of tumor cells to lymphatics seems to be the interface of the tumor and surrounding tissue with open lymphatics.

Preservation of molecular structure of arterial grafts in pulverized sodium chloride: an experimental study.

There is a desperate need for preserved human arteries to be transplanted in infected areas of ischemic lower limbs. Cryopreserved arteries are fragile evoking strong allogeneic reactions. We have been searching for a preservation method that spares arterial wall structure and decreases its antigenicity. LEW and BN rat segments of aorta were harvested, immersed in pulverized dehydrated sodium chloride, and stored for 1 week to 3 months. Thereafter, they were desalinated and transplanted side-to-side to the abdominal aorta and side-to-side to the aorta-IVC (AV fistula). We performed staining for actin, elastic fibers, collagen (trichrome), CD31, endothelial cells (HIS52), MHC class II. Popliteal lymph node reactions were tested after subcutaneous implantation of an aortic fragment into the paw. We measured tensile strength and maximum intraluminal pressures. The preserved fragments of syngeneic aorta retained their cellular, stromal, and molecular structures. After transplantation, they did not evoke any reaction around the graft. The allogeneic fragments brought about only minor recipient responses, remaining patent and pulsatile for 3 months. The tensile strength and maximum intraluminal pressures did not significantly differ from freshly harvested, transplanted aortic segments.

Hepatocyte transplantation-biology and application.

Transplantation of liver has been remarkably effective in the treatment of liver failure and liver-based inherited metabolic diseases and has revolutionized the care of patients with end-stage liver disease. Unfortunately demand for transplantable livers is progressively outpacing the supply of donated cadaver organs, resulting in longer waiting times and increased mortality for prospective transplant recipients. Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. The current knowledge on this method has been review in this article. Now the two-step collagenase perfusion technique is widely used for isolation of hepatocytes. Liver has been considered as an optimal site for hepatocyte transplantation, however, even in this organ the survival rate of transplanted hepatocytes in extremely low. The main obstacle for wider usage of hepatocyte transplantation is their rapid elimination by recipient macrophages. We tried in animal experiments to downregulate the innate process of recipient cellular attack on implanted hepatocytes by irradiation of recipient and elimination of NK cells. Ligation of bile duct and partial hepatectomies facilitated proliferation of accepted donor hepatocytes and formation of bile canaliculi. The described method is now adjusted to clinical conditions.

Recipient immature dendritic cells do not prolong allograft survival.

Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DCs exert their tolerogenic function in the graft or in recipient lymphoid tissue. To answer this question we created a chimeric heart model deprived of its own DCs and repopulated with recipient DCs. The rationale for this model was to observe whether recipient mature and immature DCs located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DCs with prospective recipient either mature or immature DCs. Replacement of the donor heart with either of these cells did not prolong graft survival. The intragraft microchimerism did not mitigate the allogeneic rejection reaction.