Induction of intestinal lesions in nu/nu mice induced by transfer of lymphocytes from syngeneic mice infected with murine retrovirus.

BACKGROUND: Murine leukemia virus, LP-BM5, induces severe immunodeficiency with abnormal lymphoproliferation in susceptible C57BL/6 mice. In a previous study, it was shown that a Sjögren's syndrome-like systemic exocrinopathy is induced in the virus infected mice. AIMS: To examine lymphocyte functions of the virus infected mice. METHODS: Four-week old mice were inoculated with the virus and their spleen cells were transferred into syngeneic nu/nu mice. Their organs were examined by light and electron microscopy. Phenotypes of the colon infiltrating cells were examined by flow cytometry. RESULTS: All nu/nu recipients had died by six weeks after cell transfer, showing runting disease like cachexia with diarrhoea and anal bleeding. Histopathological examination revealed that systemic exocrinopathy was adoptively transferable and that the colon became thickened due to mononuclear cell infiltration into the mucosal and submucosal layer with hyperplasia of intestinal epithelial cells. No virus particles were found in the colon. Flow cytometric analyses revealed that most of the infiltrating CD4+ T cells showed CD45RBlow. No intestinal lesions were observed in the virus infected mice nor in nu/nu mice inoculated with normal lymphocytes. CONCLUSION: Lymphocytes of the virus infected mice induced colitis and hyperplasia of intestinal epithelial cells as well as systemic exocrinopathy in nu/nu mice. Our experimental system may give some insight into intestinal lesions associated with virus infection.

Induction of autoimmune-like hepatic and ductal lesions by administration of lipopolysaccharide in mice undergoing graft-versus-host reaction across MHC class I difference.

In this paper, we examined the induction of autoimmune-like histologic changes in the liver and other organs of mice undergoing graft-versus-host reaction (GVHR) with MHC class I disparity by the administration of bacterial lipopolysaccharide (LPS), on the assumption that stimulation with LPS could be an exacerbating factor. Spleen cells of C57BL/6 (B6) mice were injected twice into (B6 x bml) F1 recipient mice at an interval of 7 days to induce MHC class I GVHR and then challenged with 1 microg of LPS intravenously on the next day of the cell transfer. The hepatic lesions of the group of MHC class I GVHR mice challenged with LPS showed marked cellular infiltration at the portal area and focal necrosis was observed in the hepatic lobule. The major infiltrating cells were CD8+, and others including CD4+ cells being of minor populations. In addition, ductal lesions in extrahepatic organs, including the pancreas and salivary glands also showed marked cellular infiltration. Thus, we have demonstrated that LPS induced ductal lesions in mice with MHC class I disparity. CD8+ cells were detected at the destructive hepatic lesions, which might be effector cells. These findings indicate that LPS might be one of the potential factors which augment autoimmune-like lesions.

[Quantitative analysis of organophosphorous pesticide residues in Chinese drugs].

This paper reports the GC determination of 13 organophosphorous pesticides in Chinese drugs Flos Ionicerae and Moluodan etc by the present method of Japan for determining pesticide residues. The results suggest that the determined samples do not contain the above said 13 organophosphorous pesticides.

[Quantitative analysis of organochlorine pesticide residues in Chinese drugs].

This paper reports the GC determination of 20 organochlorine pesticides in Chinese drugs Flos Ionicerae and Moluodan etc by the present method of Japan for determinaing pesticide residues. The results suggest that except Folium Isatidis, Radix Codonopsis and Sanqi Pian all accord with the for provisions pesticide residues in Japanese foodstuffs.

Concentric fibrosis and cellular infiltration around bile ducts induced by graft-versus-host reaction in mice: a role of CD8+ cells.

We report in this paper on obvious fibrotic lesions in the liver of mice undergoing specified graft-versus-host reaction (GVHR). B6 CD8+ splenocytes were transferred into (bm 12 x bm 1)F1 mice to induce GVHR. Recipient mice had been thymectomized and administrated with anti-CD8 monoclonal antibody (mAb) to deplete CD8+ cells from the hosts. Two weeks after the mAb administration, recipient mice were injected with B6 CD8+ cells and sacrificed further two or four weeks later for analyzing hepatic lesions histopathologically. Light microscopic analyses revealed the presence of concentric fibrosis around both small and large duct levels and the infiltration of mononuclear cells into portal areas. Focal necrosis of hepatocytes was also detected electron-microscopically. These findings suggest that CD8+ T lymphocytes might play an important role in the induction of fibrotic lesions in the liver.

Mechanism of the induction of autoimmune disease by graft-versus-host reaction. Role of CD8+ cells in the development of hepatic and ductal lesions induced by CD4+ cells in MHC class I plus II-different host.

BACKGROUND: We previously reported that primary biliary cirrhosis (PBC)-like hepatic lesions were induced in (bm12xB6)F1 mice undergoing graft-versus-host reaction with major histocompatibility complex class II disparity. In this paper, we report on a new experimental system, enabling establishment of more progressed stages of PBC-like lesions and clarification of the role of CD8+ cells in the development of the lesions. EXPERIMENTAL DESIGN: Recipient (bm12xbm1)F1 mice were thymectomized and administered anti-Lyt-2 monoclonal antibodies intraperitoneally to deplete host CD8+ cells as completely as possible. The treatment was necessary to induce autoimmune hepatic lesions in this type of F1 mice. Recipients were divided into five groups: group 1 received B6 CD4+ cells on day 0 and 2 weeks later, B6 CD8+ cells; group 2, only B6 CD4+ cells on day 0; group 3, only B6 CD8+ cells on day 14; group 4, B6 CD4+ on day 0 plus F1 CD8+ cells 14 days later. Cell transfer was not done in group 5. All of the mice were killed on day 28 for examination by light and electron microscopy and also by immunohistochemistry. RESULTS: PBC-like hepatic lesions without tissue destruction were induced in the mice of group 2, as was previously reported. In addition to similar lesions to group 2, destruction of hepatocytes and bile duct epithelia was induced in the mice of group 1. Weak lymphocytic infiltration and periductal concentric fibrosis were observed in the mice of group 3. PBC-like hepatic lesions without tissue destruction were induced in mice of group 4 as were those of group 2. However, the cellular infiltration was much weaker. CONCLUSIONS: For the animal model of PBC, we have devised a new experimental system in which the role of donor or host type CD8+ cells is assessable. Tissue-destructive lesions were induced only in mice that received donor CD4+ cells followed by CD8+ cells. The PBC-like lesions were suppressed by host type CD8+ cells. These results suggest that destructive hepatic lesions of PBC might progress through CD8+ cells in cooperation with CD4+ cells, and that host type CD8+ cells could act as "regulatory" T cells for the progression of the lesions.

Exocrinopathy resembling Sjögren's syndrome induced by a murine retrovirus.

BACKGROUND: Sjögren's syndrome (SS) is characterized by lymphocytic infiltration into, and destruction of exocrine glands, resulting in dryness of the mouth and eyes. The disease is considered to have an autoimmune etiology, however, its etiopathogenesis remains largely unknown. Recently, retrovirus is suggested to participate in the pathogenesis of SS, because SS-like lesions are reported in HIV infection or in human T cell leukemia virus type I infection. Moreover, human intracisternal A-type retroviral particles are reported to be detected in SS patients. During the course of our study on the histopathology of mice infected with a murine retrovirus, we happened to find SS-like exocrinopathy in those mice. EXPERIMENTAL DESIGN: Four-week-old C57BL/6 (B6) mice were injected intraperitoneally with LP-BM5 murine leukemia virus. This virus is known to induce splenomegaly, lymphadenopathy followed by lymphoid malignancy, and profound immunodeficiency in sensitive strains of mice. From 4 to 16 weeks after the virus inoculation, the infected mice were sacrificed and their submandibular and lacrimal glands were analyzed light and electron microscopically and immunohistochemically. The existence of the virus in the lesion in situ was also analyzed by the same method, and additionally by a polymerase chain reaction method. RESULTS: Periductal lymphocytic infiltration into the submandibular and lacrimal glands was observed in all the virus-infected mice at 4 weeks after the infection and progressed with time. Extraglandular lymphocytic infiltration was also observed in liver, kidney, lung, and pancreas. Immunohistochemical examination revealed that most infiltrating cells into the glands were composed of CD3+ T cells (CD4-dominant), Mac-1+ cells, and B220+ cells. The virus genome was detected in submandibular glands by immunohistochemistry or by polymerase chain reaction. In addition, retroviral particles were secreted into the lumen of exocrine ducts of submandibular glands. CONCLUSIONS: This might be an SS animal model that is induced by a certain defined retrovirus. This experimental system might provide us with valuable information for analyzing the mechanisms of how a retrovirus could induce SS.

Inhibition of intracellular topoisomerase II by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors.

In the accompanying paper (K. Tanabe, Y. Ikegami, R. Ishida, and T. Andoh, Cancer Res., 51: 4903-4908, 1991), we showed that ICRF-154 and -193, dioxopiperazine derivatives, inhibited the activity of purified topoisomerase II, without formation of a cleavable DNA-protein complex. In order to see whether ICRF-154 and ICRF-193 affect cellular topoisomerase II in situ or not, we examined the effect of these drugs on etoposide (VP-16)-induced, topoisomerase II-mediated DNA breaks in RPMI 8402 cells by alkaline sedimentation analysis. When RPMI 8402 cells were exposed to VP-16 in the presence of ICRF-154 or ICRF-193 for 1 h, VP-16-induced DNA strand breaks were greatly inhibited by both ICRF compounds. In parallel with this observation, VP-16-induced growth inhibition was also reversed by ICRF-193. Exposure of cells to ICRF-154 resulted in a progressive accumulation of cells with 4C DNA content. Although mitotic index did not significantly increase, mitotic abnormalities were seen in cells exposed to ICRF-193 or ICRF-154: all mitotic cells exhibited early mitotic figures with fewer condensed and entangled chromosomes. The most sensitive phase of the cell cycle to ICRF-154 was the G2-M. ICRF-154 did not affect the spindle formation. However, abnormally oriented spindles were observed in drug-treated cells in parallel with the appearance of multinucleated cells. The results suggest that ICRF-154 and -193 inhibit topoisomerase II activity in RPMI 8402 cells, and this effect resulted in the appearance of cells in G2 and early M phase with fewer condensed and entangled chromosomes and of cells with multilobed nuclei.

Maintenance of embedded pig pancreatic pseudo-islets in a collagen gel matrix: study of the effect of hydrocortisone, a collagenase inhibitor, and nicotinamide on collagenolysis and the morphogenesis of pancreatic islet-cells in collagen gel matrix.

We describe a method for maintaining neonatal pig pancreatic isletlike cell clusters (as pseudo-islets) embedded in a collagen gel matrix for long periods. The pseudo-islets were formed from single cells of pig pancreas maintained in a suspension culture and then embedded in pepsin-solubilized type I collagen. When the pseudo-islets were cultured in the collagen matrix, the amount of collagen in the culture decreased gradually during the culture period as soluble hydroxyproline-containing material accumulated in the medium. A low concentration of collagen (0.16%) degraded the collagen gels more rapidly than did high concentrations of collagen (0.64%). The degradation of collagen depended both on the number of pseudo-islets embedded in the gel matrix and on the culture conditions used to maintain them. With added nicotinamide, the accumulation of hydroxyproline decreased in the medium and the structure of the gel matrix was well maintained. Hydrocortisone or a specific inhibitor of collagenase did not decrease the solubilization of embedded pseudo-islet cultures and did not help to maintain their structure. These observations indicate the possible utility of long-term maintenance of pseudo-islets in collagen gel matrix in the presence of nicotinamide.

Immunocytochemical studies on the islet and the gut of the arctic lamprey, Lampetra japonica.

The endocrine cells and nerves in the islet and the gut of the arctic lamprey Lampetra japonica were examined immunocytochemically by using antisera against brain-gut peptides and amine. The cellular composition of the islets as reported by previous researchers in European species of the lamprey was confirmed in the present study. The islet consisted exclusively of insulin immunoreactive cells in the larvae (ammocoetes), whereas in the adult somatostatin immunoreactive cells were added to the insulin immunoreactive cells; the gut epithelium in the adult was now devoid of somatostatin cells. In the gut of the lamprey, the endocrine cells--which were flask-shaped with a cytoplasmic process extended to the lumen--were classified into three types in the larvae, but were represented by a single type in the adult. In the larval lamprey, the first type was immunoreactive for somatostatin, the second one for gastrin/cholecystokinin (CCK) and the third cell type was immunoreactive for glucagon, pancreatic polypeptide and FMRFamide, simultaneously. In the gut of the adult lamprey, the single type of endocrine cell reacted simultaneously to C-terminal specific anti-glucagon serum, N-terminal specific anti-glucagon serum, anti-bovine PP serum, anti-neuropeptide Y serum and anti-FMRFamide serum. These cells occurred most frequently in the upper intestine, their distribution decreasing from the middle to the lower intestine. Two types of peptide containing nerves were identified in the islet and the gut of the larval and adult lamprey. The first type of neurons (perikarya and fibers) was immunoreactive for serotonin and calcitonin gene-related peptide (CGRP), and was located in the mucous and muscular layer of the intestine and in the islet. The second type of neurons contained both serotonin- and gastrin releasing peptide (GRP)-like immunoreactivities and was scattered exclusively in the muscular layer of the gut. In larval and adult lampreys, a few serotonin/CGRP immunoreactive nerve cell bodies and beaded fibers were found in the connective tissue around the islet cell cords. These nerve fibers were sometimes closely apposed to the blood capillaries and to the islet cells. These findings indicate that a neuroendocrine correlation comparable with that in mammals may have been established in the islet of this most primitive vertebrate.

Monolayer-forming islet cell culture from neonatal pig pancreas: using sequential treatment with EDTA-dispase and monoiodoacetic acid for preparation and purification.

A new method for the preparation and purification of a monolayer-forming islet cell culture from the neonatal pig pancreas is described. Single cell preparations of the pig pancreas were obtained by gently stirring the chopped pancreases in a medium containing sequential EDTA-dipase. Exposure of the cells to monoiodoacetic acid (10 micron) and BSA-gradient resulted in improved preparation, highly enriched in the endocrine cells. The cells were maintained in tissue culture medium 199 containing 5.5 mM D-glucose, with or without 0.1 mM 3-isobutyl-1-methylxanthine, or 16.7 mM D-glucose and 20% fetal calf serum. During a 6-day culture period, many small cell aggregates appeared in the media. The cell clusters attached to the bottom of the dish and formed monolayers. Provocative stimulation and radioimmunoassay for insulin showed the existence of numerous and viable B cells in the cell clusters. The other three types of islet cells were also demonstrated immunohistochemically in the cell cluster. It is concluded that this improved culture technique provides a useful tool for morphological and biochemical studies of islet cells and a potential source of material for transplantation of B-cells from the pancreas.

Serotonergic efferent nerve fibers in the retinal plexiform layer of the abalone.

Serotonin (5-HT)-immunohistochemistries at light- and electron-microscopic levels, using rabbit anti-5-HT serum (#1234), were applied to the whole head and only to the eye of the abalone, respectively. Peroxidase-antiperoxidase and fluorescein isothiocyanate methods were used for the light-microscopic immunohistochemistries. Many immunoreactive nerve fibers were demonstrated in the outer zone of the retinal plexiform layer, small optic nerve fiber bundles, the optic nerve trunk and the cerebral ganglion. Immunoreactive somata were observed only in the cerebral ganglion. Accordingly the immunoreactive fibers in the retinal plexiform layer are considered to be efferent. Cored vesicles in the retinal plexiform layer demonstrated by both conventional chemofixation and a rapid-freeze-substitution method showed strong immunoreactivities localized within their limiting membrane. The same fibers also contained small clear vesicles. They are considered to be different from larger clear vesicles in non-immunoreactive fibers reportedly containing acetylcholine. The function of the efferent fibers remains to be elucidated.

Immunocytochemical studies on the pancreatic islets of the ratfish Chimaera monstrosa.

The islet cells and nerves in the pancreas of the ratfish Chimaera monstrosa were examined immunocytochemically by using antisera against mammalian brain-gut peptides. Five types of islet cells were recognized. The B and D cells reacted to anti-insulin and anti-somatostatin sera, respectively. The A and X cells exhibited glucagon-like immunoreactivities. The N-terminal anti-glucagon serum reacted both to the A and the X cells, while the C-terminal anti-glucagon serum bound specifically to the X cells. These results suggested that the X cells contained pancreatic-type glucagon, whereas the A cells, an enteroglucagon (glicentin)-like substance. A fifth type of endocrine cell was scattered in the islets and contained serotonin-like immunoreactivity. Two kinds of peptide nerves were identified. Vasoactive intestinal polypeptide (VIP)-immunoreactive neuronal somata were located in the intrapancreatic ganglia. Nerve fibers and terminals containing VIP-like immunoreactivities occurred in the pericapillary space surrounding the islets. Gastrin releasing peptide (GRP)-immunoreactive nerve somata and fibers were scattered along the margins of the islets. The pericapillary arrangement of these nerve terminals suggests a hemocrine release of the peptides.

Glucagon-related peptides in the rat hypothalamus.

Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.

PHI-like immunoreactivity in the nervous system of the cockroach (insect) and Aplysia (mollusc) with special reference to its relationship to VIP-like immunoreactivity.

Studies in mammals have indicated that PHI (peptide histidine isoleucine) and VIP (vasoactive intestinal polypeptide) share a prepropeptide, and that both peptides are contained in the same neurons. The present study proposes that this relationship may not hold true in protostomian invertebrates. In the nervous system of the cockroach (Insecta, Arthropoda) and Aplysia (Gastropoda, Mollusca) distribution of PHI and VIP was examined by immunocytochemistry. In both animals neurons containing PHI-like immunoreactivity were numerously found and only a small part of them in the cockroach contained VIP-like immunoreactivity. These findings suggest that in these invertebrates, PHI may play a more important regulatory role than VIP. Possible reasons for the difference in the distribution of immunoreactivities for PHI and VIP are discussed.

Neuropeptide immunocytochemistry in protostomian invertebrates, with special reference to insects and molluscs.

In some molluscs (Aplysia and Fusitriton) and insects (silkworm and cricket), occurrence and distribution of neuropeptides in the nervous system and gut were studied with following results: in these invertebrates and also in planaria, PP-like immunoreactivity is extensively distributed in neurons and (in insects) in gut endocrine paraneurons. These cells are negative for NPY, the mammalian neuropeptide related to PP in molecular structure. PHI-like immunoreactivity is widely distributed in the neurons of those invertebrates; it occurs also in gut endocrine paraneurons in insects. The PHI-immunopositive cells are immunonegative for VIP and the coexistence of both peptides due to the common precursor in mammals cannot be recognized in these invertebrates. Immunoreactivity for urotensin I, the neuropeptide derived from teleostean urophysial neurons, is widely distributed in the neurons of the invertebrates. In insects (cricket) it occurs in gut endocrine cells.

Co-existence of enkephalin and adrenalin in the frog adrenal gland.

By means of immunohistochemistry combined with formaldehyde-induced fluorescence microscopy, met-enkephalin-like immunoreactivity was found to occur in the adrenalin -cells of frog adrenal glands. Immunoelectron microscopy demonstrated that the immunoreactive material is confined to the secretory granules. These findings suggest the concomitant release of enkephalin and adrenalin by exocytosis.

An electron microscopic study on enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs.

Enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs were characterized by a high population of large granular vesicles mixed with small clear vesicles. The immunoreactive material is confined to the large granular vesicles. The immunoreactive nerve fibers formed many axo-dendritic as well as axo-somatic synapses and also formed a few synapses with presumed preganglionic axons containing numerous vesicles. The immunoreactive fibers were regarded as presynaptic at these synapses. These findings suggest that enkephalin might play a role as a neurotransmitter or neuromodulator in the ganglionic transmission of this prevertebral ganglion.