Search

The WASH Sector Development Plan (2011–2025) recognizes the absence of harmonized approaches as the root challenge to scaling up drinking water safety in Bangladesh. UNICEF is supporting the Government of Bangladesh (GoB) through the Department of Public Health Engineering (DPHE) and Policy Support Branch to re-engineer its approach to drinking water safety by integrating policy, systems strengthening & sustainable services delivery at national and sub-national levels. Some of the key contributions to the sector include the implementation of the UNICEF-DPHE Arsenic Safe Union model with the declaration of 106 Arsenic Safe Villages, reduction in arsenic contamination rates of new tubewells, a system for preventing elite capture of water points, and the adoption of the ASU model in a $240 million arsenic mitigation drinking water project using domestic resources. Relatedly, a Policy Support Branch has been established, the sector coordination mechanisms revised, WASH bottleneck analysis is ongoing & the National Implementation Plan on Arsenic has been reviewed: Next steps include the professionalisation of drilling by local drillers through a partnership with KTH, Sweden, the GoB and UNICEF.

This is a retrospective longitudinal review.The purpose of this review was to identify predictors of developing clinical scoliosis and compare between traumatic and neurological aetiologies of SCI.This study was conducted at the Midland Centre of SCI.Case notes of all patients injured at an age up to 18 years and admitted between 1971 and 2013 were reviewed.Sixty-nine individuals were identified, of which seven were excluded: three with pre-existing scoliosis and four with spina bifida. The remaining 62 (44 males, 18 females) had a median age at injury of 17 years (inter quartile range 13-17). Of these, 51 (82%) had traumatic and 11 (18%) had neurological injury. Most (42/51; 82%) of the children who had a traumatic injury were older than 13 years. The risk of developing scoliosis was lower for older patients (RR 0.68 per year, 95% CI 0.52-0.83) or following a traumatic injury (RR 0.36, 95% CI 0.20-0.66). A multivariable analysis based on age and trauma showed that only older age decreased the risk. A robust Receiver Operator Curve analysis suggested 14.6 years as the optimal threshold to predict development of scoliosis within 10 years (Area Under the Curve; AUC 0.83 (95% CI 0.73-0.93), sensitivity 70% (95% CI 50-89%), specificity 89% (95% CI 74-100%).Our results suggest that age below 14.6 years was a predictor for scoliosis. Once adjustment is made for age, the incidence of scoliosis does not differ between traumatic and neurological aetiologies of paediatric SCI injury.

Retrospective review of findings during cystourethroscopic surveillance of symptomatic and asymptomatic patients with indwelling urethral catheters (IUC) and suprapubic catheters (SPC) monitored between January 2003 and December 2008.To audit and compare findings between symptomatic and asymptomatic patients and between SPC and IUC population. To systematically review the literature including the recent National Institute for Health and Clinical Excellence guidelines on cystoscopic surveillance.Theater management system and the electronic patient records used to retrieve demographics, injury details and operative findings.Of 925 cystoscopies performed in 507 patients, 449 were performed in 277 patients with IUC/SPC. Only 419 procedures (SPC 264; IUC 155) in 262 patients fit the inclusion criteria. Thirty procedures in fifteen non traumatic patients were excluded. Statistically there was no significant difference in incidence of findings between the symptomatic and asymptomatic group. Recurrent blockage of catheter was predominant in the SPC group and symptomatic urinary tract infections (UTIs) were the most common indications in the IUC group. In the asymptomatic group, there were 44 squamous metaplastic changes in 27 patients. Two of these patients had keratinizing variants. The duration of catheterization ranged from 20 months to 27 years and mean of 13.7 years. The average duration between two cystoscopies in the symptomatic group was 16 months compared with an average 21 months in the asymptomatic group.Cystourethroscopic surveillance in high-risk patients with IUC/SPC is essential to diagnose and manage at an early-stage complications associated with IUC/SPC, minimize symptomatology, mitigate aggravation of complications, maintain good health and probably good quality of life.

SUMMARY The clinician-patient relationship is more intense in the field of psychiatry than in other specialities of medicine. In this context, an understanding of the personality assets and liabilities of a psychiatrist takes on special significance. In the present comparative cross-sectional study, the personality profile, positive mental health characteristics and empathy were studied in two groups of residents in psychiatry and one in general medicine. Residents in psychiatry had greater amount of positive mental health characteristics and identifiable personality traits. However, in empathy the three groups did not show any significant difference. The utility and implications of the results are discussed.

SUMMARY A study was conducted to ascertain how often GPs encountered patients with psychological crisis and their means of tackling such cases. A questionnaire was administered to 47 GPs attending a training course on drug de-addiction orientation program. It was observed that failure in an examination (22.9%), break up of a love affair (16.8%) and the death of someone very close to the patient (14.9%) were among the common causes precipitating a crisis. Regarding the treatment techniques, reassurance, tranquilizers and referral to a psychiatric set-up were most commonly resorted to. 68.1% of the GPs were not at all satisfied with the treatment administered by them and 78.9% of them felt the need for further training in psychiatry. Implications of these findings are discussed.

Familial hypercholesterolemia is an inherited disease in humans, caused by a deficiency of low density lipoprotein (LDL) receptors, that we have used as a model for developing liver-directed gene therapies. Our strategy is to reconstitute hepatic LDL receptor expression in vivo by administering a DNA-protein complex that is capable of targeting the delivery of functional LDL receptor genes to hepatocytes. Infusion of this DNA-protein complex into the peripheral circulation of a rabbit animal model for familial hypercholesterolemia resulted in hepatocyte-specific gene transfer and a temporary amelioration of hypercholesterolemia. This noninvasive approach to gene therapy should have applications in the treatment of a wide spectrum of human diseases.

Familial hypercholesterolemia is an inherited disease in humans that is associated with coronary artery disease and is caused by a deficiency of the receptor that mediates the internalization of low density lipoprotein (LDL). We have used an animal model for familial hypercholesterolemia, the Watanabe heritable hyperlipidemic (WHHL) rabbit, to design a therapeutic approach for this disease, which attempts to correct the hepatic defect in LDL receptor expression. Hepatocytes were harvested from WHHL rabbits, plated in primary cultures, and exposed to recombinant retroviruses capable of efficiently transferring a functional human LDL receptor gene. Genetically modified cells were harvested and infused into the portal vein of WHHL recipients, who were analyzed for metabolic consequences of human LDL receptor expression. Each animal exhibited a statistically significant decrease in total serum cholesterol 2-6 days after transplantation, with an eventual return to pretreatment levels. Proviral DNA sequences and virus-directed transcripts were detected in liver tissue 24 hr after transplantation. In situ hybridization demonstrated provirus expression in a small population of hepatocytes distributed in periportal sections of the liver. This study illustrates the potential of somatic gene therapy in ameliorating hyperlipidemia associated with familial hypercholesterolemia.

Hepatocyte cell lines are beginning to be developed as universal donors for isolated liver cell transplantation, which is a less invasive method than orthotopic liver transplantation for treatment of metabolic liver disease. The immune response to isolated liver cell transplantation and its modification by costimulatory blockade are as yet not well delineated.Adenovirus expressing CTLA4Ig was used to study blockade of the costimulatory CD28/B7 pathway in murine models of hepatocyte transplantation, and the effects on alloreactive T and B cells were studied.CTLA4Ig delayed rejection of subcutaneously administered C57L-derived murine hepatoma cells in CBA/J recipients for50 days. Activation and cytokine secretion by allospecific CD4+ and CD8+ T cells were initially blocked by CTLA4Ig; delayed rejection was associated with tumor infiltration by CD8+ T cells that did not secrete interferon-gamma. CTLA4Ig failed to block transplant rejection in primed mice, indicating that memory effector T cells were resistant to its action. In contrast, CTLA4Ig suppressed both naive and memory alloreactive B cells. High levels of CTLA4Ig mediated acceptance of hepatoma cells delivered directly into the spleen. However, isolated primary C57BL/6 mouse hepatocytes delivered into the spleen were rejected with only moderately delayed kinetics.Transplant antigenicity, transplant site, and CTLA4Ig dose all affected the survival of transplanted liver cells. CD8+ T cells are significant mediators of hepatocyte transplant rejection and are relatively resistant to costimulatory blockade with CTLA4Ig. Strategies to specifically antagonize CD8+ T cells or to modulate MHC class I expression in association with costimulatory blockade by CTLA4Ig may enhance the clinical feasibility of transplanting allogeneic hepatocytes.

Uridine-diphosphoglucuronate glucuronosyltransferases (UGTs) are a family of enzymes that conjugate various endogenous and exogenous compounds with glucuronic acid and facilitate their excretion in the bile. Bilirubin-UGT(1) (UGT1A1) is the only isoform that significantly contributes to the conjugation of bilirubin. Lesions in the gene encoding bilirubin-UGT(1), lead to complete or partial inactivation of the enzyme causing the rare autosomal recessively inherited conditions, Crigler-Najjar syndrome type-1 (CN-1) and type 2 (CN-2), respectively. Inactivation of the enzyme leads to accumulation of unconjugated bilirubin in the serum. Severe hyperbilirubinemia seen in CN-1 can cause bilirubin encephalopathy (kernicterus). Kernicterus can be fatal or may leave behind permanent neurological sequelae. Here, we have compiled more than 50 genetic lesions of UGT1A1 that cause CN-1 (including 9 novel mutations) or CN-2 (including 3 novel mutations) and have presented a correlation of structure to function of UGT1A1. In contrast to Crigler-Najjar syndromes, Gilbert syndrome is a common inherited condition characterized by mild hyperbilirubinemia. An insertional mutation of the TATAA element upstream to UGT1A1 results in a reduced level of expression of the gene. Homozygosity for the variant promoter is required for Gilbert syndrome, but not sufficient for manifestation of hyperbilirubinemia, which is partly dependent on the rate of bilirubin production. Several structural mutations of UGT1A1, for example, a G71R substitution, have been reported to cause mild reduction of UGT activity toward bilirubin, resulting in mild hyperbilirubinemia, consistent with Gilbert syndrome. When the normal allele of a heterozygote carrier for a Crigler-Najjar type structural mutation contains a Gilbert type promoter, intermediate levels of hyperbilirubinemia, consistent with the diagnosis of CN-2, may be observed.

The liver offers specific opportunities, as well as challenges, as a target for cell transplantation and gene therapy. Hepatocyte transplantation and hepatocyte-directed gene therapy are mutually augmenting complementary strategies that are rapidly developing for the treatment of inherited and acquired disorders. Both non-viral and viral vectors are being rapidly improved for exploiting the advantages and overcoming the challenges of liver-directed gene therapy. Rapid development in these areas suggests that vectors that will be used a decade from now have not yet been developed. Nevertheless, hepatocyte transplantation already has been successful in ameliorating at least some inherited liver diseases, and it is safe to state that successful liver-directed gene therapy will be achieved during the early years of the new millennium. The nine articles in this issue of the Seminars review the promise and limitations of the current methods of hepatocyte transplantation and liver-directed gene therapy and attempt to provide a glimpse of the anticipated developments.

The shortage of human livers available for hepatocyte isolation limits its clinical application. The availability of cloned, conditionally immortalized hepatocytes that could be grown in culture but would lose their transformed phenotype and provide metabolic support upon transplantation would greatly facilitate the treatment of acute liver failure. Toward this goal, we transduced isolated Lewis rat hepatocytes using a replication-defective recombinant retrovirus capable of transferring a gene encoding a thermolabile mutant simian virus 40 T antigen (SV40ts). The cloned, immortalized hepatocytes proliferate at 33 degrees C. At the nonpermissive temperatures (37-39 degrees C), they stop growing and exhibit characteristics of differentiated hepatocytes. These cells did not produce tumors when transplanted in mice with severe combined immunodeficiency disease or in syngeneic rats. To induce acute liver failure, Lewis rats were subjected to 90% hepatectomy (Hpx) and given 5% oral dextrose. All rats that did not undergo hepatocyte transplantation died within 96 hr. Fifty percent of rats that received intrasplenic injection of 10 x 10(6) primary Lewis rat hepatocytes (G2, n=6) or 10 x 10(6) SV40ts-conditionally immortalized (SV40ts-ci) hepatocytes (G3, n=8) 1 day before 90% hepatectomy survived, whereas 80% of rats that received an intraperitoneal injection of 200 x 10(6) primary Lewis rat hepatocytes (G4, n=10) or 200 x 10(6) SV40ts-ci hepatocytes (G5, n=10) on the day of hepatectomy survived. Survival after intraperitoneal injection of a cellular homogenate of 200 x 10(6) primary Lewis rat (G7, n=9) or SV40ts-ci hepatocytes (G8, n=10) on the day of Hpx was 33% and 40%, respectively, whereas survival after intraperitoneal injection of 200 x 10(6) Lewis rat bone marrow cells (G6, n=7) was 29%. Thus, transplanted, conditionally immortalized hepatocytes can be as effective as primary hepatocytes in supporting life during acute liver insufficiency. This work represents the first step in developing an hepatocyte cell line that would partially alleviate the organ-donor shortage and could be of potential clinical value.

Die Lebertransplantation ist heutzutage fur das Leberversagen die Therapie der Wahl. Dennoch stimulieren der Spenderorganmangel, die postoperative Letalitat und die erheblichen Kosten zur Suche nach Therapiealternativen. Die Transplantation von Leberzellinien (HTx) wurde aufgrund der funktionellen Uniformitat und der Verfugbarkeit der Zellen sowie aufgrund des geringen transplantationschirurgischen Aufwandes, insbesondere beim akuten Leberversagen, indiziert erscheinen.

Advances in orthotopic liver transplantation have improved the survival rate of both acute and chronic liver failure patients to nearly 70%. However, the success of this treatment modality has created an international organ shortage. Many patients die while awaiting transplantation in part due to the minimal capacity to store viable transplantable livers beyond 24 h. Additionally, for many areas of the world, routine use of whole liver transplantation to treat liver disease is impractical due to the demands on both financial and technical resources. Potentially, these issues may be alleviated, at least in part, by the use of liver cell transplantation or cellular-based liver assist devices. The well-documented regenerative capacity of the liver may obviate the need for whole organ transplantation in some instances of acute failure, if the patient may be provided temporary metabolic support. Although other patients ultimately may require transplantation, a longer period of time to find a suitable organ for transplantation may be gained by that supportive therapy. The field of liver cell transplantation may offer solutions to patients with inherited metabolic deficiencies or chronic liver disease. The potential to treat an hepatic disorder by using only a fraction of the whole liver would increase the number of whole organs available for orthotopic liver transplantation. Research in the fields of hepatocyte based intra- and extra-corporeal liver support is providing evidence that these therapeutic modalities may ultimately become routine in the treatment of severe liver disease. A historic overview of that technology along with its current status is discussed.

The data indicate that partial hepatectomy results in a decrease in degradation of targeted DNA. This appears to be due to an inhibition of the endocytotic pathway shortly after hepatectomy and is associated with the accumulation of targeted DNA within a population of light endosomal vesicles. It is likely that these vesicles serve as a reservoir from which targeted DNA gradually escapes and can be found in the nucleus. The DNA targeted to liver is capable of expressing the marker gene, and the DNA in the vesicles is transfection competent, suggesting that a substantial portion is intact. Overall, the receptor-mediated delivery system is highly efficient in transporting foreign DNA to liver cells. Partial hepatectomy and the ensuing cellular events provide a means of inhibiting the degradative portion of the endocytotic pathway. Pharmacological agents that can mimic the cellular processes that occur after partial hepatectomy may be useful in increasing the duration of foreign gene expression without the trauma of surgery.

In a patient with celiac sprue, abdominal complaints were ascribed to acute pancreatitis in the setting of hyperamylasemia and choledocholithiasis. The problem turned out to be macroamylasemia associated with elevated serum immunoglobulin A. The patient remained stable for 4 years with no specific intervention except gluten restriction. An association between celiac sprue or other malabsorptive states and macroamylasemia requires more study.

Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)-mediated endocytosis are being developed to transfer genes for the correction of bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin-UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33 degrees C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone-UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione-S-transferase Yp (GST-Yp), an oncofetal protein, was expressed in these cells at 33 degrees C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39 degrees C or 37 degrees C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone-UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 was expressed in these cells. The immortalized cells were not tumorigenic after transplantation into severe combined immunodeficiency mice. These conditionally immortalized cells will be useful for ex vivo evaluation of bilirubin-UGT gene transfer vectors.

Apolipoprotein (apo) B is the essential structural protein of the atherogenic lipoproteins, most notably low density lipoproteins (LDL) and lipoprotein (Lp) a. The apo B mRNA is subject to a unique mRNA modification which is termed RNA editing: at nucleotide position 6666 the genomically encoded cytidine is post-transcriptionally deaminated to uridine. This renders amino acid residue 2351 from glutamin CAA into a translational stop codon UAA and therefore leads to the synthesis of the truncated isoform apo B48. Apo B mRNA editing with production of apo B48 has been previously thought to be limited to the small intestine. However, recent results have suggested that in contrast to man in many species apo B mRNA editing is not intestine specific but does occur also in liver. Since apo B48 apparently has an accelerated plasma turnover, apo B mRNA editing in liver limits the plasma concentration of the atherogenic LDL and Lpa. It is therefore proposed that the reconstitution of apo B mRNA editing in liver by gene transfer of the apo B mRNA editing enzyme is an attractive approach for lowering high plasma cholesterol. Current work in our laboratory is addressing the feasability, efficiency and physiological impact of hepatic gene transfer of the apo B mRNA editing enzyme. In the following article the background and rationale for this approach are outlined in detail.

As the genes encoding the glucuronidating enzymes are discovered, it is evident that glucuronidation is a magnificent example of how in evolution, man became adapted to his "intoxicating" environment. A superfamily of genes is necessary to dispose of the toxins and carcinogens that are encountered by inhalation and ingestion. The enzymes that glucuronidate endogenous compounds are members of this large family. For the clinician, it is important to remember that jaundice may sometimes be the result of interactions at the level of bilirubin glucuronidation. When jaundice results from inactivation of members of the UGT1 family, conjugation of certain phenols, such as the anesthetic propofol, or synthetic estrogens, such as ethinylestradiol, can also be impaired. In the case of severe bilirubin glucuronidation deficiencies, such as the Crigler Najjar syndrome type I, there are exciting prospects for a possible cure by gene therapy.

Dietary antioxidants protect laboratory animals against the induction of tumors by a variety of chemical carcinogens. Among possible mechanisms, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes. Therefore, we investigated the effects of two commonly used food preservatives, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), on the expression of UDP-glucuronosyltransferase isoforms in rat liver. Male Wistar rats were fed a control diet or diets containing BHA (0.75%) or BHT (0.5%) for 2 weeks. BHT and BHA increased UDP-glucuronosyltransferase activities in liver microsomes for p-nitrophenol (236 and 218%, respectively), 3-hydroxybenzo(a)pyrene (246 and 175%, respectively), and androsterone (269 and 152%, respectively). Immunoblots showed changes in the amounts of UDP-glucuronosyltransferase isoforms corresponding to the changes in enzyme activities. Northern blot analysis showed that the concentration of UDP-glucuronosyltransferase mRNA paralleled the concentration of enzyme proteins and their respective levels of enzyme activity. BHT, for example, caused about a 250% increase in mRNA using a probe that recognizes the common 3'-domain of bilirubin/phenol UDP-glucuronosyltransferase mRNAs. In addition to inducing hepatic enzyme activities, BHT and BHA increased the activity of UDP-glucuronosyltransferase in the small intestine and kidney.

Asialoglycoproteins (ASG) are internalized by hepatocytes by ASG receptor (ASGR)-mediated endocytosis. We have shown previously that when a plasmid DNA, pAlb(9-12)CAT (expressing chloramphenicol acetyltransferase driven by an albumin promoter enhancer), was complexed with an ASG-polylysine conjugate and injected intravenously in rats, 80% of the DNA was internalized by the liver. In normal recipient rats, over 95% of the internalized DNA was degraded in 4 h; the plasmid was undetectable after 48 h. In contrast, when 66% hepatectomy was performed 20 min after DNA administration, the internalized DNA persisted for several weeks in cytoplasmic vesicles (Chowdhury, N. R., Wu, C. H., Wu, B. Y., Yerneni, P. C., Bommineni, V. R., and Chowdhury, J. R. (1993) J. Biol. Chem. 268, 11265-11271). Since microtubules are required for the translocation of ligand-containing endosomes to lysosomes, the site of ligand degradation, we hypothesized that persistence of the endocytosed DNA might be related to changes in microtubular structure and function. To test this hypothesis, we examined hepatocellular microtubules by immunofluorescence confocal microscopy. Liver from untreated rats or sham-operated controls showed a network of fibrillar microtubules throughout the cytoplasm. The extent of the microtubular network was substantially reduced 3-6 h after 66% hepatectomy. By 24 h, microtubules had regenerated. Intraportal infusion of cycloheximide (250 mg/kg body weight) 15 min before 66% hepatectomy, prevented microtubular disruption, indicating that protein synthesis is required for this process. Immunotransblot analysis showed that hepatic alpha-tubulin concentration remained unchanged through microtubular disassembly and subsequent reassembly, which is consistent with conservation and reutilization of tubulin released by depolymerization of microtubules.

The copper content and ceruloplasmin activity were determined in mice bearing benzo(a)pyrene induced fibrosarcoma. The copper level and ceruloplasmin activity in different organs of fibrosarcomatous mice varied when compared to their controls. Significant changes in copper and ceruloplasmin concentration were observed in the liver and tumor tissue of host mice bearing fibrosarcoma compared to controls. Disturbed copper metabolism at the hepatic level may account for the hypercupremia observed during malignancy.

After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is endocytosed by hepatocytes via asialoglycoprotein receptors and is expressed transiently. Long term persistence and expression occurs when partial hepatectomy is performed after gene delivery. To determine the intracellular location of the persisting DNA, we transferred a plasmid expressing bacterial chloramphenicol acetyltransferase into the liver of rats in vivo by asialoglycoprotein receptor-mediated endocytosis. The internalized DNA was measured by Southern blot. Twenty min after administration, 80-85% of the plasmid appeared in the liver, 80% of which was within hepatocytes (12,000-18,000 copies/hepatocyte). In sham-operated control rats, the transgene concentration decreased to 8-12 and 2-4% of the initial levels in 4 and 24 h, respectively, and became undetectable at 7 days. In rats subjected to 66% hepatectomy 20 min after DNA administration, 20, 9, and 7% of the plasmid in the residual liver persisted at 4 h, 24 h, and 7 days, respectively. Liver homogenates were fractionated by differential centrifugation and Percoll gradient centrifugation. In 66% hepatectomized rats, the plasmid persisted in an undegraded, transfection-competent form in plasma membrane/endosome-enriched fractions throughout the duration of the experiment (7 days), indicating that cytoplasmic vesicles are the main site of persistence of the endocytosed DNA.

Experiments in Gunn rats have provided important insights into physiological, biochemical, and molecular mechanisms of bilirubin conjugation and disposition. The Gunn rat is a natural model for bilirubin encephalopathy, and much of our knowledge of bilirubin toxicity and its treatment has come from studies performed in Gunn rats. The genetic lesion in Gunn rats closely parallel those in Crigler-Najjar syndrome, Type I. Presently, the Gunn rat model is being used to develop methods for gene therapy for inherited bilirubin-UGT deficiency. Thus, Gunn rats continue to be a valuable model for the investigation of inherited UGT deficiency and severe nonhemolytic unconjugated hyperbilirubinemia.

We developed a new hybrid mutant rat (athymic Gunn hybrid strain) that does not form and excrete conjugated bilirubin into bile and accepts xenografts because of T-lymphocyte deficiency. Cryopreserved isolated normal human, dog, or rat liver cells attached to collagen-coated microcarriers were transplanted into the athymic Gunn hybrid rats. Transplantation of human or rat liver cells resulted in biliary excretion of bilirubin conjugates predominantly as glucuronides, whereas isolated dog liver cells likewise transplanted resulted in the excretion of 40% to 50% of the conjugates as a glucoside-glucuronide diconjugate. The biliary bilirubin conjugates of the transplanted athymic Gunn hybrid rats paralleled those of the donor species. The data indicate that the types of bilirubin conjugates excreted by each species are determined by intrinsic properties of the respective hepatocytes rather than nutritional or other environmental factors. The findings also show that conjugation of bilirubin after liver cell transplantation results from functioning of the engrafted cells rather than activation of endogenous uridine diphosphatelucuronosyltransferase activity of the host.

Hepatocytes from donor transgenic mice that produce an easily assayable circulating marker have been used to develop a novel hepatocyte transplantation system. Isolated G7 HBV transgenic donor hepatocytes secreting HBsAg were transplanted into congeneic or allogeneic mouse recipients. Serum HBsAg was present three days after hepatocyte transplantation in congeneic animals and persisted indefinitely when hepatocytes were transplanted into the spleen. Transplanted hepatocytes within the splenic pulp were identified by morphologic and histochemical analysis. Migration of hepatocytes injected into the spleen to the liver was demonstrated by in situ hybridization using an RNA probe for HBsAg. Transplantation into nonimmunosuppressed allogeneic recipients resulted in disappearance of detectable hepatocytes in the spleen within two weeks. This novel transplantation system should facilitate studies of hepatocyte engraftment and survival, modulation of allograft rejection, and development of hepatocyte-directed gene therapy.

Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. 4-Nitrophenol glucuronidation is mediated by several UDPGT isoforms that are distinct from bilirubin-UDPGT, one of which is induced after 3-methylcholanthrene (3-MC) administration in normal, but not in Gunn rats. In normal rats, 3-MC-inducible UDPGT mRNA concentration increased 15-fold in the liver and 3-fold in kidney after 3-MC (140 mg/kg) administration. Concentration of this mRNA is much lower in Gunn rat liver and kidney compared to normal. However, this mRNA was normally induced after 3-MC administration. By RNA blot hybridization, the mRNA in Gunn rat liver and kidney appeared to be of normal size. Nuclear run-on studies showed that the transcription rate for 3-MC-inducible UDPGT was 3-fold higher in Gunn rat liver and kidney than in normal and increased 3- to 5-fold after 3-MC administration. Immunotransblot studies revealed an Mr = 56,000 3-MC-inducible UDPGT in liver and kidney of normal, but not in Gunn rats. However, a new immunoreactive UDPGT band (Mr = 43,000) was present in Gunn, but not in normal rats. Cell-free translation of kidney mRNA from 3-MC-treated Gunn rats showed that the Mr = 43,000 UDPGT is synthesized as an Mr = 45,000 protein. Prior hybridization of the mRNA with an isoform-specific oligonucleotide spanning the initiation codon abolishes synthesis of this protein. These results suggest that a sequence abnormality in the 3-MC-inducible UDPGT mRNA in Gunn rats results in reduced mRNA concentration and synthesis of a truncated, enzymically inactive UDPGT.

A total of 1232 female subjects including 100 prostitutes from Calcutta and adjoining areas in the age group of 21-70 years were screened for cervical pathology according to the method of Papanicolaou. Of 1232 examined, 815 were found to have smears with normal cellular cytology (66.15%). The others were grouped into dysplasia (18.4%) and inflammatory (15.4%) according to the cytomorphological characteristics of the exfoliated cells. The findings though revealed more cases of cervical dysplasia in women with early marriage, early childbirth, high parity and multiple sexual partners, it failed to support the smegma theory of cervical carcinoma. This study also showed a number of inflammatory cervical smears in women coming from low socio-economic group.

The ceruloplasmin concentration was determined in 145 cancer patients prior to and after treatment with different radiotherapeutic and chemotherapeutic regimes. The ceruloplasmin concentration was observed to be higher in patients with malignancies than in healthy controls. There was a positive correlation of the values with the clinical condition of the patients. The ceruloplasmin concentration was noted to stop increasing and subsequently fall in patients who responded to therapy, and, in contrast, to remain high or become higher in those who did not respond to therapy. The diagnostic and prognostic value of ceruloplasmin determination is discussed.

This study demonstrates that for the isolated cat gall bladder a smaller molar dose of the sulfated form of OP-CCK and gastrin is required to produce contraction as compared to the respective non-sulfated forms. For OP the D50 for the sulfated form versus the non-sulfated form was 1.94. For gastrin it was 1.10.

Erythrocyte Survival. Osmotic Fragility, Fibrosarcom a Red cell survival studies in mice with benzo(a) pyrene [B(a)p]-induced fibrosarcoma indicate marked reduction in RBC 51Cr rW2. In contrast, the t w2 of erythrocytes from B(a)p injected but tumor-free mice was not declined. Increasing tumor burden resulted in increment in the severity of this disorder, and preincubation of normal red cells with tumor supernatant caused significant reduction (p < 0.05) in 51Cr t > /2 . It is suggested that the observed alteration in RBC life span may be due to the effect of tumor. Apparently, both intrinsic and extrinsic cellular defects could be implicated for this abnormality. However, the shortened survival cannot be attributed to altered osmotic fragility, since the erythrocytes of the fibro-sarcomatous mice were more resistant to hypotonic hemolysis in vitro.

The technique of side-to-side mesocaval anastomosis with the interposition of a prosthetic graft was described in 1970. We report the result of venographic evaluation of patency of this type of shunt in our patients. Demonstration of an unoccluded graft; resistance to retrograde injection of dye against the blood stream into the superior mesenteric vein; rapid clearing of dye from superior mesenteric vein following injection and "lamellar flow defect" in the vena cava above the site of anastomosis during vena cavogram, were considered criteria of patency of the interposition mesocaval shunt.

Effect of tumor growth on the survival of circulating erythrocytes was studied in mice bearing a wide spectrum of experimental tumors. RBC half-life (t1/2), measured by 51Cr-labeling technique, decreased significantly (p less than 0.05) in all the tumor types studied, particularly in transplantable Sarcoma-180 and benzo(a)pyrene-induced primary fibrosarcoma. Changes in erythrocyte morphology like anisopoikilocytosis were also observed in the tumor hosts. Cross-transfusion of 51Cr-labeled RBCs between normal and tumor-bearing animals revealed that both intrinsic and extrinsic factors are responsible for shortened RBC survival. As far as the cellular abnormalities are concerned, the decrease in RBC t1/2 was not attributable to increased osmotic fragility as the cells were observed to be osmotically more resistant. Similarly, membrane sialic acid content was markedly elevated in the tumor hosts, thus the shortened erythrocyte life-span cannot be attributed to decrease in sialic acid content of the erythrocyte membrane.

The erythropoietic response of Strain A mice with benzo(a)pyrene-induced fibrosarcoma has been studied. The rate of erythropoiesis, expressed in terms of 59Fe incorporation into circulating erythrocytes, was slightly increased in the fibrosarcomatous mice compared to the matched controls. The femoral marrow of the tumor hosts became hypocellular with reduced number of erythroblasts. The spleen, on the other hand, was hypercellular with an increased number of erythroid progenitor cells. Quantitative assessment of erythropoietic activity in the hematopoietic organs by measuring the 6-hr organ uptake of 59Fe revealed erythropoietic suppression in the marrow but enhancement in the spleen following tumor development. While the number of CFU-s in the femoral marrow of the tumor-bearing animals was slightly increased, marked increase in the concentration as well as absolute number of CFU-s was found in the spleen. Bioassay for erythropoietin revealed appreciable increase in the level of plasma erythropoietin in the tumor-bearing animals.

The occurrence and distribution of glycogen was investigated in cervical cells exfoliated under different physiologic and pathologic conditions using cytochemical parameters. The conditions studied included the menstrual cycle, menopause, the reproductive cycle and neoplastic lesions of the cervix in both premenopausal and postmenopausal and postmenopausal situations. The results obtained on the localization of this carbohydrate substance offer interesting information with respect to the absence and presence of glycogen in various degrees in different cell types. The presence of glycogen seems to be linked with cellular maturation; it disappears with the loss of differentiation during neoplasia. The high glycogen content during the menstrual cycle and reproductive cycle and its absence in postmenopause indicates a specific hormonal influence. The discrepancies noted in glycogen distribution in the same or different exfoliated cells obtained from different biologic states assume significance in light of various factors governing the synthesis and utilization of this polymer.

A mammary tumor-associated antigen (MTAA) of the murine mammary tumor virus (MuMTV)-induced spontaneous mammary tumors of C3H/J mice was purified and partially characterized. The crude extract of the mammary tumor, when subjected to DEAE-cellulose chromatography and eluted with a discontinuous NaCl gradient, provided three major protein peaks, of which only the first (F1) possessed the MTAA activity. The antigen was further purified by subjecting F1 to polyacrylamide gel electrophoresis. The MTAA was a glycoprotein with a molecular weight of approximately 83,000. The antigen was localized in the plasma membrane and was different from the MuMTV structural antigens. Circulating antibodies against the MTAA were observed in the sera of tumor-bearing mice but not in that of tumor-free mice.

Formation of bilirubin monoglucuronide from unconjugated bilirubin requires a microsomal enzyme, UDP-glucuronate glucuronyltransferase (EC 2.4.1.17). Conversion of bilirubin monoglucuronide to bilirubin diglucuronide, the major bilirubin conjugate in bile, was studied in subcellular fractions of rat liver. The highest specific activity for bilirubin diglucuronide formation occurred in a fraction highly enriched in plasma membranes. Studies of reaction stoichiometry and utilization of UDP-D-[14C]glucuronic acid revealed that conversion of bilirubin monoglucuronide to bilirubin diglucuronide is not catalyzed by UDP-glucuronyltransferase, and results from transglucuronidation of bilirubin monoglucuronide, with formation of bilirubin diglucuronide and unconjugated bilirubin. When unconjugated bilirubin was infused intravenously into rats at rates exceeding the maximal hepatic excretory capacity, bilirubin monoglucuronide accumulated in serum and bilirubin diglucuronide was found exclusively in bile as the predominant bilirubin metabolite. These results suggest that formation of bilirubin diglucuronide occurs at the surface membrane of the liver cell. Conversion of bilirubin monoglucuronide to bilirubin diglucuronide may play a role in the transport of bilirubin glucuronides from liver to bile.

After oral administration of sulfasalazine, the majority of the administered dose reaches the colon, where it is split into 5-aminosalicylic acid (5-ASA) and sulfapyridine. 5-ASA is believed to be the effective component in the treatment of inflammatory bowel disease. After intraduodenal administration of 5-ASA (20 mg) in rats, 91% of the drug was absorbed in the proximal small intestine. Peak serum 5-ASA concentration (55 micrograms/ml) was reached in 1 h. Approximately 61 and 6% of the administered dose were excreted in the urine and bile, respectively, in 24 h, almost exclusively in the acetylated form. When sulfapyridine (20 mg) was administered in addition to 5-ASA, 70% of the sulfapyridine was absorbed in the small intestine, peak serum concentration (50 micrograms/ml) was reached in 1 h, and 30% of the administered dose was excreted in the urine in 24 h. The results indicate that after oral administration of 5-ASA, a therapeutically significant concentration of the drug is not expected in the terminal ileum which is a common site of involvement in Crohn's disease. The therapeutic implications of these findings are discussed herein.

A comprehensive study was made of the patterns of lipid granules in vaginal cells exfoliated during different hormonal states as well as under neoplastic conditions. The influence of in vitro incubation with estrogen and progesterone on the lipid patterns in cells exfoliated from normal and cancerous cervices was also investigated. The results clearly indicated the influence of sex hormones on the size and distribution of the lipid granules. While estrogen increased the fine lipid granules, as evident during the estrogenic phase of the menstrual cycle, progesterone influenced the coarse granules, which increased during the progestational phase and pregnancy. Similar responses were observed in the in vitro studies. The significance of the fine and coarse granules in different conditions is discussed in light of both the alteration of the metabolic process governing the synthesis of these components and the conversion of fine granules to coarse granules under such conditions.