Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats.

Graft-versus-host disease (GvHD), a major complication of allogeneic bone marrow transplantation, has been ascribed to mature T cells in the graft. Because T cells play an important role in engraftment of the bone marrow and decrease the probability of relapse of leukemia, a treatment strategy was developed to preserve the benefits of T cells in the graft and to control the severe complications of GvHD. This can be accomplished by the genetic modification of donor T cells with a suicide gene that allows their selective in vivo elimination and subsequently the abrogation of GvHD. For clinical benefit the alloreactivity of herpes simplex virus thymidine kinase (HSV-TK) gene-transduced T cells should be retained. Therefore, we investigated the influence of gene transduction and the selection procedure on T cells. We demonstrated that activation and culturing of T cells reduce their capacity to induce lethal GvHD in an allogeneic rat bone marrow transplantation model. Furthermore, positive immunomagnetic selection of gene-transduced T cells resulted in loss of the GvHD-inducing capacity of HSV-TK(+) T cells directly after MACS (magnetic cell sorting) selection; this loss could be recovered by a 1-day expansion of the selected T cells. No effect on alloreactivity was observed to be caused by the gene transduction procedure. Our study resulted in the development of an optimized culture and gene transduction protocol with preservation of T cell alloreactivity. Treatment of transplanted rats with ganciclovir resulted in a rapid reduction in the number of HSV-TK(+) T cells in the peripheral blood and in increased survival of the animals.     

Renal function in tyrosinaemia type I after liver transplantation: A long-term follow-up

Summary Hereditary tyrosinaemia type I is an autosomal recessive inborn error of tyrosine catabolism caused by a deficiency of the enzyme fumarylacetoacetase that results in liver failure, hepatocellular carcinoma, renal tubular dysfunction and acute intermittent porphyria. When treated with liver transplantation, tyrosinaemia type I was considered to be cured. Some years after the first liver transplantations in these patients, some reports focused on the renal function after transplantation. These reports showed that urinary succinylacetone excretion remained but that tubular function normalized. In this report we discuss the long-term renal follow-up (mean follow-up time 11 years, range 7–14 years) after liver transplantation in 9 patients with tyrosinaemia type I treated by liver transplantation in our centre. An evaluation was made of renal function and succinylacetone excretion in urine. In all patients we found a persistent excretion of succinylacetone in the urine. With respect to the glomerular function, we can conclude that there is no clear change in GFR. At the same time, tubulopathy persisted in some patients. We consider that excretion of metabolites such as succinylacetone will be an important contributing factor to tubular dysfunction after liver transplantation in patients with tyrosinaemia type I. Therefore, notwithstanding the major effect of liver transplantation on tyrosine metabolism, renal tubular dysfunction remains at risk and needs careful monitoring. Progressive tubular dysfunction can cause glomerular damage. The use of low-dose NTBC might be considered after liver transplantation in case of tubulopathy to prevent progression of tubular and glomerular dysfunction. An erratum to this article is available at .     

Ganciclovir-mediated elimination of HSV-Tk+ T cells and cure of graft-vs-host disease in an allogeneic bone marrow transplantation model in the rat

OBJECTIVE: Suicide gene therapy for leukemia aims to benefit from T cells in the BM graft, by reducing the probability of leukemia relapse (GVL), while severe complications of graft-vs-host disease (GVHD) may be avoided. In an allogeneic rat BMT model we defined the conditions to induce a lethal GVHD with HSV-Tk gene-transduced T cells. We studied the feasibility to rescue the animals by conditional elimination of the T cells with ganciclovir (GCV) treatment. METHODS: Allogeneic T cells transduced with a retroviral vector encoding the HSV-Tk suicide gene were added in varying numbers to a BM graft. Expression of HSV-Tk strongly increases the cytolytic effect of GCV, thereby allowing elimination of overreactive T cells at will. Various experimental conditions were tested in the rat model. RESULTS: A relation between the number of HSV-Tk(+) T cells added to the BM graft and GVHD development was found. GCV treatment resulted in selective HSV-Tk(+) T-cell elimination in blood and tissues but not in abrogation of GVHD due to persistence of HSV-Tk(-) T cells. T cells in unmanipulated rat BM normally have a low risk to induce GVH but when they are administered in combination with high numbers of HSV-Tk(+) T cells there is an apparent increase in their GVH-inducing potential. When HSV-Tk(+) T cells are added to T cell-depleted BM a consequently developing GVH can be controlled by GCV treatment with 60-70% of the animals surviving. CONCLUSIONS: We show that T cell-mediated suicide gene therapy within the context of allo-BMT can be applied with success. The apparent limitation in the number of transduced as well as nontransduced T cells that can be safely added to the BM graft should be taken into consideration when designing human suicide gene therapy protocols.     

Effect of BH4 on blood phenylalanine and tyrosine variations in patients with phenylketonuria

BackgroundIn patients with phenylketonuria, stability of blood phenylalanine and tyrosine concentrations might influence brain chemistry and therefore patient outcome. This study prospectively investigated the effects of tetrahydrobiopterin (BH4), as a chaperone of phenylalanine hydroxylase on diurnal and day-to-day variations of blood phenylalanine and tyrosine concentrations.MethodsBlood phenylalanine and tyrosine were measured in dried blood spots (DBS) four times daily for 2 days (fasting, before lunch, before dinner, evening) and once daily (fasting) for 6 days in a randomized cross-over design with a period with BH4 and a period without BH4. The sequence was randomized. Eleven proven BH4 responsive PKU patients participated, 5 of them used protein substitutes during BH4 treatment. Natural protein intake and protein substitute dosing was adjusted during the period without BH4 in order to keep DBS phenylalanine levels within target range. Patients filled out a 3-day food diary during both study periods. Variations of DBS phenylalanine and Tyr were expressed in standard deviations (SD) and coefficient of variation (CV).ResultsBH4 treatment did not significantly influence day-to-day phenylalanine and tyrosine variations nor diurnal phenylalanine variations, but decreased diurnal tyrosine variations (median SD 17.6 μmol/l, median CV 21.3%, p = 0.01) compared to diet only (median SD 34.2 μmol/l, median CV 43.2%). Consequently, during BH4 treatment diurnal phenylalanine/tyrosine ratio variation was smaller, while fasting tyrosine levels tended to be higher.ConclusionBH4 did not impact phenylalanine variation but decreased diurnal tyrosine and phenylalanine/tyrosine ratio variations, possibly explained by less use of protein substitute and increased tyrosine synthesis.     

Use of sapropterin dihydrochloride in maternal phenylketonuria. A European experience of eight cases

Sapropterin dihydrochloride (SD) is the first drug treatment for phenylketonuria (PKU), but due to the lack of data, its use in maternal PKU must be undertaken with caution as noted in the FDA and EMEA labels. We collected data from eight pregnancies in PKU women treated with SD and we analysed the phenotypes of these patients, their tetrahydrobiopterin (BH4) responsiveness, the indications for SD treatment, the efficacy (metabolic control, phenylalanine (Phe) tolerance and offspring outcome) and the safety data. Results showed that in the seven patients known to be responsive to BH4, the use of SD during pregnancy was efficient in terms of metabolic control and Phe tolerance. The indications for giving SD included the failure of the low-Phe diet (n?=?3), the fact that some of these women had never experienced the low Phe diet (n?=?2), one unexpected pregnancy in a woman currently on SD and one pregnancy where the foetus was known to have PKU. The offspring of these seven pregnancies were all normal babies with normal birth measurements and outcomes. No side effect related to SD was observed in these seven cases. In the eighth case, SD was prescribed as a rescue treatment without previous knowledge of the BH4 responsiveness to a woman who was already 8 weeks pregnant without diet. The birth occurred at 33 weeks of gestational age with Potter syndrome (probably related to the absence of metabolic control during the first trimester) and the baby died in the first hours of life. In conclusion, the data presented here provides the first evidence that treatment with pharmacological doses of SD appears to be efficient and safe in women with PKU during pregnancy. Its use should, however, be restricted to those women previously identified to be clear responders to BH4.     

PKU—What is daily practice in various centres in Europe?

Background:  

Since the start of the European Society of Phenylketonuria and Allied Disorders Treated as Phenylketonuria (ESPKU) in 1987, an increasing number of parental organizations of member countries have joined. Treatment varies widely within Europe. A survey among professionals was done to determine goals and practice.     

Information processing in patients with early and continuously-treated phenylketonuria

A total of 33 patients with early and continuously-treated phenylketonuria (PKU) between 7 and 16 years of age and 33 matched controls participated in a study examining perceptual, central, and response-related mechanisms of information processing. The specific mechanisms studied were: perceptual filtering, memory search, response selection, response execution, and motor presetting. In addition, groups were compared on mean intelligence level and task oriented behaviour. The performance of the PKU patients practically matched that of the controls on all three tasks, suggesting that PKU patients who are continuously maintained on a well-controlled phenylalanine-restricted diet are not impaired in the elementary mechanisms of information processing. Furthermore, groups did not differ in mean IQ or task-oriented behaviour.     

Measuring growth of residual cholesteatoma in subtotal petrosectomy

Background: Little is known about the growth rate of cholesteatoma in patients.

Objective: Investigate the growth of residual cholesteatoma in subtotal petrosectomy based on volume measured in MRI scans.

Materials and methods: Retrospective case series in a Tertiary Medical Centre. Thirteen residual cholesteatomas were identified in 10 patients after subtotal petrosectomy for which a wait-and-scan policy was adopted. Volume of the residual cholesteatoma was calculated by manual segmentation as well as the ‘box method’.

Results: Mean growth rate was 27.9 mm3/month (SD 22.8), with a large individual variation ranging from 2.2 to 69.8 mm3/month. No complications were reported in 10 patients with a wait-and-scan policy for residual cholesteatoma in subtotal petrosectomy. The box method overestimates growth rate compared to the reference method manual segmentation and a linear increase of this systematic error was seen with increasing size of the cholesteatoma.

Conclusions: Residual cholesteatoma growth rate shows a large individual variation. A wait-and-scan policy could be considered in case of a (small) residual in subtotal petrosectomy with ample room to grow before destroying any remaining structures. Furthermore, the clinically more applicable and less time-consuming box method can be used to accurately measure volumes of small cholesteatomasup to a volume of 500?mm³.