Mucosa cell regeneration following intestinal ischemia/reperfusion injury in rats.

OBJECTIVE: To evaluate regeneration and proliferation of the mucosa cells following intestinal ischemia/reperfusion (I/R) injury. METHODS: Sixty rats (200-250 g) were examined after intestinal I/R injury with immunohistochemical (BrdU) method and flow cytometric analysis. RESULTS: Severe damage to the villi was evident at 2 hours reperfusion and the rate of cell proliferation was lower (P < 0.01). All the specimens has healed mucosa with re-epithelialized villi tips after 2 days regeneration, but the villi were scarce and tiny. After two and four days regeneration, the rate of cell proliferation was significantly higher than that of controls (P < 0.01). CONCLUSIONS: Our study suggests that the intestinal regeneration following I/R injury in rats occurs early. The immunohistochemical method with BrdU-labeled technique is a rapid method for quantifying the rate of cellular DNA synthesis.

Oncogene expression in patients with familial polyposis coli/Gardner's syndrome.

We evaluated whether c-myc and ras P-21 oncogene expression could identify familial polyposis coli/Gardner's syndrome patients at risk for colon cancer. Monoclonal antibodies recognizing c-myc and ras P-21 proteins were used in immunohistochemistry to stain 26 paraffin-embedded tissue specimens collected over 12 years from 5 familial polyposis coli/Gardner's syndrome patients at various stages of their disease. Differences in staining intensity between specimens were noted with each of the two tissues markers; however, c-myc showed also a distinct cellular staining pattern in patients with advanced histologic features. The c-myc oncogene exhibited strong homogeneous cytoplasmic staining in all adenocarcinoma specimens; weak cytoplasmic staining was found in normal biopsies and in 5/10 familial polyposis coli/Gardner's syndrome specimens in the early stage of disease. In contrast, a heterogeneous staining pattern with a strong supranuclear and weak nuclear and cytoplasmic staining was demonstrated in specimens with advanced histologic features of familial polyposis coli/ Gardner's syndrome and also in postoperative ileal specimens. Anti-ras P-21 antibody, on the other hand, demonstrated a homogeneous cytoplasmic staining pattern in all specimens. We feel that the c-myc oncogene expression, in distinction to that of ras P-21, has potential as a genetic tissue marker to distinguish early from more progressive disease.

Small intestinal mucosa changes, including epithelial cell proliferative activity, of children receiving total parenteral nutrition (TPN).

We examined the small intestinal histology disaccharidase activities as well as the incorporation of [3H]thymidine into DNA of biopsies maintained in organ culture from seven children (ages 9 months to 5 years) receiving total parenteral nutrition (TPN). Three children suffered from inflammatory bowel disease and received TPN for one month (short term). Four required long-term TPN (> 9 months) for short-bowel syndrome. DNA was extracted from the samples following serial precipitation with perchloric acid. Results were compared to those from 22 age-matched children investigated for abdominal pain or chronic diarrhea. Short-term TPN resulted in slightly lower lactase, sucrase, and palatinase activities that were not statistically different from controls. Long-term TPN resulted in focal mild villus atrophy and a decrease in disaccharidase activity in two patients. Biopsies from long-term TPN patients incorporated less thymidine compared to those of controls (P < 0.001) when data was expressed per total biopsy (3.6 +/- 1.1 vs. 8.4 +/- 1.1 fmol) or per milligram of tissue (1.0 +/- 0.12 vs 2.7 +/- 0.7 fmol). The above data are in general agreement with the hypoplastic effect of TPN in animals. However, in children, much longer periods of TPN are required to realize the changes.

Stromal cells derived from spleen or bone marrow support the proliferation of rat natural killer cells in long-term culture.

Rat nylon wool nonadherent bone marrow cells were propagated for up to 75 days in co-culture with stromal cells derived from either spleen or bone marrow. Interleukin (IL) 1 enhanced the ability of spleen stroma to support the long-term culture of natural killer (NK) cells, ostensibly by inducing these support cells to synthesize other cytokines. Flow cytometry studies indicated that the nylon wool separation procedure enriched the concentrations of mature NK cells from 7.9% to 38.1% for splenocytes and from 3.8% to 19.5% for bone marrow cells. Analyses of the adherent zones of suspended nylon screen NK cell cultures revealed substantial numbers of large granular lymphocytes that expressed NK 323+/MOM/3F12/F2- phenotypes. The presence of both mature and immature cells of the NK lineage in this matrix was inferred by the presence of both IL-2 receptor (IL-2R) positive and IL-2R negative, and OX-8+ and OX-8- NK 323+ cells over the greater than 4-month experimental period. Suspended nylon screen cultures displayed a greater potential for producing cytolytic cells than either co-cultures of bone marrow nonadherent cells on stroma monolayers or suspension cultures. The large granular lymphocytes produced in suspended nylon screen cultures could be transformed into active killers of YAC-1 targets by IL-2. In contrast to bone marrow nonadherent cells, more splenic nylon-wool-passed cells displayed a mature NK phenotype, but their proliferative potential and ability to be transformed into cytolytic cells by IL-2 decreased rapidly in culture. In the suspended nylon screen culture system, NK cells migrate from the underlying stroma in stages as they mature, retain their cytolytic potential, and manifest a capacity for self-renewal. Cultured cells were routinely dissociated into single cell suspensions via enzyme treatment and were reinoculated onto "fresh" nylon screen/stromal cell templates after passage through nylon wool columns. These co-cultures continued to generate cytolytic cells in numbers greater than those of the initial inoculum.

Adoptive immunotherapy using lymphokine-activated killer cells and recombinant interleukin-2 in preventing and treating spontaneous pulmonary metastases of syngeneic Dunning rat prostate tumor.

Lymphokine-activated killer (LAK) cells were generated from splenocytes of rats bearing a weakly immunogenic Dunning prostate tumor (R-3227 AT-3) and activated with recombinant interleukin-2 (rIL-2). The maximal LAK activity was obtained from splenocytes of rats bearing tumors for 10 to 14 days after incubation with 1000 U/ml./day of rIL-2 for five to eight days. The majority of these LAK cells expressed high levels of asialo GM1 (89%), laminin (83%), OX-19 (80%) and OX-8 (88%) surface markers. LAK cells exhibited higher cytotoxicity to rat prostate tumor cells and mouse lymphoma in vitro than to other non-prostate tumor cells or normal rat splenocytes and thymocytes. Splenocytes of rats bearing prostate tumors have higher LAK activity than normal splenocytes. The Winn type assay showed that Dunning prostate tumor growth was inhibited effectively by LAK cells at a tumor cell:LAK cell ratio of 1:50. The therapeutic efficacy of LAK cells in the treatment of primary solid prostate tumors and pulmonary metastases of Dunning rats was evaluated. LAK cells in combination with rIL-2 showed a greater therapeutic benefit in 1) prevention of prostate tumor metastases to lung, 2) retardation of the primary tumor growth, 3) regression of spontaneously established pulmonary metastases, and 4) prolongation of survival as compared to untreated controls or those groups treated with LAK cells or rIL-2 alone. The results of this study indicate that the conjunctive therapeutic approach of using surgical therapy to remove primary solid tumors followed by adoptive immunotherapy with LAK cells plus in vivo administration of IL-2 may be potentially valuable in the treatment of prostate tumors, particularly for the spontaneous pulmonary metastases.

Murine monoclonal antibodies reactive with a variety of androgen independent Dunning rat prostate adenocarcinoma sublines also reactive with human prostate adenocarcinoma.

Murine hybridoma-derived monoclonal antibody (MCA) to Dunning rat prostate adenocarcinoma R-3327HIS (androgen independent type) has been produced by fusing P3x63 Ag8-653 myeloma cells with splenocytes of BALB/c mice which were immunized with R-3327HIS tumor cell membranes. One monoclonal antibody designated MCA-R1 (IgG2a subtype) produced an intense immunostaining of various androgen independent Dunning rat prostate tumor sublines (HIS, HIM, HIF, AT-1, AT-2, AT-3, MAT-Lu and MAY-Ly-Lu), but did not stain other tumors of rat origin or normal rat tissues. Marginal immunostaining was detected in the androgen responsive R-3327H and R-3327G tumor subline. Although MCA-R1 antibody did not react with the regressed prostate tumor of R-3327H or R-3327G, it strongly reacted with the relapsed prostate tumor from either R-3327H or R-3327G tumor derived from rats were treated with diethyl stilbestrol (DES) or castration. MCA-R1 antibody also produced a strong cross-reaction with human prostate adenocarcinoma. Like the Dunning rat tumor, human adenocarcinoma exhibited distinct immunostaining patterns with respect to intracellular localization among well differentiated, moderately differentiated and poorly differentiated tumor. Benign prostatic hyperplasia or other normal tissues did not stain. Immunofluorescence, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautographic analysis revealed that the Dunning rat prostate tumor antigen recognized (m.wt. 50 and 120 Kd) by MCA-R1 antibody are localized on both cell surface and in the cytoplasm. This MCA may represent a potential reagent for the study of tumor biology and immunotherapy of prostate tumor.

Hematopoiesis on suspended nylon screen-stromal cell microenvironments.

A three-dimensional culture system for the growth of primate and rodent bone marrow was developed in our laboratory. This method involves the seeding of stromal cells onto a nylon screen and the inoculation of fresh or cryopreserved bone marrow hematopoietic cells after stromal cell processes had extended across 3 to 4 out of every 5 mesh openings. Stromal cells attach, grow, and secrete matrix proteins which contribute to an intricate microenvironment for the support of multilineage hematopoiesis, which was observed for greater than 270 days in the rat model and for greater than 12 weeks in the human system, as evidenced by flow cytometry analysis and in vitro clonogenic assays. The adherent zones of these suspended nylon screen cultures consisted primarily of immature cells. These cultures could also be used as substrates for cytotoxicity measurements; treatment of rat bone marrow cultures of various ages with cytosine beta-D arabinofuranoside, cyclophosphamide, 5-fluorouracil, or methotrexate resulted in a dose-dependent decrease in CFU-C numbers and altered the phenotypic distribution of hematologic cells in the adherent zone. The use of a modification of this method to generate large numbers of active cytolytic cells after greater than 75 days culture of rat bone marrow-derived natural killer cells is described also. Suspended nylon screen bone marrow culture also has potential uses in genetic insertion and graft vs. host disease studies, blood component therapy, the evaluation of ex vivo purging programs, and in marrow expansion for transplantation.