Marika Arthur

Cryopreserved microencapsulated hepatocytes--transplantation studies in Gunn rats.

Hepatocyte transplantation has been shown to provide significant metabolic support in several animal models of liver diseases. However, for it to be a viable alternative for supplementation of liver function in disease, large quantities of isolated hepatocytes would be necessary. At the present time there are no inexpensive routine methods for cryopreservation of hepatocytes. Existing procedures are cumbersome and require expensive programmable freezers. Hepatocyte cultures are sensitive and easily damaged in handling. By utilizing techniques of microencapsulation and cryopreservation we have attempted to overcome these problems. We have developed a simple, convenient, and inexpensive technique for the long-term storage of hepatocytes. Biological activity of the nonfrozen isolated encapsulated hepatocytes (IEH) and cryopreserved IEH (cIEH) was assessed both in tissue culture and by transplantation in Gunn rats. Significant urea and protein syntheses were detectable during the 10-day culture period even in the 30-day cIEH. Additionally, transplanted IEH and cIEH significantly reduced hyperbilirubinemia in Gunn rats for up to 30 days posttransplantation. Control (empty) microcapsules did not lower serum bilirubin levels. Thus we conclude: (1) cryopreservation of IEH is a convenient and cost-effective method for preserving and storing hepatocytes; (2) cryopreserved IEH function as well as nonfrozen IEH both in vitro and in vivo; (3) microencapsulation may protect hepatocytes from the adverse effects of cryopreservation.

Preliminary report on isolation of mycobacteria from patients with Crohn's disease

Several investigators have recently described the isolation of slow growing mycobacteria from the tissues of patients with Crohns disease (CD). The primary purpose of this study was to culture and identify mycobacteria from the intestines of patients with CD and other intestinal diseases (control tissues). The culture methods were designed to eliminate most rapidgrowing mycobacteria and to enhance the isolation of slow growing mycobacteria. Eighty-two surgically resected intestinal tissue samples were cultured over a four-year period: 27 tissues were from CD patients and 55 from patients with other intestinal diseases. After 4–12 months of culture, five mycobacteria were isolated, but only two have been identified thus far. Both of these organisms appeared to have initially grown as spheroplasts, but revertant bacteria were cultivated after transfer into fresh media. Four of the mycobacteria were from CD tissues, and one isolate was from a control tissue. Two of the isolates have been identified as M. cheloneisubsp. abscessus,strain 390, and M. paratuberculosisstrain 410. This M. paratuberculpsisis similar to the previously identified M. paratuberculosisstrains isolated from other human intestinal tissues from patients with CD. Both strains 390 and 410 were inoculated into neonatal goats, but they failed to reproduce a CD-like disease. The isolation of four mycobacteria from 27 CD tissues and only one from 55 control tissues strengthens the findings of previous investigators and supports the hypothesis that mycobacteria may be etiologically associated with some cases of Crohns disease.

Evidence for the isolation of a new virus from ulcerative colitis patients. Comparison with virus derived from Crohn's disease.

To assess the possible role of viral infection in the development of ulcerative colitis (UC) and of Crohns disease (CD), electron-microscopic, physical, and chemical studies were performed on viral agents cultivated in tissue culture following inoculation with surgical specimens obtained from patients with CD, UC, and from control patients with other gastrointestinal diseases. Viral isolates obtained from CD patients were compared with those from UC patients. Each of the agents produced cytopathic change (CPE) in the tissue culture systems used, rabbit ileum (RI), Peking duck, and Riff free chick embryo, and had similar physical and chemical properties including ether resistance, temperature stability, and lack of inhibition by methotrexate. The three tissue culture systems supported viral growth and produced virus-like particles from CD filtrates with similar morphologic appearance, including a central core and an outer coat. Electron-microscopic studies revealed that the particle from CD has a diameter of 60 nm (range 42–71 nm). The agent isolated from UC patients also has a mean particle diameter of 60 nm (range 45–75 nm). The CD and UC agents produced different CPE in RI tissue culture. Agents different from CD-and UC-derived viruses were isolated from one patient with necrotizing enterocolitis and from three patients with carcinoma of the colon. Ileal homogenates of other control patients failed to reveal evidence of viral contaminants and evaluation of the tissue culture systems revealed no adventitious agents. Virus was found in diseased as well as in adjacent histologically normal CD tissue. Guinea pig antibody prepared against CD-derived agents inhibited the growth of each of the CD agents but not of the agents from UC, necrotizing enterocolitis, or colon carcinoma. Antibody directed against the agents from UC failed to inhibit growth of the agents from CD, necrotizing enterocolitis, or colon carcinoma. The current studies therefore suggest that a virus has now been cultivated from UC patients which produced CPE different from the viruses previously cultivated from CD patients and which lacks cross-reactivity with the CD derived virus. Since each filtrate produced viruses in the three different tissue culture systems, these viruses are not tissue culture contaminants but are derived from the human filtrates. These studies also suggest an association between viruses and inflammatory bowel disease but do not establish an etiologic relationship.

De novo liver tissue formation in rats using a novel collagen-polypropylene scaffold.

In experimental and clinical settings hepatocyte transplantation has provided limited benefit to patients with chronic liver disease because the transplanted hepatocytes were short-lived and were merely maintained for a brief period within the body. Except for whole-liver transplantation, creation of de novo liver tissue is necessary to treat this condition on a long-term basis. The aim of this study was to facilitate the formation of new tissue by actual self-regeneration, rather than by compensatory hypertrophy, or scar formation, with our collagen-polypropylene composite scaffold. Collagen-polypropylene composite scaffolds, not containing hepatocytes, were implanted into the median liver lobe and the dynamics of new liver tissue formation was analyzed immunohistochemically over a 6-month period. Control scaffolds consisted of polypropylene scaffolds without collagen matrix. The control scaffold implants remained hollow throughout the study period and became encapsulated with a hard connective tissue capsule 1 week after implantation. In contrast, the collagen-polypropylene composite scaffold was filled with regenerating tissue structures 3 weeks after implantation. At this time, the predominant cell type within the scaffold was sesmin-positive stellate cells. A week earlier, oval cells were identified using monoclonal antibody staining (OV-6). Subsequently, these cells differentiated into α-fetoprotein-positive immature hepatocytes. After 6 months, mature liver tissue, juxtaposed with bile ducts and blood vessels, was seen within the polypropylene scaffolds. We report the first evidence of de novo formation of liver tissue within a polypropylene scaffold, following implantation in the liver. This scaffold may play a role in treating chronic liver diseases requiring organ replacement therapy.

A Morphological and Functional Evaluation of Transplanted Isolated Encapsulated Hepatocytes Following Long-Term Transplantation in Gunn Rats

In this study we investigated the fate of microencapsulated hepatocytes following long-term (6 months) transplantation in Gunn rats. Isolated hepatocytes were microencapsulated with a collagen matrix within an alginate-poly L-lysine composite membrane. Isolated, encapsulated hepatocytes (IEH) or free (unencapsulated) isolated hepatocytes were intraperitoneally transplanted into homozygous Gunn rats that exhibit congenital hyperbilirubinemia. Control Gunn rats received empty microcapsules. Total serum bilirubin was measured at weekly intervals for one month post-IEH transplantation, every two weeks for the next month, and monthly thereafter for up to six months. IEH samples were biopsied from the Gunn rats at monthly intervals and analyzed by light and electron microscopy. A significant (p < 0.01) decrease in total serum bilirubin was observed in IEH transplanted animals during the first month of transplantation. Thereafter, total serum bilirubin levels gradually returned to pre-transplantation levels. A mild, transient decrease in total serum bilirubin was seen in animals transplanted with free (unencapsulated) hepatocytes. No decrease in total serum bilirubin levels was seen in the Gunn rats transplanted with control (empty) microcapsules. Transplanted IEH retained its normal ultrastructure for up to one month and intact microcapsules showed no evidence of hepatocyte rejection, at this time. Degenerative changes observed in the IEH beginning at 2 months post-transplantation, suggests that repeated transplantations may be necessary for long-term effectiveness of IEH therapy.

Post-Transfusion Hepatitis: The Role of Hepatitis B Antibody

Aliquots from units of blood previously transfused as part of a prospective post-transfusion hepatitis (PTH) study were rescreened for the presence of hepatitis B antibody (anti-HBS) to determine the effect of transfusion of such material. Anti-HBS was more commin in commercial blood. Infusion of anti-HBS was not associated with an increased or decreased risk of PTH, hepatitis B, or hepatitis B (HB) exposure. Receipt of anti-HBS did not modify the hepatitis which occurred. Receipt of large amounts of anti-HBS may be associated with an increased incidence of HB events. Preexisting anti-HBS was not only not protective against PTH, but more PTH (67% versus 40%) and hepatitis B (47% versus 12%) were seen in those patients with it.