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Histone deacetylase inhibitors are a new and promising drug family with a strong anticancer activity and potent modulation of connexin expression. The restoration of connexins in cancer cells has been suggested as a possible mechanism to control tumor progression. The aim of this study was to investigate the effects of 4-phenylbutyrate (4-PB) on the growth of human pancreatic cell lines in vitro and in vivo with a focus on connexin modulation. The proliferation of tumor cells was determined using an MTT assay, and the effect of 4-PB in vivo was studied in a chimeric mouse model. The expression and localization of connexin 43 (Cx43) were assessed by Western blot, immunofluorescence microscopy, and immunohistochemistry. Antitumoral activity was assessed by immunohistochemistry for Ki-67 and histone H4. Treatment with 4-PB resulted in the significant in vitro and in vivo growth inhibition of pancreatic tumor cells. The reduction of the xenograft tumor volume was associated with the inhibition of histone deacetylation and decrease in cell proliferation. Treatment with 4-PB caused a significant increase in the Cx43 expression in T3M4 cells (up to 2.8-fold). The newly synthesized Cx43 was localized in the cytoplasm but not on the cell membrane. Treatment with 4-PB inhibited the proliferation of human pancreatic tumor cells in vitro and in vivo and increased the expression of Cx43. Therefore, 4-PB might be useful in the therapy of pancreatic cancer.

The response of the oesophageal microcirculation to luminal damaging agents may play an important role in reflux-induced mucosal injury. We characterized the microcirculatory consequences of exposure to bile with or without hydrochloric acid, and determined the changes in the constitutive nitric oxide synthase and inducible nitric oxide synthase activities in a canine model of acute reflux oesophagitis.Group 1 served as a saline-treated control, while groups 2-4 were exposed for 3 h to bile alone, to hydrochloric acid, or to bile + hydrochloric acid, respectively. The mucosal microcirculation was observed continuously by means of intravital videomicroscopy with an orthogonal polarization spectral imaging technique. Myeloperoxidase, constitutive and inducible nitric oxide synthase activities were measured via tissue biopsies, while the degree of mucosal damage was evaluated histologically.Bile evoked deep tissue damage and leucocyte accumulation in the mucosa and muscle layer. The capillary red blood cell velocity and the relative vessel area increased significantly (P0.05). The constitutive NO synthase activity was decreased, and the inducible NO synthase activity was increased significantly. In the hydrochloric acid-treated group the functional capillary density decreased, the mucosal damage was less severe, the constitutive NO synthase activity did not change, whereas the inducible NO synthase activity was increased significantly. The constitutive NO synthase activity did not change after the bile + hydrochloric acid treatment either.Reflux components induce characteristic microcirculatory alterations. The structural damage and leucocyte invasion are accompanied by bile-induced constitutive NO synthase inhibition when hydrochloric acid production is suppressed.

Nicotine adenine dinucleotide phosphate-dia- phorase staining was used to study nitric oxide synthase activity and distribution in the midbrain visual structures of white leghorn chick embryos and post-hatched chick- ens. Enzyme staining first appeared in the isthmic region at the tenth embryonic day (E10) in the neuropil of the nucleus isthmi, pars parvocellularis. At E11 faint enzyme positivity appeared also in the nucl. isthmi pars magnocellularis, the nucl. semilunaris and the nucl. isthmo-opticus. The staining intensity of the isthmic nuclei dramatically increased between the 12.5th and the 13th days of incubation. The nucl. isthmi, pars parvocellularis showed the strongest enzyme reaction throughout embryonic life. A day before hatching all the isthmic nuclei were heavily stained, however, nicotine adenine dinucleotide phosphate-diaphorase-positive cells occurred exclusively in the nucleus isthmo-opticus. In the tectum opticum, intensely stained cells occupied the stratum fibrosum et griseum superficiale. The layer con- taining the projection neurons to the isthmo-optic nucle- us was unstained. In the isthmic region, the intensity of staining surpassed that of the tectum and reached its maximum at E17 and then slowly decreased till the end of the experimental period (120 days post-hatched). The tractus isthmo-opticus showed nicotine adenine dinucle- otide phosphate-diaphorase activity throughout the in- vestigated period of life of the chicken, but the tractus tectoisthmo-opticus was unstained. Our results suggest that in the isthmic nuclei, nicotine adenine dinucleotide phosphate-diaphorase-positive neurons occur only in the isthmo-optic nucleus and optic tectum. The other posi- tively stained structures are the fibers and terminals of tectal cells. In most brain areas nicotine adenine dinucle- otide phosphate-diaphorase indicates nitric oxide syn- thase that produces nitric oxide. The transient appear- ance of this molecule is probably necessary for neuronal differentiation or the establishment of synaptic connec- tions in the isthmic nuclei, and these developmental changes are under the control of the optic tectum.

Gadolinium chloride (GdCl 3 ), a rare earth metal salt, depresses macrophage activity, and is commonly used to study the physiology of the reticuloendothelial system. In the present work, the effect of GdCl 3 -induced Kupffer cell blockade on the humoral immune response in mice to sheep red blood cells (SRBC) was investigated. Kupffer cell phagocytosis blockade was found to increase both the primary and secondary immune responses to SRBC. The primary immune response was significantly augmented in animals injected intravenously with GdCl 3 2, 3 or 4 days before injection of the cellular antigen, but GdCl 3 injected 7 days before the antigen did not modify the immune response. Increased secondary humoral immune responses were also observed. When GdCl 3 was injected 2 days before the second dose of antigen, the numbers of both IgM and IgG-producing plaque forming cells were augmented. GdCl 3 injected 2 days before the first dose of SRBC did not modify the humoral immune response. Earlier studies with 51 Cr-labelled foreign red blood cells suggested that the augmentation of the humoral immune response in GdCl 3 -pretreated mice is a consequence of the spillover of the antigen from the liver into the spleen and other extrahepatic reticuloendothelial organs.

Objective and Design: Rats treated with aggregated IgG (Aggr.) become 'refractory' to the hypotensive action of a second dose of Aggr. The objective of this study was to assess the responsiveness of animals pretreated with Aggr. to bacterial LPS and vice versa.¶Material or Subjects: Female Wistar rats (250–300 g) were used. Each experiment was carried on at least 4 animals.¶Treatment: A human IgG preparation containing 30% aggregates (10–16 mg/100 g) or E. coli serotype 0111.B4 (0.005–1 mg/100 g) was administered i.v. Certain groups of animals were pretreated with 1 mg/100 g GdCl3 or with 10 mg/100 g pentoxyphylline (PTX).¶Methods: Arterial blood pressure was monitored in the carotis using a polyethylene cannula and an electronic tension meter. Tumor necrosis factor alpha (TNF-α) activity was estimated by the use of an L-929 cell cytotoxicity assay.¶Results: Pretreatment of rats with a sublethal dose of LPS impaired the hypotensive reaction of the animals to Aggr. Rats made 'refractory' to Aggr. reacted to the injection of LPS with hypotension and a second phase milder than in the controls. Hypotension could not be elicited by Aggr. in rats pretreated with GdCl3. The same pretreatment had no effect on the first phase of hypotension induced by intravenous injection of LPS, whilst a mitigation of the second phase was observed. Infusion of PTX immediately prior to Aggr. administration prevented the drop of blood pressure. A sizeable level of TNF-α was detected only later than blood pressure had reached its minimum level following Aggr. administration.¶Conclusions: Hypotension induced by LPS may involve a macrophage population broader than that responsible for the vascular action of Aggr. The data presented do not support a primary role for TNF-α in Aggr. induced hypotension.

Objective and design: The consequences of Kupffer cell phagocytosis blockade were studied in endotoxemic rats with obstructive jaundice.¶Material or Subjects: 159 male Wistar rats.¶Treatment: Obstructive jaundice was induced by bile duct ligation (BDL). Gadolinium chloride (1 mg/100g iv) was given 6 days after BDL to inhibit Kupffer cell activity and the animals were challenged with 1 mg/g endotoxin 24 h later.¶Methods: Endotoxin sensitivity, tumor necrosis factor-α and interleukin-6 production were studied, liver and lung injury were assessed by neutrophil infiltration assay, tissue adenosine triphosphate, aspartate aminotransferase, alanine aminotransferase level determinations and histology, respectively. For statistics non-parametric methods were used.¶Results: BDL sensitized the animals to endotoxin, increased endotoxin-induced tumor necrosis factor-α and interleukin-6 production and reduced ATP contents of the liver and the lung. Kupffer cell blockade significantly increased the resistance against endotoxin, diminished the inflammatory cytokine release and reduced endotoxin-induced tissue injury in BDL animals.¶Conclusion: Attenuation of Kupffer cell function decreases endotoxin-induced lethality and morbidity in obstructive jaundice.

Data in the literature concerning the role of Kupffer cells in anaphylaxis are contradictory. In the present study the effect of Kupffer cell blockade induced by gadolinium chloride (GdCl3) on anaphylactic shock was investigated. Our observations show that GdCl3 prevents lethal anaphylactic shock in mice sensitized to ovalbumin. Gadolinium chloride given i.v. in a dose of 1 mg (100 g)−1 body weight 24 or 48 h before the elicitation of anaphylactic shock resulted in 80% survival, compared with the 43% survival in the control group. The same dose of gadolinium chlorde and also other rare earth metal salts, like lanthanum-, praseodymium-, neodymium-, and european chlorides given i.v., one day before the elicitation of anaphylactic shock, greatly reduced the mortality in mice sensitized with ovalbumin and Bordatella pertussis vaccine. However, gadolinium chloride given, per os, in a dose of 2 mg (100 g)−1 body weight, one or two days before the elicitation of anaphylactic shock were ineffective. Gadolinum chloride similarly abrogated the symptoms of anaphylaxis, including the accumulation of serotonin and histamine in the liver. The results suggest that Kupffer cells play an important role in mouse anaphylaxis.

Although the pathogenesis of cervical inlet patch (CIP) is not fully understood, most authors consider it as a congenital abnormality, whereas others surmise it to be related to gastroesophageal reflux disease (GERD). We aimed to evaluate esophageal function and the prevalence of GERD and Barrett's esophagus in patients with CIP. GERD is defined by the presence of erosive esophagitis or an abnormal pH monitoring. Seventy-one consecutive patients with endoscopic and histological evidence of CIP were prospectively evaluated. Esophageal symptom analysis, 24-hour simultaneous biliary reflux and double-channel pH-monitoring, and esophageal manometry were carried out in 65/71 (92%) patients and in 25 matched controls. Six patients were not suitable for testing and were, therefore, excluded. The histological evaluation of the heterotopic islands showed cardia and/or oxyntic mucosa in 64/65 (98%) patients and specialized intestinal metaplasia (SIM) in one patient (2%). The cardia and/or oxyntic mucosa was accompanied by focally appearing pancreatic acinar metaplasia and pancreatic ductal metaplasia in 7/64 (11%) and in 1/64 (2%), superficial mucous glands in 6/64 (9%), and SIM in 2/64 (3%) cases. In total, SIM was present in three patients (5%), and one of them had low-grade dysplasia. At the gastroesophageal junction, 28 (43%) patients had columnar metaplasia, including nine (14%) patients with SIM. Erosive esophagitis was present in 37 (57%) cases. Thirty-two patients (49%) had abnormal acid reflux in the distal and 25 (38%) in the proximal esophagus. Abnormal biliary reflux was present in 25 (38%) cases. On the basis of endoscopic and pH studies, GERD was established in 44/65 (68%) patients. Typical reflux symptoms were common (33/65, 51%). The combined 24-hour biliary and double-channel pH-monitoring detected significantly more significant acidic reflux at both measurement points and significantly longer bile exposure time in the distal esophagus in patients with CIP. Acid secretion in the CIP was detected in three (5%) cases. Esophageal manometry revealed decreased LES pressure and prolonged relaxation with decreased peristaltic wave amplitude, and an increased number of simultaneous contractions in the esophageal body. The detailed evaluation of the esophageal morphology and function in subjects with CIP showed a high prevalence of GERD and Barrett's esophagus. Further studies are needed to evaluate whether combined acidic and biliary reflux is able to promote similar histomorphological changes in the CIP, as it is shown distally in patients with Barrett's esophagus.

The hypotensive side effect of four IgG preparations was studied in a rat model. An unmodified, chromatographically isolated preparation (prepn. C) appeared to induce a transient drop in blood pressure via the kininogen kinin system. A preparation made by ethanol fractionation (prepn. E) and an ethanol-fractionated, modified product (prepn. M) produced an action which does not depend on kininogen activation. No blocking of kininase was required for this effect and it was brought about by prepn. M was devoid of any sizeable prekallikrein activator or kallikrein like proteinase content. The effect was associated by the aggregate fraction of prepn. E, but not with IgG monomers of the same preparation. Animals given these latter types of IgG preparations were refractory to a second dose 24 hours later. The involvement of PAF in the kinin-independent hypotensive action is suggested by the finding that PAF-receptor antagonist BN 52021 prevented the effect and returned the blood pressure close to normality when given during hypotension. A blockade of Kupffer cells brought about by administration of GdCl3 was also found to prevent the effect pointing to the involvement of these type of cells in the reactions.

Alloantigens are recognized by T-cells in the context of both class I and class II antigen, but class II antigens predominate in the recognition of xenoantigens. Since class II molecules bind peptides derived from exogenous proteins that have been phagocytized and digested into small fragments by antigen presenting cells, in the present studies the effect of gadolinium chloride (GdCl 3 )-induced Kupffer cell blockade on the survival of discordant insulinoma cell xenografts was investigated. Insulinoma cells isolated by means of collagenase from human insulinoma and cultured were transplanted through the v. portae into the liver of streptozotocin-induced diabetic, male, CFY inbred rats. In the control, streptozotocin-treated rats, the decrease in blood glucose level was only transitory, in contrast with the GdCl 3 -pretreated diabetic rats, which remained normoglycaemic during the 2-week observation period. Histologically, in the liver and lung of rats pre-treated with GdCl 3 , large areas of extensively proliferating insulinoma cells were seen, whereas no insulinoma cells were seen in either the liver or the lung of diabetic-control rats, not-treated with GdCl 3 . These studies suggest that the Kupffer cells play significant roles in the recognition of xenoantigens and the induction of xenograft rejection.

Intramural gene expression changes may be critically involved in tissue damage, defense and repair after esophageal regurgitation. The aims were to characterize the consequences of short-term exposure to luminal bile, acid, or bile mixed with acid on the beta-ATPase, keratinocyte growth factor 1 (KGF-1) and KGF receptor (KGF-R) expressions within the mucosa and the muscle layer in a large animal model.Esophageal segments of anesthetized dogs were exposed to saline (n = 3), diluted canine bile (n = 6), hydrochloric acid (n = 5) or bile + hydrochloric acid (n = 5), and tissue biopsies were taken at the end of the 180-min observation period. Semiquantitative reverse transcriptase polymerase chain reactions were carried out and the degree of histological damage was evaluated on the 0-16-grade Geisinger scoring scale.Acid exposure was followed by a significant decrease in the level of beta-ATPase expression in the mucosa, and parallel increases in KGF-1 and KGF-R expression. Corresponding changes in the muscle layer were not significant. Bile alone evoked more severe tissue damage, with significantly decreased beta-ATPase levels in both the mucosa and the muscle, whereas the KGF-1 expression did not change significantly. The bile + acid treatment induced an intermediate state, with significant beta-ATPase transcription level decreases in both layers, while the mucosal KGF-1 expression was lower than that following acid treatment alone.The acid-induced transcriptional level downregulation of mucosal beta-ATPase gene expression in the smooth muscle layer was exacerbated by bile, but the concomitant KGF and KGF-R gene expression changes may indicate the start of a consecutive repair process.

The effects of glucocorticoids on acute pancreatitis (AP) have remained contradictory. The aim of this study was to investigate the time courses of the effects of the exogenous glucocorticoid agonists dexamethasone (DEX) and hydrocortisone (HYD) and a glucocorticoid antagonist (RU-38486) and to characterize the local and systemic responses in AP in rats. The glucocorticoid antagonist and agonists were administered just before AP induction. Serum amylase activity determinations, IL-6 bioassays, pancreatic weight/body weight ratio measurements, and survival analysis were performed. Liver and lung injuries were assessed via neutrophil leukocyte infiltration in myeloperoxidase (MPO) assays, tissue adenosine triphosphate (ATP) level determinations, and histology. In the glucocorticoid agonist groups, the survival rate increased, while the serum amylase level, the IL-6 activity, and the pancreatic weight/body weight ratio decreased significantly as compared with the control and RU-treated groups. AP resulted in significant decreases in tissue ATP levels in both the liver and the lung. In the DEX- or HYD-treated groups, the liver ATP levels were significantly elevated, while both the liver and the lung MPO levels were attenuated as compared with the AP and RU-treated groups. These results suggest that glucocorticoids may play important roles in mitigating the progression of the inflammatory reaction during the early phases of AP.

Several recent data suggest that in severe injuries, such as a shock state, the gradual activation of Kupffer cells and the excessive release of destructive and immunosuppressive products from macrophages may contribute to the development of “multiple organ failure”. The effect of Kupffer cell phagocytosis blockade on the correlation between the tissue distribution of bacterial lipopolysaccharide (LPS), endotoxin sensitivity and LPS-induced tumor necrosis factor (TNF) production was investigated in in vivo experiments in mice. To depress the activity of the Kupffer cells, gadolinium chloride (GdCl3) or carrageenan was used. The studies indicated the dissociation of the tissue localization of 51Cr-labeled endotoxin and endotoxin lethalithy. Both GdCl3 and carrageenan depressed the Kupffer cell activity, but the endotoxin sensitivity was enhanced only by carrageenan treatment. However, there was a close correlation between the sensitivity to LPS and LPS-induced TNF production as measured in the serum, since LPS-induced TNF production was enhanced only by carrageenan treatment.

THE IMMUNOLOGIC consequences of xenogenic islet transplantation remain unsolved. Gadolinium chloride (GdCl3) is known to depress reticuloendothelial activity by selectively blocking the function of hepatic Kupffer cells. We previously reported that xenotransplantation of insulinoma cells normalized blood glucose levels in diabetic rats after a single intravenous GdCl3 injection. 1 In the present work, we investigate the effects of GdCl3-induced long-term Kupffer cell blockade on the survival of human fetal islet xenografts.

The possibility that brain phagocytes may empty remnants of degenerated neurons into capillaries has been studied in frogs. Degeneration of nerve fibers was brought about by transectioning the optic tract, the tectothalamic and tectoisthmic tracts, the postoptic commissure or the radial nerve. To help identification of phagocytozed degenerated neuronal elements, the transected fibers were filled either with horseradish peroxidase (HRP) or cobaltous-lysine complex. The survival times were 3, 4, 7, 27, 47 and 70 days after the application of the markers. The HRP-labeled structures were identified in 60 microm thick sections using diaminobenzidine as chromogen, while cobalt was precipitated in the form of cobaltous sulfide. Small pieces of these sections were further processed for electron microscopy. In each area of the brain and spinal cord investigated, microglial cells and astrocytic processes containing fragments of degenerated neuronal elements could be seen close to capillaries. In some cases a microglial or astrocytic process pierced the capillary basal lamina and seemingly delivered inclusion bodies into the cytoplasm of capillary endothelial cells and pericytes. In the inclusion bodies, which were usually large vesicles, fragments of HRP or cobalt-labeled or unlabeled membranes with a foamy appearance, or condensed myelin lamellae could be observed. These vesicles protruded the luminal membrane of the endothelial cell that was disrupted in some cases suggesting that the content of the inclusion body was discharged into the lumen of the capillary. These results give support to Penfield's hypothesis (1925) that glial cells may empty phagocytozed materials into capillaries.

Vision is the most important sensory modality to anurans and a great deal of work in terms of hodological, physiological, and behavioral studies has been devoted to the visual system. The aim of this account is to survey data about the distribution of peptides in primary (lateral geniculate complex, pretectum, tectum) and secondary (striatum, anterodorsal and anteroventral tegmental nuclei, isthmic nucleus) visual relay centers. The emphasis is on general traits but interspecies variations are also noted. The smallest amount of peptide-containing neuronal elements was found in the lateral geniculate complex, where primarily nerve fibers showed immunostaining. All peptides found in the lateral geniculate complex, except two, occurred in the pretectum together with four other peptides. A large number of neurons showing intense neuropeptide thyrosine-like immunoreactivity was characteristic here. The mesencephalic tectum was the richest in peptide-like immunoreactive neuronal elements. Almost all peptides investigated were present mainly in fibers, but 9 peptides were found also in cells. The immunoreactive fibers show a complicated overlapping laminar arrangement. Cholecystokinin octapeptide, enkephalins, neuropeptide tyrosine, and substance P (not discussed here) gave the most prominent immunoreactivity. Several peptides also occur in the tectum of fishes, reptiles, birds, and mammals. Peptides in various combinations were found in the striatum, the anterodorsal- and anteroventral tegmental nucleus, and the isthmic nucleus that receive projections from the primary visual centers. The functional significance of peptides in visual information processing is not known. The only exception is neuropeptide tyrosine, which was found to be inhibitory on retinotectal synapses.