Expression of advanced glycation end-products and NFκB in chick embryos exposed to dioxins and treated with acetylsalicylic acid and α-tocopherol.

Dioxins have adverse and multifaceted effect on body functions. They are known to be carcinogens, immunotoxins, and teratogenic agents. In vivo, transformation of dioxins occurs after their interaction with the aryl hydrocarbon receptor (AhR) and leads to formation of proinflammatory and toxic metabolites. The aim of this study was to verify whether α-tocopherol (vitamin E) and acetylsalicylic acid (ASA), could reduce the damage caused by the action of dioxins. Fertile chicken eggs were injected with a solution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), followed by the injection of α-tocopherol or acetylsalicylic acid. Organs such as heart and liver were dissected from the chick embryos at d 13 and 19 of development and subjected to immunohistochemical analysis of presence of advanced glycation end products (AGEs) and nuclear factor kappa B (NFκB) in tissues. The AGEs were used as the marker for exposure to dioxins, since it is well established that their level increases in dioxin-damaged tissues. Formation of AGEs was evaluated in embryos exposed to dioxin and treated with vitamin E and/or ASA (against dioxin-exposed, untreated controls). We have found that TCDD causes developmental disorders and increases the level of AGEs in chick embryo tissues. The use of such pharmacological agents as vitamin E, ASA, and combination of ASA and vitamin E, inhibited formation of the AGEs in 13-day-old embryos and reduced the AGEs level in embryos after 19 d of the development.

[Strategies for hepatocyte transplantation]. / Strategie transplantacji hepatocytów.

Hepatic parenchymal cell transplantation is a promising method for managing patients with the acute liver failure and may create a bridge to whole organ grafting. Elimination or reduction of the immunogenicity of the hepatocytes would permit long-term graft survival without the need of non-specific immunosuppression. The presented experimental evidence suggests that modulation of hepatocyte immunogenicity by purification and cryopreservation reduces the alloresponse to hepatocytes both in vitro and in vivo. Identification of inoculated cells facilitates long-term monitoring of their localization and metabolic activity.

Hepatocyte transplantation in acute liver failure.

The majority of patients with acute liver failure (ALF) die waiting for orthotopic liver transplantation (OLT). No other treatment modality is shown to improve survival. This study was conducted to assess the safety and feasibility of hepatocyte transplantation (HT) and subsequent engraftment and function of donor cells. Functional and structural integrity of cryopreserved and thawed human hepatocytes were assessed by their morphological characteristics, induction of P-4501A1 transcription, and survival in vivo by xenotransplantation into rats. Five patients with severe ALF underwent intrasplenic (4 patients) and/or intrahepatic (2 patients) HT through angiography under cyclosporine immunosuppression. All patients had grade III to IV encephalopathy and factor V levels less than 0.5 U/mL, were ventilator and dialysis dependent, and were not OLT candidates. Three of the 5 patients who survived 48 hours after HT had substantial improvement in encephalopathy scores, arterial ammonia levels, and prothrombin times. Clinical improvement was paralleled by an increase in aminopyrine and caffeine clearances. All 3 patients lived substantially longer than expected based on clinical experience after HT (12, 28, and 52 days) but eventually died. Postmortem examination showed the presence of transplanted hepatocytes in liver and spleen by light microscopy and fluorescent in situ hybridization (FISH). Cryopreserved and thawed human hepatocytes can be transplanted into recipients with ALF with some acceptable but definite complications. Engraftment of donor hepatocytes was proven by histological examination and FISH by both transjugular biopsy and at autopsy. Improvement in brain edema, encephalopathy grade, and clearance of antipyrine and caffeine suggested function, albeit with a 24- to 72-hour delay posttransplantation.

Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.

Histological identification of purified and cryopreserved allogeneic hepatocytes following transplantation in a murine model without host immunosuppression.

Hepatocyte transplantation is a conceptually attractive alternative to whole organ grafting for some inborn metabolic errors and for fulminant liver failure. However, studies of the immunogenicity of transplanted allogeneic hepatocytes have yielded contradictory results. In these experiments, the effect of purification and cryopreservation of the hepatocytes on the ability of these cells to engraft in the mouse allogeneic recipients without immunosuppression was studied. BALB/cByJ mouse crude (unpurified), modified (purified or cryopreserved), or dead (irradiated) hepatocyte preparations labeled with fluorescein dye CFSE were infused either into the portal vein or into the spleen parenchyma of the recipient CBA mice. A histological examination revealed normal appearance of engrafted modified hepatocytes with no signs of acute rejection up to 21 days posttransplant. Many of the intrasplenically implanted hepatocytes migrated into the hepatic sinusoids. The modified hepatocytes showed intact ultrastructural appearance 7 days after transplantation. The numbers of inoculated crude hepatocytes rapidly declined with signs of dense infiltration of mononuclear cells in the graft indicating destructive response. The fluorescence of dead hepatocytes was undetectable. These results suggest that reduced immunogenicity may be responsible for the longer survival time of inoculated, purified or cryopreserved hepatocytes with no adverse morphological effects.

Characterization of human small intestinal cytochromes P-450.

Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P-450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. The CYP composition of enterocytes could thus affect the potential toxicity or therapeutic efficacy of xenobiotics by modifying systemic uptake. We have characterized human enterocyte CYP composition to enable assessment of its functional roles. An isolation method for enterocytes from human small intestine was developed using EDTA buffer-mediated elution. Villous enterocytes were isolated in high yield, separated from crypt cells. Reverse transcriptase-polymerase chain reaction of total RNA from enterocytes revealed that CYP1A1, 1B1, 2C, 2D6, 2E1, 3A4, and 3A5 mRNA were expressed, but only CYP2C and 3A4 were detectable by Western immunoblotting in enterocyte microsomes from 10 human small intestines, whereas CYP1A1 was weakly detectable in two of eight intestines tested. Microsomal protein content decreased markedly along the small intestine from the duodenum to the ileum, whereas total CYP content and CYP3A4 erythromycin N-demethylase activity increased slightly in progressing from the duodenum to the jejunum and then decreased markedly toward the ileum. Levels of CYP3A4 and 2C protein did not decrease in concert as a function of length along the intestine distally. Maximal CYP content for the 10 intestines varied from 0.06 to 0.18 nmol/mg microsomal protein and maximal CYP3A4 erythromycin N-demethylase activity varied from 0.30 to 0.76 nmol/min/mg microsomal protein. In conclusion, CYP3A4 is the major form of CYP expressed in human small intestine enterocytes, CYP3A5 expression was not detected, CYP2C and, in some intestines, CYP1A1 were expressed. The highest metabolic activity occurred in the proximal intestine.

Autoimmune destruction of islet grafts in the NOD mouse is resistant to 15-deoxyspergualin but sensitive to anti-CD4 antibody.

Islet allografts transplanted into Type I diabetic recipients may be destroyed by allorejection or recurrent autoimmune diabetes. We studied islet transplantation in three murine models in order to determine the relative sensitivity of autoimmunity and alloimmunity to two immunosuppressive agents that may be useful in clinical islet transplantation: 15-deoxyspergualin (DSG) and anti-CD4 antibody (GK 1.5). In the model in which only allorejection occurs (BALB/c islets transplanted into streptozotocin-induced diabetic CBA or streptozotocin-induced diabetic NOD recipients), both DSG and anti-CD4 antibody treatment led to indefinite survival of allogeneic islets (>100 days in both treatments). In the second model in which only recurrent autoimmunity can destroy islet grafts (islets from NOD donors transplanted into spontaneously diabetic NOD recipients), only anti-CD4 treatment caused prolonged graft survival [MST 36.7 +/- 6.8 days vs 9.8 +/- 1.8 days (controls), P < 0.0002]. Treatment with DSG did not cause any increase in graft survival (MST 12.6 +/- 5.4 days, NS). Finally, using a model in which both autoimmunity and allorejection may occur (BALB/c to spontaneously diabetic NOD mice), treatment with anti-CD4 caused marked graft prolongation [42.0 +/- 14.5 days vs 7.2 +/- 0.8 days (control), P < 0.002] while DSG again did not prolong graft survival with respect to untreated recipients (9.8 +/- 3.0, NS). We conclude that recurrent autoimmunity in the NOD mouse involves a CD4+ T cell that is not sensitive to DSG. Anti-CD4 antibody may be useful in human clinical islet transplantation trials because it seems to prevent both allorejection and recurrent autoimmunity.

Prolongation of islet allograft survival with an antibody to vascular cell adhesion molecule 1.

BACKGROUND: The purpose of this study was to determine whether an antibody to vascular cell adhesion molecule 1 (VCAM1) prolongs the survival of neovascularized pancreatic islet allografts. METHODS: We treated CBA (H-2k) recipients of BALB/c (H-2d) islet allografts with anti-VCAM1 antibody (400 micrograms/day for 20 days). Sensitized recipients of islet grafts also were treated with anti-VCAM1. To study mechanism we performed mixed lymphocyte reactions (MLRs) with anti-VCAM1 and studied the graft infiltrate in treated recipients. RESULTS: Anti-VCAM1-treated CBA recipients showed prolonged graft survival with indefinite survival in five of nine cases. Anti-VCAM1 prevented proliferation in an MLR but not when added 36 hours after the beginning of the MLR. Anti-VCAM1 did not prolong allograft survival in sensitized recipients and did not prevent lymphocytic infiltration of the graft at 7 days. CONCLUSIONS: Anti-VCAM1 prolongs allograft survival in neovascularized islets in which the donor vascular endothelium plays little or no role in immunogenicity. VCAM1 appears to be important in the afferent phase (lymphocyte activation) of the allograft response. Once activated, either late in an MLR or in sensitized recipients, lymphocytes are not dependent on VCAM1 for function. Finally, anti-VCAM1 does not appear to affect the homing of lymphocytes to the allograft.

[Incidence of ischemic heart disease symptoms among residents of Plock and Kutno]. / Wystepowanie objawów niedokrwiennej choroby serca wsród mieszkanców Plocka i Kutna.

An incidence of IHD in Plock and Kutno inhabitants has been analysed. Five hundred eighty men and seven hundred forty nine women from Plock, and 475 men and 579 women from Kutno, aged 18-79 years, have been examined. Ischemic heart disease has been diagnosed with Rose's test and resting ECG evaluated according to Minnesota code. The symptoms of IHD have been found in 18.8% of men and 27.3% of women from Plock, and in 17.5% of men and 22.6% of women in Kutno. In Plock population the diagnosis has been based mainly on anamneses while in Kutno--on ECG findings. More frequent ischemic disorders in ECG recordings in Kutno population has been reflected in IHD indices registered in the available documentation, eg. morality, interventions of emergency service, hospitalizations, and active counselling.

The activation of protein kinase A by cyclic AMP is influenced by oestradiol and progesterone in supernatants from the anterior pituitary but not from hypothalamus of the female rat.

The activation of protein kinase A (PKA) by cAMP was estimated in supernatant fractions from the hypothalamus (Hyp) and anterior pituitary (AP) of the female rat during the oestrous cycle and in ovariectomized and ovariectomized, ovarian steroid hormone treated animals. In both structures, the largest activation of PKA was found in dioestrus-2, while the lowest one was in Hyp in dioestrus-1 and in AP in oestrus. Ovariectomy had no influence on cAMP-dependent activation of PKA from Hyp and AP. Treatment of ovariectomized rats with 17-beta-oestradiol (E2), progesterone (P) or both abolished the activation of PKA by cAMP from AP and had no effect on hypothalamic PKA. These results indicate that ovarian steroids act specifically on AP processes via cAMP dependent pathway and regulation of PKA activity.

Superantigen-like effects and incidence of diabetes in NOD mice.

The population of T-cells that develops in any individual can be divided into families based on sequence differences in the beta-chain variable region of the T-cell receptor heterodimer. Major histocompatibility complex products and endogenous retroviral gene products have both been shown to exert powerful influences on the frequency distribution of T-cell receptor beta-chain variable region families in the mouse. In most mouse strains, these repertoire modifiers appear to be fully functional early in mouse development and shape a repertoire of antigen specificities that remains essentially unchanged from the first weeks of life until old age. In NOD mice, an inbred mouse model of type I diabetes, puberty in males coincides with a beta-chain variable region-specific T-cell expansion that mimics the results of exposure to exogenous superantigens in immunologically mature animals. The subsequent behavior of this subset indicates that it may play a role in the relative protection of male NOD mice from complete pancreatic beta-cell destruction and overt diabetes.

The dentatorubral projection in the rabbit with emphasis on distinction from the interpositorubral connectivity: an HRP retrograde tracer study.

The projection from the lateral (dentate) cerebellar nucleus (NL) to the red nucleus in the rabbit was investigated using free horseradish peroxidase (HRP). Following injections of this tracer into the red nucleus (RN), the distribution patterns of retrogradely labeled cells were checked in the NL. The projection from the NL was found to extend rostrocaudally throughout the contralateral RN except for the predominant part of the rostral one-quarter and the caudalmost part of the RN, i.e. the regions described to receive projections exclusively from the interpositus nuclear complex (NI). On the other hand, the only region of the RN receiving projection exclusively from the NL appeared to be rostralmost parvocellular region of the RN (RN-PA). This study suggested that, in contrast to other species, the projection from the NL is not confined to the most rostrally localized RN-PA but is distributed throughout predominant part of the nucleus and to a great extent overlapped with projection from the NI. This overlapping projection pattern parallels the absence of clear differentiation between parvo- and magnocellular parts of the rabbit RN.

I-E-independent deletion of V beta 17a+ T cells by Mtv-3 from the nonobese diabetic mouse.

Analysis of TCR beta-chain V region (V beta) frequency among NOD lymphocytes reveals a profound depletion of V beta 3+ T cells, and a recent study has linked this phenomenon to the Mtv-3 insertion on chromosome 11. When the V beta 17a gene segment is introduced into mice with an nonobese diabetic mouse background, T cells bearing the TCR encoded by this gene segment are also dramatically reduced in frequency. Deletion of V beta 17a+ T cells segregates with deletion of T cells bearing V beta 3 and occurs in the absence of I-E, which had been shown in previous studies to be a major deleting element for V beta 17a+ thymocytes.

Spatial arrangement of the interpositorubral projection in the rabbit. A retrograde HRP study.

The objective of this investigation was to analyse the organization of rubral afferents from the cerebellar interpositus nuclear complex (NI) in the rabbit. Free HRP was employed as a retrograde tracer to identify the distribution patterns of labeled cells in the anterior and posterior interpositus nuclei. The interpositus projections distribute throughout nearly the entire contralateral red nucleus (RN) except for its rostralmost parvocellular region (RN-PA) which does not correspond to the usually distinguished parvocellular subdivision of the red nucleus (RN-P). The central and lateral regions of the posterior interpositus nucleus (NIP) project (nearly exclusively) to the medial region of the RN. The lateral region of the anterior interpositus nucleus (NIA) sends projections to both the lateral and medial regions of the caudal three-quarters of the RN. Thus, the lateral region of the RN appears to be under nearly exclusive control of NIA. The rostral one quarter of the RN receives cerebellar afferents from the NIP only and caudal pole of RN from the NIA exclusively. Comparison of labelling patterns indicates absence of topogrophic differentiation of projections from the NIP, whereas dorsoventral topography in the NIA can correlate to the rostrocaudal and mediolateral arrangement of the RN. Collectively, interpositorubral projections in the rabbit are similar to those in other species only in basic features.

The dynamics of beta-endorphin release by the hypothalamic nuclei and pituitary gland in sheep under physiological and stress condition.

The secretion of beta-endorphin (beta-End) by the infundibular nuclei--median eminence (IN-ME) and paraventricular nuclei (PVN) of the hypothalamus and the anterior lobe (AL) of the pituitary gland was determined in 18 anestrous ewes by RIA assay of this opioid in perfusates collected from these formations. Perfusion with Ringer-Locke solution was carried out on the animals under resting and stress-full conditions, using a pushpull cannula method. Electrical footshocks were used as stress stimuli. Two series of perfusion experiments were performed. In the first, the perfusions were carried out over two hours and perfusates were collected before and during stress for 1 h. In the second series of experiments the dynamics of the release of the opioid by the IN-ME and the pituitary gland were followed by collecting five or six perfusates in 20 min fractions before and during stress, respectively. The first series of experiments showed that the concentrations of beta-End in perfusates from the pituitary gland were much higher than those from the hypothalamic nuclei and that the concentrations of this opioid during stressing rose significantly only in perfusates from the IN-ME nuclei. In the second series of experiments the release of beta-End from the IN-ME and pituitary gland was altered by stress. The shift was very characteristic in that the concentrations of the opioid during stimulation initially significantly rose and then gradually declined. The concentration of the opioid in the peripheral blood also rose at the beginning of stimulation and declined as stimulation continued.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-reciprocal connections between the vestibular nuclei and the cerebellar paramedian lobule, with special reference to the climbing fiber zones: an analysis in the rabbit using the retrograde horseradish peroxidase method.

The organization of the secondary vestibular projections onto the cerebellar paramedian lobule (PML) and possible reciprocal corticovestibular connections were investigated by the retrograde horseradish (HRP) technique in the rabbit. Following injections of the tracer into the vestibular nuclear complex (VNC), an ill-defined, sagittal band composed of numerous labelled Purkinje cells was found ipsilaterally throughout the length of the lateral portion of the vermis, the ventral paraflocculus and the flocculus. However, no labelled Purkinje cells were found in the cortex of the PML. The results indicate that zone B, considered to give rise to cerebellar corticovestibular projections, is not present in the rabbit PML. After injections of HRP into the PML, the retrograde labelling pattern in the VNC was analyzed, in relation to the climbing fiber zones identified by retrograde labelling in the inferior olive. No clear-cut correspondence could be found between the vestibular subdivisions and climbing fiber zones in the PML, except that only the interstitial nucleus of the vestibular nerve projects into zone D1 in sublobules e and d. There were no vestibular projections to zone D2. The only salient feature was that cells projecting onto zones C2, C1 and C3 of the PML were arranged rostrocaudally in the inferior vestibular nucleus and the caudal portion of the medial vestibular nucleus. In addition, a topical relationship was found between parts of the VNC and sublobules of the PML.

The neurons of origin of non-cortical afferent connections to the oculomotor nucleus. A retrograde labeling study in the rabbit.

The cellular origin of the brainstem projections to the oculomotor nucleus in the rabbit has been investigated by using free (HRP) and lectin-conjugated horseradish peroxidase (WGA-HRP). Following injections of these tracers into the somatic oculomotor nucleus (OMC), retrogradely labeled cells have been observed in numerous brainstem structures. In particular, bilateral labeling has been found in the four main subdivisions of the vestibular complex, predominantly in the superior and medial vestibular nuclei and the interstitial nucleus of Cajal, while ipsilateral labeling was found in the rostral interstitial nucleus of the medial longitudinal fascicle (Ri-MLF), the Darkschewitsch and the praepositus nuclei. Neurons labeled only contralaterally have been identified in the following structures: mesencephalic reticular formation dorsolateral to the red nucleus, abducens internuclear neurons, group Y, several areas of the lateral and medial regions of the pontine and medullary reticular formation, ventral region of the lateral cerebellar nucleus and caudal anterior interpositus nucleus. This study provides also information regarding differential projections of some centers to rostral and caudal portions of the OMC. Thus, the rostral one-third appears to receive predominant afferents from the superior and medial vestibular nuclei, while the caudal two-thirds receive afferents from all the four vestibular nuclei. Finally, the group Y sends afferents to the middle and caudal, but not to the rostral OMC.