Scoliosis in paediatric onset spinal cord injuries.

STUDY DESIGN: This is a retrospective longitudinal review. OBJECTIVE: The purpose of this review was to identify predictors of developing clinical scoliosis and compare between traumatic and neurological aetiologies of SCI. SETTING: This study was conducted at the Midland Centre of SCI. METHOD: Case notes of all patients injured at an age up to 18 years and admitted between 1971 and 2013 were reviewed. RESULTS: 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%). CONCLUSION: 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.

Use of antibiotics and the prevalence of antibiotic-associated diarrhoea in patients with spinal cord injuries: an international, multi-centre study.

BACKGROUND: Little is known about the use of antibiotics and the extent of antibiotic-associated diarrhoea (AAD) in patients with spinal cord injuries (SCIs). AIMS: To record the use of antibiotics, establish the prevalence of AAD and Clostridium difficile infection (CDI), and assess if there was any seasonal variation in antibiotic use and incidence of AAD in patients with SCIs. METHODS: A retrospective study was conducted in six European SCI centres between October 2014 and June 2015. AAD was defined as two or more watery stools (Bristol Stool Scale type 5, 6 or 7) over 24 h. FINDINGS: In total, 1267 adults (median age 54 years, 30.7% female) with SCIs (52.7% tetraplegia, 59% complete SCI) were included in this study. Among the 215 (17%) patients on antibiotics, the top three indications for antibiotics were urinary tract infections (UTIs), infected pressure ulcers and other skin infections. Thirty-two of these 215 (14.9%) patients developed AAD and two patients out of the total study population (2/1267; 0.16%) developed CDI. AAD was more common in summer than in spring, autumn or winter (30.3% vs 3.8%, 7.4% and 16.9%, respectively; P<0.01). AAD was associated with age ≥65 years, tetraplegia, higher body mass index, hypoalbuminaemia, polypharmacy, multiple antibiotic use and high-risk antibiotic use. Summer and winter seasons and male sex were identified as independent predictors for the development of AAD. CONCLUSION: This survey found that AAD is common in patients with SCIs, and UTI is the most common cause of infection. Summer and winter seasons and male sex are unique predictors for AAD. Both AAD and UTIs are potentially preventable; therefore, further work should focus on preventing the over-use of antibiotics, and developing strategies to improve hospital infection control measures.

To cystoscope or not to cystoscope patients with traumatic spinal cord injuries managed with indwelling urethral or suprapubic catheters? That is the question!

STUDY DESIGN: 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. OBJECTIVES: 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. METHODS: Theater management system and the electronic patient records used to retrieve demographics, injury details and operative findings. RESULTS: 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. CONCLUSION: 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.

Tuberculosis and female reproductive health.

Tuberculosis (TB) is an important cause of mortality and morbidity all over the world and is particularly relevant in developing countries like India where the disease is endemic. Female reproductive system is very vulnerable to this infection and clinical presentation of this disease in female reproductive tract is protean in nature and in a large majority of patients could be completely silent. This disease is an important cause of infertility, menstrual irregularity, pregnancy loss, and in association with pregnancy, morbidity to both the mother and child increases. Some of the effects of TB infection on female genital tract could be remote in nature due to infection elsewhere. Medicines used to treat TB infection can also have adverse effects on contraception and other areas of female reproductive health. HIV coinfection and multidrug-resistant tuberculosis (MDR-TB) and increased population migration from developed to developing countries have now added a whole new dimension to this infection. Though new, finer diagnostic tools of detection of TB are increasingly available in the form of bacterial cultures and polymerase chain reaction (PCR) based diagnostics, suspicion by clinicians remains the main tool for diagnosis of the condition. Hence, doctors need to be properly trained to become "Tuberculosis Minded".

Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications.

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.

The effect of CD28/B7 blockade on alloreactive T and B cells after liver cell transplantation.

BACKGROUND: 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. METHODS: 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. RESULTS: CTLA4Ig delayed rejection of subcutaneously administered C57L-derived murine hepatoma cells in CBA/J recipients for >50 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. CONCLUSIONS: 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.

Genetic lesions of bilirubin uridine-diphosphoglucuronate glucuronosyltransferase (UGT1A1) causing Crigler-Najjar and Gilbert syndromes: correlation of genotype to phenotype.

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.

A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats.

BACKGROUND & AIMS: In the quest for a recombinant viral vector for liver-directed gene therapy that would permit both prolonged and efficient transgene expression in quiescent hepatocytes in vivo and repeated administration, we evaluated a recombinant simian virus 40 (rSV40). METHODS: The rSV40 was generated through replacement of the DNA encoding for the T antigens (Tag) by the coding region of human bilirubin-uridine 5'-diphosphate-glucuronosyl-transferase (BUGT) complementary DNA (SV-hBUGT). Helper-free rSV40 units were generated at infectious titers of 5 x 10(9) to 1 x 10(10) infectious units (IU)/mL in a Tag-producing packaging cell line (COS-7 cells). RESULTS: After 1, 3, or 7 daily infusions of 3 x 10(9) IU of SV-hBUGT through an indwelling portal vein catheter in bilirubin-UGT-deficient jaundiced Gunn rats, mean serum bilirubin concentrations decreased by 40%, 60% and 70%, respectively, in 3 weeks and remained at those levels throughout the duration of the study (40 days). Results of liver biopsies from SV-hBUGT-treated Gunn rats, but not from controls, were positive for human BUGT DNA, messenger RNA, and protein. Bilirubin-UGT activity in liver homogenates was 8%-12% of normal, and bilirubin glucuronides were excreted in bile. Immunostaining showed that >50%-60% of hepatocytes stably expressed the transgene. Portal vein infusion of an rSV40 expressing hepatitis B surface antigen (HBsAg) in a naive Gunn rat and a Gunn rat that had received 7 injections of SV-BUGT resulted in approximately equal levels of hepatic expression of HBsAg, indicating that multiple inoculations of SV-BUGT did not elicit neutralizing antibodies. Plasma alanine aminotransferase levels and liver histology remained normal despite repeated injections of rSV40. CONCLUSIONS: rSV40 vectors may represent a significant advance toward gene therapy for metabolic diseases.

Enhancement of retrovirus-mediated gene transfer to rat liver in vivo by infusion of hepatocyte growth factor and triiodothyronine.

BACKGROUND/AIMS: Gene transfer using recombinant Moloney murine leukemia viruses (rMoMuLV) requires mitosis of the target cell. Previously, we and others have used partial hepatectomy for induction of hepatocellular proliferation for gene transfer to the liver in vivo by exsanguineous perfusion with rMo-MuLV. We hypothesized that induction of hepatocellular proliferation by combined administration of two hepatocellular mitogens, hepatocyte growth factor (HGF) and triiodothyronine (T3), should permit rMo-MuLV-mediated gene transfer into liver without invasive approaches. METHODS: HGF (1 mg/kg) was perfused continuously into the portal vein of Wistar male rats and T3 (2 mg/kg) was injected subcutaneously. Twenty-four hours after injecting HGF and T3, the state of proliferation of hepatocytes was estimated from the incorporation of 5'-bromo-2'-deoxy-uridine (BrdU). The amphotropic retroviral receptor (Ram-1) expression of liver was evaluated at different time points after injecting HGF and T3 by means of Northern blotting using Ram-1 cDNA probe. In order to evaluate the role of hormone treatment on gene transfer, the liver was perfused exsanguineously with rMoMuLV 24 h after injection with hormones. RESULTS: Rats treated with a combination of HGF and T3 expressed BrdU and beta-galactosidase in 8.3% and 0.7% of hepatocytes, respectively. On the other hand, there was near absence of gene transfer in untreated rats perfused with rMoMuLV Twenty-four hours after the initial manipulation, abundant expression of Ram-1 mRNA was observed in rat hepatocytes treated with HGF plus T3. CONCLUSIONS: Stimulation of hepatocellular mitosis and upregulation of Ram-1 expression by HGF and T3 augment retrovirus-mediated gene transfer into hepatocytes.

Treatment of experimental colitis by oral tolerance induction: a central role for suppressor lymphocytes.

OBJECTIVE: Inflammatory bowel diseases (IBD) are immune-mediated disorders wherein an imbalance between proinflammatory (Th1) and antiinflammatory (Th2) cytokines is thought to play a role in the pathogenesis. The aim of this study was to test whether induction of oral tolerance to proteins extracted from inflammatory colon alleviates experimental colitis, and whether oral tolerization mediated by suppressor cells can induce immune tolerance. METHODS: Colitis was induced in rats by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Rats received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Splenocytes harvested from tolerized and control rats were transplanted into irradiated naive rats. RESULTS: Feeding of colitis-extracted proteins ameliorated colonic inflammation, as shown by reduction of colonic ulcerations, as well as decreased diarrhea, intestine and peritoneal adhesions, wall thickness, and edema. A marked reduction of the fraction of injured colonic area and colon weight, and decrease in colon weight, were observed in tolerized rats versus controls. Histological parameters for colitis were markedly improved in tolerized animals that showed significant reduction in inflammatory response and mucosal ulcerations. Tolerized rats developed an increase in TGFbeta1 and a decrease in IFNgamma serum levels. TNBS-induced colitis was significantly attenuated in naive recipients of splenocytes from tolerized rats, compared with rats that received splenocytes from control donors. CONCLUSIONS: Induction of oral tolerance to colitis-extracted proteins downregulates the anticolon immune response, thereby ameliorating experimental colitis. Suppressor lymphocytes mediate the tolerance by induction of a shift from a proinflammatory to an antiinflammatory immune response.

Liver-directed gene therapy: promises, problems and prospects at the turn of the century.

Although liver-directed gene therapy arrived later than gene therapy directed at bone marrow cells, intrinsic advantages of the liver as a target organ make it likely that gene therapy for liver diseases will be among the first therapeutically relevant applications of this treatment modality at the onset of the 21st century. Vectorology for gene transfer to the liver is advancing rapidly, and it is safe to predict that gene therapy vehicles that will be in clinical use a decade from now, have not yet been developed. None of the currently available modes of gene transfer to the liver is optimal for all types of applications. Nonetheless, the concerted effort of many investigators has provided a wide choice of non-viral and viral vectors for gene transfer to the liver for use in specific situations. Original strategies for liver-directed gene therapy included substitution of missing gene products, overexpression of intrinsic or extrinsic genes and inhibition of expression of specific genes. To the list is now added the possibility of site-specific correction or generation of mutations within specific genes in somatic cells of living adult animals. Thus, despite some initial faux pas, liver-directed gene therapy is poised to make an important impact on health care in the year 2000 and beyond.

Oral tolerization ameliorates liver disorders associated with chronic graft versus host disease in mice.

In chronic graft versus host disease (cGVHD), an immune attack by transplanted donor lymphocytes results in damage of host target organs. A disbalance between proinflammatory (Th1) and anti-inflammatory cytokines (Th2) plays an important role in the pathogenesis. Immune hyporesponsiveness induced by oral antigen administration has been shown to suppress autoimmunity. We evaluated the efficacy of oral tolerization in preventing cGVHD in a mouse model. cGVHD was generated by infusing 2.5 x 10(7) splenocytes from B10.D2 donor mice, to sublethally irradiated (6 Gy) BALB/c recipient mice, which differ in minor histocompatibility antigens. The transplantation resulted in cGVHD, with characteristic hepatic and small bowel inflammation, and increased skin collagen content and fibrosis. Oral tolerance was induced by feeding donor B10.D2 mice with proteins extracted from BALB/c splenocytes at 50 microg/d per mouse for 11 days before transplantation. Tolerization was evidenced by reduction in mixed lymphocyte response of effector splenocytes from tolerized B10.D2 mice against BALB/c target splenocytes. Liver and small bowel biopsy specimens revealed much less inflammation. Oral tolerization prevented weight and subcutaneous fat loss, reduced thickening, and skin collagen deposits. Reduction of collagen alpha1 (I) gene expression was shown by in situ hybridization. Serum interleukin 10 (IL-10) levels measured significantly higher in tolerized mice than in controls, whereas interferon gamma (IFN-gamma), IL-2, and tumor necrosis factor alpha (TNF-alpha) were reduced significantly. Oral tolerization of splenocyte donors towards recipient-strain splenocytes ameliorated cGVHD of the liver, small intestine, and skin. A cytokine shift from a proinflammatory to an anti-inflammatory pattern may play a role in down-regulation of the immune-mediated target organ damage.

Correction of the UDP-glucuronosyltransferase gene defect in the gunn rat model of crigler-najjar syndrome type I with a chimeric oligonucleotide.

Crigler-Najjar syndrome type I is characterized by unconjugated hyperbilirubinemia resulting from an autosomal recessive inherited deficiency of hepatic UDP-glucuronosyltransferase (UGT) 1A1 activity. The enzyme is essential for glucuronidation and biliary excretion of bilirubin, and its absence can be fatal. The Gunn rat is an excellent animal model of this disease, exhibiting a single guanosine (G) base deletion within the UGT1A1 gene. The defect results in a frameshift and a premature stop codon, absence of enzyme activity, and hyperbilirubinemia. Here, we show permanent correction of the UGT1A1 genetic defect in Gunn rat liver with site-specific replacement of the absent G residue at nucleotide 1206 by using an RNA/DNA oligonucleotide designed to promote endogenous repair of genomic DNA. The chimeric oligonucleotide was either complexed with polyethylenimine or encapsulated in anionic liposomes, administered i.v., and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, and DNA sequencing, and confirmed by genomic Southern blot analysis. DNA repair was specific, efficient, stable throughout the 6-month observation period, and associated with reduction of serum bilirubin levels. Our results indicate that correction of the UGT1A1 genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, with consequent improvement in the metabolic abnormality.

Characterization and evaluation of detoxification functions of a nontumorigenic immortalized porcine hepatocyte cell line (HepLiu).

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TAI (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Induction of tolerance to hepatitis B virus: can we 'eat the disease' and live with the virus?

Hepatitis B virus (HBV) is a major health problem, with over 300 million HBsAg carriers worldwide. The HBV itself is non-cytopathic, and it is widely accepted that the mechanism of hepatocellular injury is the host anti-viral immune response. Current treatments, including the newly developed therapeutic modalities, are either based on antiviral drugs or focus on attempts to augment the anti-viral immune response. The results of these approaches have been largely disappointing. There is evidence, however, that subjects with a natural tolerance to HBV or a down-regulated immune response develop little or no liver injury, despite chronic viremia. Lately, it has been shown that it is possible to induce tolerance toward viruses by oral administration of major viral structural proteins. Here, we discuss the pathogenesis of HBV-mediated liver disease and approaches to down-regulate the immune response directed against liver cells by orally inducing tolerance toward the virus. We hypothesize that this acquired tolerance should turn chronic active hepatitis patients with deteriorating disease into 'healthy' virus carriers. The proposed new treatment strategy would redirect the focus from augmenting anti-viral immune response to inducing host tolerance towards the virus.