Pubertal development of male African catfish (Clarias gariepinus). Pituitary ultrastructure and responsiveness to gonadotropin-releasing hormone.

Pubertal development was studied in male African catfish by immunocytochemical examination of pituitary gonadotrophs and by monitoring the responsiveness of gonadotropin (GTH) secretion to salmon GnRH analogue (sGnRHa) in vitro. Experiments were carried out with fish from 9 to 28 wk of age. Fish were assigned to four groups, according to the stage of spermatogenesis: I, spermatogonia alone; II, spermatogonia and spermatocytes; III, spermatogonia, spermatocytes, and spermatids; IV, all germ cell stages, including spermatozoa. Basal and sGnRHa-stimulated secretion of the LH-like GTH II increased 3- to 4-fold from stage I to II and from stage II to III, whereas a 15-fold increase was recorded from stage III to IV. The ED50 values of sGnRHa varied between 0.08 and 0.49 nM, stages II and III being less sensitive. The highest dosage of sGnRHa (100 nM) led to a reduction of GTH secretion. In the first three stages, the pituitary secreted large amounts of free alpha-subunit while free GTH II beta-subunit was not detected at any stage of development. Antisera against GTH II and its alpha- and beta-subunits were used for immunocytochemical studies. In stages I and II, two subtypes of gonadotrophs, which differed in the size and labeling intensity of their secretory granules, were present. Both types of granules were immunopositive for the two subunits of GTH II. In stages III and IV, only gonadotrophs of the subtype with the larger granules were found. Globules and irregular, membrane-bound masses (IMs), probably arising through fusion of secretory granules, appeared in the gonadotrophs in stage III and became more prominent in stage IV. Globules and IMs were immunopositive for the beta-subunit but negative for the alpha-subunit. We conclude that the two subtypes of gonadotrophs represent different developmental stages of GTH II-producing cells, as they shared immunolabeling for the alpha- and the beta-subunits of GTH II. The scarcity of GTH II beta-subunit may be rate-limiting for the amount of intact GTH II available for secretion, particularly at early stages of development. In contrast, at more advanced stages when the readily releasable pool of GTH II has greatly increased, the amount of GTH II also appears to be controlled by modification or elimination of the alpha-subunit from globules and IMs.

Local low-dose interleukin-2 induces systemic immunity when combined with radiotherapy of cancer. A pre-clinical study.

Tumor recurrence and outgrowth of metastases limit the therapeutical effect of radiotherapy. We have tested whether these problems can be overcome by supplementing radiotherapy with locoregional interleukin-2 (IL-2) treatment. The SL2 lymphoma and the M8013 mammary carcinoma were used. Mice bearing a 10-day-old s.c. tumor were locally irradiated and were treated daily with IL-2 peritumorally for 5 or 10 days. Low-dose IL-2 therapy improved local response (LR) and increased disease-free survival (DFS) in both tumor models following either single-dose irradiation or fractionated irradiation. For example, 93% of SL2-bearing mice treated with single-dose irradiation and 10 days of IL-2 experienced long-term DFS, compared with 17% for irradiation alone (p < 0.0001). Additionally, treatment of one tumor with irradiation +IL-2 led to anti-tumor effects in a second, untreated tumor in 80% of SL2-bearing mice. LR was increased to 100% and DFS to 70% when the second, non-irradiated tumor was also treated with peritumoral IL-2. We conclude that supplementing local radiotherapy with low doses of IL-2 results in increased local tumor control and regression of distant, non-irradiated tumors. This type of radioimmunotherapy is a promising new approach for the clinic.

Anti-tumor effects of local irradiation in combination with peritumoral administration of low doses of recombinant interleukin-2 (rIL-2).

The aim of this work was to improve radiotherapy results by immune stimulation. We tested the effects of a combination of radio- and immunotherapy, i.e., local low dose recombinant interleukin-2 (rIL-2) treatment, in two murine tumor models. Syngeneic tumors (SL2 lymphoma or M8013 mammary carcinoma) were induced subcutaneously on one or both flanks of mice. Irradiation was given either as a single dose (20 Gy) or fractionated (25 Gy) in 2 weeks. One or two cycles of rIL-2 were given concurrent with or subsequent to radiotherapy. One cycle of rIL-2 consisted of peritumoral injections administered on 5 consecutive days. Treatment effects were measured in terms of local tumor response and disease-free survival (DFS). The combined treatment modality was significantly better than treatment with either irradiation alone or rIL-2 alone. When tumors were inoculated on both flanks of the mice, combined radioimmunotherapy of one of the tumors also resulted in regression of the contralateral untreated tumor, indicating that a systemic anti-tumor immune reaction was induced. Additional rIL-2 injections did not enhance radiation toxicity. In conclusion supplementing irradiation with locally administered low doses of rIL-2 results in better local anti-tumor responses and DFS rates than either treatment alone without enhanced treatment toxicity. Furthermore, the local treatment induces a systemic anti-tumor reaction, influencing the growth patterns of a second, untreated tumor.

Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats.

Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.

Gastrointestinal expression and partial cDNA cloning of murine Muc2.

To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM resembled other mucins in physicochemical properties. Anti-MCM recognized MCM as well as rat and human MUC2 on Western blots, interacting primarily with peptide epitopes, indicating that MCM was identical to murine Muc2. Using anti-MCM and previously characterized anti-human and anti-rat MUC2 antibodies, we identified a murine Muc2 precursor in the colon of approximately 600 kDa, which appeared similar in size to rat and human MUC2 precursors. Western blotting, immunoprecipitation of metabolically labeled mucins, and immunohistochemistry showed that murine Muc2 was expressed in the colon and the small intestine but was absent in the stomach. To independently identify murine Muc2, we cloned a cDNA fragment from murine colonic mRNA, encoding the 302 NH2-terminal amino acids of murine Muc2. The NH2 terminus of murine Muc2 showed 86 and 75% identity to the corresponding rat and human MUC2 peptide sequences, respectively. Northern blotting with a murine Muc2 cDNA probe showed hybridization to a very large mRNA, which was expressed highly in the colon and to some extend in the small intestine but was absent in the stomach. In situ hybridization showed that the murine Muc2 mRNA was confined to intestinal goblet cells. In conclusion, by two independent sets of experiments we identified murine Muc2, which appears homologous to rat and human MUC2. Because Muc2 is prominently expressed in the colon, it is most likely to be the predominant mucin in the colonic mucus layer.