Pharmacokinetic evaluation of liposomal encapsulated ampicillin in male and female rats

The dispositions of free and liposomal entrapped ampicillin were compared in male and female rats after i.v. administration. Serial blood samples were collected for 2 h in the free drug study and 12 h for the liposomal formulation. Pharmacokinetic parameters obtained with free drug were not significantly different between genders. However, gender significantly influenced the disposition of liposomal encapsulated ampicillin. While no difference was observed in distribution t1/2 between genders, female rats had a shorter MRT, smaller Vss and Vt, and faster clearance as compared to male rats. In a second study, spleen, liver, kidney, heart, and lung were harvested post-injection of free and liposomal entrapped ampicillin. Free ampicillin did not distribute extensively into the tissue compartment and no gender difference was noted. In contrast, liposomal encapsulation resulted in a substantial tissue uptake. In general, female rats had higher concentrations in the spleen and lung as compared to male rats. In vitro plasma stability was not significantly different, suggesting that destabilization of the liposomes does not play a large role in the dispositional differences observed in these studies. However, in vivo interaction of liposomes and plasma lipoproteins may influence the disposition of encapsulated drug.     

Influence of acinar tissue contamination on encapsulated pancreatic islets: morphological and functional studies

BACKGROUND: The present study concerns the influence of acinar contamination on pancreatic islets encapsulated in an artificial membrane (AN 69). METHODS: Pure, handpicked pancreatic islets were contaminated by the addition of acinar tissue (ratio, 1:1). The morphological aspect and insulin release of both pure (n=12) and contaminated (n=12) encapsulated islets were assessed after 10 days of culture or implantation in the peritoneal cavity of rats. RESULTS: After implantation, the encapsulated islets, irrespective of their purity, were totally altered, whereas the morphological aspect of the cultivated islets remained intact only in the absence of acinar tissue contamination. This contamination induced a significant decrease in the stimulation index of insulin release. The stimulation index decreased by 42% for fresh islets and by 52% and 34% for cultivated and implanted islets, respectively, without modification in their basal release of insulin. CONCLUSIONS: The acinar tissue proved detrimental to the encapsulated implanted and cultivated islets.     

Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats

To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.     

Effect of hepatocyte growth factor on the proliferation of intrasplenically transplanted hepatocytes in rats

Expression of genes encoding insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II) and type I insulin-like growth factor receptor (IGFr) was measured in theca and granulosa cells from the ovary of the laying hen, using an RNase protection assay. Expression of genes encoding IGF-I and -II was confined to theca tissue and expression was not detected in granulosa cells. In contrast, expression of genes encoding IGFr in granulosa cells was significantly greater than that in theca tissue. The 98 base IGF-II probe was similar to a region of the second coding exon of chicken IGF-II and produced multiple RNase-protected RNA hybrids. Theca RNA from follicles at all stages of development produced RNase-protected hybrids of size 98, 96 and 90 bases; however, an additional band (66 bases) was also observed in theca RNA from small yellow follicles. The stage of follicular development during which maximum amounts of the 66 base RNase-protected fragment was detected correlates with the stage at which small follicles are selected for recruitment into the follicular hierarchy. The results provide evidence for the involvement of IGFs in the intraovarian control of ovarian function in a non-mammalian species, and highlight the importance of IGF-II in this process.     

Regulation of intracellular pH in periportal and perivenular hepatocytes isolated from ethanol-treated rats

The aim of this study was to gain information on intracellular pH (pHi) regulation in periportal (PP) and perivenular (PV) hepatocytes isolated from rats pair-fed liquid diets with either ethanol (T rats) or isocaloric carbohydrates (C rats). pHi was analyzed by the pH-sensitive dye BCECF in perfused subconfluent hepatocyte monolayers. Cells were acid-loaded by pulse exposure to NH4Cl and were alkali-loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHout. In cells from C rats: (a) steady-state pHi was higher in PP than in PV hepatocytes in the presence, but not in the absence, of bicarbonate; (b) pHi recovery from an acid load was 35% higher in PP than in PV cells in the presence of HCO3-, whereas it was similar in HCO3(-)-free experiments; and, on the contrary, (c) pHi recovery from an alkaline load was 30% higher in PV than in PP cells. In cells from T rats: (a) steady-state pHi was always lower than in cells isolated from pair-fed animals; (b) steady-state pHi was similar in PP and PV hepatocytes either in the presence or absence of bicarbonate in the perfusate; (c) pHi recovery from an acid load was not significantly different in PP and PV cells either in the presence of HCO3- or in HCO3(-)-free experiments; and (d) pHi recovery from an alkaline load was similar in PP and PV cells. Our data suggest that chronic ethanol treatment selectively modifies pHi by affecting the activity of ion transport mechanisms regulating pHi in PP and PV hepatocytes isolated from rat liver.     

Multiple esterase forms in isolated hepatocytes and Kupffer cells of partially hepatectomized rats

The esterase patterns of isolated parenchymal liver cells of rats consisted of 6 bands of enzymatic activity, whereas the patterns of iron-loaded Kupffer cells showed 5 bands. Both patterns become simpler in the early prereplicative period of liver regeneration. Dukring simultaneous replication of DNA, i.e. 24 h after partial liver removal, an additional band of esterase activity appears in patterns of hepatocytes and Kupffer cells. At the moment of maximum hepatocyte mitotic rate, i.e. 36 h after partial hepatectomy, both esterase patterns lose the single band of activity again. 2 or 3 days after surgery the initial esterase patterns in hepatocytes return whereas the patterns of Kupffer cells remain incomplete.     

Meal patterns prior to and following liver transplantation in rats

As the second part of a community based educational campaign to convey the risk of HIV/AIDS to commercial sex workers in Jakarta, from May-July 1995, a total of 253 male transvestites (WARIA) were questioned about their sexual behavior patterns and their knowledge and attitude toward HIV/AIDS. In the previous report, 1991-1993, there was one out of 830 WARIA found HIV positive in November 1993. In this study, 1995, a total of two out of 253 WARIA were confirmed of HIV infection in July 1995. Most of them still have incorrect knowledge on HIV/AIDS transmission mode, they are still practicing high risk sexual attitudes such as an exchange partner rate of 5 men per three weeks, low condom use (1.2 out of the last 5 sexual contacts). The reasons for not using condoms were forgetfulness 35.3% and partner does not like condom 38.2% Most of WARIA know about condoms (94.5%), but it is difficult to access condom use from small shops around them. Therefore, to prevent further spread of HIV/AIDS in WARIA, condom should be used constantly and properly. It has been shown from another study, that more information, better availability and better promotion of condoms can increase condom use. Thus, attention should be placed on various ways of distributing condoms for WARIA in Jakarta, especially community-based distribution by peer leaders, social marketing and commercial sales.     

Bronchoscopic evaluation of pulmonary complications following kidney transplantation

The authors evaluated the efficiency of bronchoscopy in kidney transplanted patients with late pulmonary complications (mean 370 days after transplantation). The bronchoscopy was made meanly 9.5 days after recognizing pulmonary diseases, in that time 7 patients were mechanically ventilated. In 15 cases therapy guided by the cytological, histological and microbiological results has been hoped to improve outcome. This study suggest that bronchoscopy might be important in the diagnosis of the late pulmonary complications of kidney transplanted patients.     

Enzymatic activity and expression of cytochrome P450 LA omega within intrasplenically transplanted fetal hepatocytes in spontaneously hypertensive rats

We examined the expression and enzymatic activity of cytochrome P450 LA omega within transplanted hepatocytes. Fetal hepatocytes were harvested at day 20 of gestation from spontaneously hypertensive rats (SHRs) and transplanted into recipient adult SHR spleens. Microscopic examination of the recipient spleens at 4 and 10 wk after transplantation revealed masses of hepatocytes with cord-like structures in the red pulp. Immunochemical studies detected cytochrome (cyto) P450 LA omega in the fetal hepatocytes before transplantation without prior induction. Although the cyto P450 LA omega was not detected by the second week after transplantation, by the 6th and 10th wk after transplantation, it was. Cyto P450-arachidonic acid omega/omega-1 hydroxylase activity (formation of 20- and 19-hydroxyeicosatetraenoic acid) was detected at 10 wk after transplantation, but not 2 or 6 wk after transplantation. These results demonstrated that fetal hepatocytes can be transplanted successfully into recipient spleens and then grow in the spleens, as in the case of the adult hepatocyte response.     

Intraperitoneal transplantation of isolated hepatocytes of the pig: the implantable bioartificial liver]

To elucidate the histopathological features of pancreatic ischemia, we examined postmortem pancreases in which cholesterol emboli were present. Cholesterol emboli were detected in 17 pancreases (6 of 36 cases of aortic aneurysm and 11 of 223 control cases). Two of the 17 pancreases had well-demarcated patchy lesions composed of degenerating acinar cells showing deeply eosinophilic cytoplasm and pyknotic nuclei, indicating fresh ischemia. In the marginal zone of the larger lesions and in the small lesions, the intralobular ductules had avoided the ischemic changes. Five of the 17 pancreases had patchy fibrotic foci containing small ductules with slightly retraction features. These ductules are considered to be the remnant intralobular ductules that have avoided the previous ischemic damage. We conclude that these patchy fibrotic foci are the healed ischemic lesions. The current findings suggest that the healed ischemic lesions can be differentiated from common pancreatic fibrosis. The existence of remnant intralobular ductules and the patchy retraction features may be useful histological markers for the determination of healed ischemic lesions.     

Bronchoscopic evaluation of pulmonary infiltrates following bone marrow transplantation

Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin alpha-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin alpha fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin alpha through an unknown mechanism, without influencing the nuclear import of the protein.     

A noninvasive functional evaluation following peripheral nerve repair with electromyography in a rat model

A new bipolar surface electrode array was designed and constructed for a noninvasive "closed" functional evaluation with electromyography following sciatic nerve transection in a rat model. This "closed" method was compared with a conventional one-shot "open" measurement. Nerve conduction velocity and distal latency were calculated. Data obtained from the recordings from different animals as well as from the same animal at different points in time yielded excellent reproducibilities. There is no difference in the mean values whether nerve conduction velocity and distal latency are obtained by "closed" or "open" measurements. Correlation was significant (p < 0.01; rNCV = 0.77, rDL = 0.63) between these two methods. The results lead to the conclusion that the noninvasive functional evaluation with the parameters of nerve conduction velocity and distal latency introduced in the present study could be employed as a reliable method for serial functional evaluations following nerve transection in a long-term study in a rat model.     

Immediate and long-term functional and morphological changes in the neuronal structures of cerebral cortex of cats subjected to callosotomy

OBJECTIVES: To investigate cerebral vasomotor reactivity in five patients with limb shaking transient ischaemic attacks by using transcranial Doppler sonography. METHOD: Attacks with transient limb shaking were unilateral in four patients and bilateral in one. Internal carotid arteries on the side opposite the abnormal limb movements showed three 90-95% stenoses and three occlusions as assessed by cerebral angiography in three and magnetic resonance angiography and ultrasound in one case each. Reactivity of cerebral resistance vessels was studied by measuring peak mean velocities in the middle cerebral artery (MCA) before and after the application of CO2 enriched air. Reference values were obtained from 25 normal subjects. RESULTS: During hypercapnia peak mean velocities slightly decreased in five MCAs (steal phenomenon) and remained unchanged in one MCA opposite the abnormal movements, whereas the other MCAs showed normal reactivities. CONCLUSION: The delineation of an exhausted cerebral vasoreactivity in all hemispheres opposite the involuntary limb movements suggests that haemodynamic failure is the cause of transient ischaemic attacks with limb shaking.     

The antioxidant LY 231617 ameliorates functional and morphological sequelae induced by global ischemia in rats

In this study the effect of LY 231617, an antioxidant, on spatial learning deficit and on neuronal damage following transient cerebral ischemia was evaluated. Global ischemia was induced by four-vessel-occlusion (4VO) for 20 min in rats. LY 231617 (20 mg/kg i.p.) was administered after onset of reperfusion. One week after surgery spatial learning was tested in the Morris water maze. LY 231617 reduced the increase in escape latency and in swim distance induced by 4VO. Neuronal damage in the CAI sector of the hippocampus produced by 4VO was significantly attenuated by LY 231617. The present data demonstrate that posttreatment with LY 231617 exerts a protective effect on hippocampal neuronal damage and deficits in spatial learning induced by 4VO.     

Potassium uptake and water content in hepatocytes isolated from rat livers preserved in Euro-Collins and UW solutions and after transplantation

Isolated hepatocytes from the rat were used to assess the maintenance of liver cell function in relation to the composition of the preservation medium. After separation by collagenase, they were incubated in Krebs-Ringer-Bicarbonate medium (KRB), Euro-Collins (EC), or University of Wisconsin (UW) solutions. Potassium influx, cell volume, and transaminase release were measured in cells freshly separated from control livers or from livers preserved in vitro up to 12 h in these media or having undergone orthotopic liver transplantation (OLT). While ion exchange levels were retained in all media, cells shrank significantly in UW but were able to restore their volume after 3 h of liver preservation. With regard to in vivo conditions, UW appears to be the best medium to prevent edema and to maintain more stable potassium exchange and enzyme production. These results are of value for liver transplantation in humans.     

Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats

Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo. However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral readministration. We tested the feasibility of prolonging gene expression by induction of central tolerance to adenoviral antigens in bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats. Tolerance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recombinant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (Ad-hBUGT1) carrying the hBUGT1 gene; a protein extract of the same virus; or viral infected hepatocytes. Controls received intrathymic injections of normal saline. After 12 d all groups were injected intravenously with 5 x 10(9) pfu of either Ad-hBUGT1 or adenovirus beta-galactosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosidase [LacZ] gene). In all three groups of tolerized rats, hBUGT1 was expressed in the liver after administration of Ad-hBUGT1, with glucuronidation of biliary bilirubin of above 95%. Serum bilirubin levels decreased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk. Similar findings were observed following repeat injections given on days 45 and 112. In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective. In all tolerized groups, but not in controls, there was a marked inhibition of appearance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus. Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response. This is the first demonstration of Ad-directed long-term correction of an inherited metabolic disease following central tolerization with thymic antigen.