Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice

We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3′ albumin enhancer (3′iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 109 vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 1010 vg/mouse. The further inclusion of an 860 base-pairs 3′iALB enhancer component in the 3′ untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production

Combined hepatic and renal transplantation in primary hyperoxaluria type I: Clinical report of nine cases

Purpose and patients and methodsThe purpose of this article is to report the experience of three centers with combined hepatic and renal transplantation for pyridoxine-resistant primary hyperoxaluria type I (alanine:glyoxylate aminotransferase [EC 2.6.1.44] deficiency), with particular emphasis on the selection criteria and timing of the operation. Nine patients with this inherited disease were treated by combined hepatic and renal transplantation. The former replaces the enzyme-deficient organ while the latter replaces the functionally affected organ.ResultsOne patient with gross systemic oxalosis died in the immediate postoperative period and another died 8 weeks postoperatively of a generalized cytomegalovirus infection, having shown evidence of biochemical correction. One patient with particularly severe osteodystrophy at the time of the operation died 14 months postoperatively from renal failure due to progressive calcium oxalate nephrocalcinosis involving the transplanted kidney, plus thromboembolic disease. He also had very extensive systemic oxalosis. An additional patient with severe osteodystrophy died 9 months postoperatively. One patient developed hyper-rejection of the kidney and died later of gastrointestinal hemorrhage. The four long-term survivors (22 to 38 months) have remained asymptomatic from the standpoint of their renal disease, with resolution of any manifestations of systemic oxalosis that they may have had. They are either employed or continuing their education.ConclusionsA prolonged period of end-stage renal failure treated by dialysis regimens that are suitable for non-hyperoxaluric renal failure and extensive systemic oxalosis, particularly oxalotic osteodystrophy, are poor prognostic features. We propose that hepatic transplantation should be considered as definitive treatment before end-stage renal failure develops. This should be supplemented by renal transplantation with vigorous pre- and perioperative hemodialysis to deplete the body stores of oxalate. Although some authorities would reserve hepatic transplantation for patients in whom renal transplantation has failed, we suggest that combined liver and kidney transplantation is appropriate in patients who have never had a renal graft. Furthermore, the time has come to consider hepatic transplantation before any irreversible renal damage has occurred in these patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29475/1/0000561.pd

Novel inhibitors of the calcineurin/NFATc hub - alternatives to CsA and FK506?

The drugs cyclosporine A (CsA) and tacrolimus (FK506) revolutionized organ transplantation. Both compounds are still widely used in the clinic as well as for basic research, even though they have dramatic side effects and modulate other pathways than calcineurin-NFATc, too. To answer the major open question - whether the adverse side effects are secondary to the actions of the drugs on the calcineurin-NFATc pathway - alternative inhibitors were developed. Ideal inhibitors should discriminate between the inhibition of (i) calcineurin and peptidyl-prolyl cis-trans isomerases (PPIases; the matchmaker proteins of CsA and FK506), (ii) calcineurin and the other Ser/Thr protein phosphatases, and (iii) NFATc and other transcription factors. In this review we summarize the current knowledge about novel inhibitors, synthesized or identified in the last decades, and focus on their mode of action, specificity, and biological effects

Alotransplante de ilhotas de Langerhans no fígado de ratos submetidos a manipulação tímica com células dendríticas

RACIONAL: A maior indicação do transplante de pâncreas ou de ilhotas de Langerhans é o diabetes mellitus do tipo I. O processo deve suprir as necessidades de insulina, mantendo os níveis glicêmicos dentro da normalidade. OBJETIVOS: Estudou-se o alotransplante de ilhotas de Langerhans no fígado de ratos Lewis, tendo como doadores de ilhotas ratos Wistar. No grupo controle (n = 8) injetava-se, no timo, solução de Hanks e no grupo de estudo (n = 9), células dendríticas. MATERIAL E MÉTODOS: No grupo controle com o método de separação e purificação das ilhotas de Langerhans obteve-se 3637 ± 783,3 ilhotas com pureza de 85% ± 3,52%. No grupo de estudo obteve-se 3268 ± 378 ilhotas de Langerhans com pureza de 87% ± 4,47% e com o método de isolamento e purificação das células dendríticas do baço obteve-se 3,34 x 105 ± 1,16 células. RESULTADOS: No grupo controle, o transplante de 3637 ± 783,3 ilhotas de Langerhans no fígado, normalizou a glicemia que chegou a 7,21 ± 0,57 mmol/L no segundo pós-operatório (diferença significativa com relação ao pré-operatório). Do pós-operatório imediato até o 8º pós-operatório a glicemia não se elevou significativamente, porém a partir do 10º pós-operatório houve aumento significativo deste parâmetro, o que pode ser compatível com rejeição aguda do enxerto. No grupo de estudo, o transplante de 3258 ± 378 ilhotas de Langerhans no fígado, normalizou a glicemia, que chegou a 9,3 ± 2,85 mmol/L no segundo pós-operatório (diferença significativa com relação ao pré-operatório). Do 4º ao 10º pós-operatório, a glicemia elevou-se significativamente, o que pode ser compatível com quadro de rejeição aguda do enxerto e certamente precoce. CONCLUSÃO: A inoculação de células alogênicas apresentadoras de antígenos (células dendríticas) no timo de ratos imunossuprimidos e diabéticos, antes do alotransplante de ilhotas de Langerhans no fígado, ao contrário de inibir a reação do receptor contra o enxerto, prolongando a sobrevida média das ilhotas e, possivelmente, levando ao estado de tolerância imunológica, induziu ao processo de rejeição aguda precoce

An analysis of cyclosporine efficacy and toxicity after liver transplantation

The use of cyclosporine long term after orthotopic liver transplantation has been analyzed in 73 adults with particular reference to the dose of drug used, either alone or in combination with other immunosuppressive agents, and the side effects observed. The first 22 patients were given cyclosporine 10 mg/kg/day for up to 2 years, but thereafter in these, and in all the other patients, the drug dose was regulated by whole blood trough levels. The proportion of patients maintained on cyclosporine alone increased from 11% at 3 months to 54.9% and 55.6% at 3 and 4 years, respectively. The dose of prednisolone used in combination with cyclosporine was lower than that used with azathioprine (P less than 0.05) up to 12 months after transplantation, but thereafter no significant difference was found. Acute cellular rejection was seen in 5 patients and in all instances was related to cessation of cyclosporine, while 10 patients developed chronic graft rejection manifested by the vanishing bile duct syndrome. At 12 months and onward, 54.5-73.3% of patients had normal serum bilirubin levels, and 47.6-80.0% had aspartate aminotransferase levels in the normal range. Cyclosporine was discontinued in 12 patients, in 8 cases because of impairment of renal function or hypertension. A trend toward rising serum creatinine levels was seen, and after 4 years on cyclosporine none of 12 patients had normal levels, and these exceeded 200 mumol/L in 5. The rise in creatinine levels was probably in part related to the higher doses used early in the study period. The incidence of hypertension progressively increased from 15.3% at 3 months to 63.6% at 4 years in patients maintained on cyclosporine