Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Melanin as a natural germ cell marker for intraspecific transplantation experiments in Ambystoma mexicanum (Urodela,Amphibia)

Summary In urodele amphibians, the lack of a reliable germ cell marker restricts the experimental study of the germ lineage. In the present work, we conducted genetic and histological analyses in order to demonstrate that melanin from oocytes constitutes a germ cell marker available for intraspecific experiments in Ambystoma mexicanum. Then, using this marker, we implanted germ cells from undifferentiated gonads (stage 48) into the blastocoel of host embryos and investigated their fate and determined state. Our results show that, from this stage on, the donor cells do not differentiate into other cell types; therefore, they are restricted in developmental capacity and irreversibly determined as germ cells. On the other hand, exogenous germ cells were found in an isotopic position until the young tail-bud stage, and then were found in an ectopic position; these results suggest that, from the middle tail-bud stage on, an active process contributes to migration of primordial germ cells to the gonadal territory.     

Pattern of cell proliferation in embryogenesis and planula development ofHydractinia echinata predicts the postmetamorphic body pattern

Summary During embryogenesis and planula development of the colonial hydroidHydractinia echinata cell proliferation decreases in a distinct spatio-temporal pattern. Arrest in S-phase activity appears first in cells localized at the posterior and then subsequently at the anterior pole of the elongating embryo. These areas do not resume S-phase activity, even during the metamorphosis of the planula larva into the primary polyp. Tissue containing the quiescent cells gives rise to the terminal structures of the polyp. The posterior area of the larva becomes the hypostome and tentacles, while the anterior part of the larva develops into the basal plate and stolon tips. In mature planulae only a very few cells continue to proliferate. These cells are found in the middle part of the larva. Labelling experiments indicate that the prospective material of the postmetamorphic tentacles and stolon tips originates from cells which have exited from the cell cycle in embryogenesis or early in planula development. Precursor cells of the nematocytes which appear in the tentacles of the polyp following metamorphosis appear to have ceased cycling before the 38th hour of embryonic development. The vast majority of the cells that constitute the stolon tips of the primary polyp leave the cell cycle not later than 58 h after the beginning of development. We also report the identification of a cell type which differentiates in the polyp without passing through a post-metamorphic S-phase. The cell type appears to be neural in origin, based upon the identification of a neuropeptide of the FMRFamide type.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

A possible contribution by glial cells to neuronal energy production enzyme-histochemical studies in the developing rat cerebellum

Summary Recent reports have revealed that certain neurons do not survive in vitro in the presence of glucose, which is the primary substrate and exclusive source of energy in the brain. But these neurons can survive in the presence of low-molecular-weight agents such as pyruvate, which are supplied by glial cells (Selak et al. 1984). To test whether this result also holds true in vivo, we investigated the distribution of hexokinase, lipoic dehydrogenase, -hydroxybutyrate dehydrogenase, and glucose-6-phosphate dehydrogenase activities in the developing rat cerebellum. Hexokinase activity was relatively higher in glial cells than in neurons. After postnatal day 8, the activity of hexokinase could hardly be detected in Purkinje cells, whereas it was highest in Bergmann glial cells. Purkinje cells were the only type of neuron with high levels of lipoic dehydrogenase at all ages tested. -Hydroxybutylate dehydrogenase activity was also high in Purkinje cells, especially in those from young rats. Relatively high glucose-6-phosphate dehydrogenase activity was demonstrated in basket and stellate cells from adult brain. Thus, it appears that, in vivo, certain neurons utilize relatively little glucose, and it is indeed possible that glial cells may supply some substance(s) other than glucose, for example pyruvate, as the primary source of energy.     

Developmental appearance of the Ca2+-binding proteins parvalbumin,calbindin D-28K,S-100 proteins and calmodulin during testicular development in the rat

Summary Calcium and intracellular Ca²⁺-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.     

Serotonin and gastrin/cholecystokinin-like immunorcactive neurons in the larval retrocerebral complex of the blowfly Calliphora erythrocephala

Summary The presence and distribution of neurons immunoreactive against antibodies to serotonin (5-HT) and gastrin/cholecystokinin (gastrin/CCK) has been studied in the larval retrocerebral complex of the blowfly Calliphora erythrocephala, a composite structure which consists of the corpus cardiacum, the corpus allatum, the thoracic gland and a portion of the cephalic aorta. Immunoreactive material was found in all these elements except in the corpus allatum. Six to eight cell bodies in the corpus cardiacum and four to eight cell bodies in the thoracic gland were 5-HT immunoreactive (5-HTi). These 5-HTi cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the cephalic aorta, corpus cardiacum and ventral part of the thoracic gland. Six to eight cell bodies in the corpus cardiacum and four to six cell bodies in the thoracic gland reacted with antibodies against gastrin/CCK. These cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the corpus cardiacum and the cephalic aorta in a pattern resembling that of the 5-HTi fibers. Additional gastrin/CCK-like immunoreactive fibers were shown to come from the central nervous system via the two nervi corporis cardiaci. An electron-microscopical analysis was performed to analyze further the morphological features revealed by the light-microscopic immunocytochemical technique. This confirmed the existence of neurosecretory-like terminals among the gland cells of the thoracic glands and the existence of neurohemal sites in several regions of the larval retrocerebral complex. Some functional aspects of the retrocerebral complex are discussed on the basis of the presented data.     

Atropine inhibits the degranulation of Paneth cells in ex-germ-free mice

Summary Previous studies have shown that the secretory products of Paneth cells contain antibacterial agents (lysozyme, IgA) that are affected by the bacterial milieu in the intestine. To investigate whether Paneth-cell secretion is controlled via cholinergic mechanisms, the ultrastructure of Paneth cells was studied in four animal groups: (1) germfree (GF) control mice (Jcl: ICR [GN], male, 13 weeks old), (2) GF mice injected subcutaneously with atropine sulfate (200 mg/kg body weight, dissolved in physiological saline 20 mg/ml), (3) ex-GF mice inoculated with feces from specific-pathogen-free (SPF) mice, and (4) ex-GF mice injected with atropine and inoculated with feces from SPF mice. In ex-GF mice inoculated with feces, 70–90% of the Paneth cells showed fewer secretory granules than those from GF mice (p<0.01). Approximately 30% of the Paneth cells had a large vacuole (3–10 m diameter) in the apical cytoplasm. Exocytosed electron-dense material from secretory granules was observed in a few crypt lumens. In ex-GF mice inoculated with feces and given atropine, about 90% of the Paneth cells contained numerous secretory granules, like those in GF control mice, but vacuolated Paneth cells and exocytotic figures were rare; thus the secretion of Paneth cells was blocked by atropine. It is therefore possible that the bacterial milieu in the intestine affects the secretory activity of Paneth cells via cholinergic mechanisms.     

Morphological studies on endocytosis of chondroitin sulphate proteoglycan by rat liver endothelial cells

Summary Endocytosis via the hyaluronic acid/chondroitin sulphate receptor of rat liver endothelial cells was studied ultrastructurally, by use of a probe consisting of chondroitin sulphate proteoglycan attached to 15-nm gold particles. The probe bound to the surface of the cells exclusively in coated regions of the plasma membrane. Internalization at 37° C took place in less than one minute during which time interval the bound probe was transferred to coated vesicles. Further transfer to lysosomes was delayed in association with an accumulation of probes in a prelysosomal compartment consisting of large vacuoles in which probes lined the inner aspect of the membrane. Transport to lysosomes occurred only after a lag phase of at least 40–60 min at 37° C.Abbreviations CS chondroitin sulphate - CSPG chondroitin sulphate proteoglycan - CSPG-Au CSPG-gold complex - EM electronmicroscopical or electron microscopy - HA hyaluronic acid - KC Kuppfer cells - LEC liver endothelial cells - PC parenchymal cells - RES reticuloendothelial system