Thiopurine methyltransferase activity in a Spanish population sample: decrease of enzymatic activity in multiple sclerosis patients

The present study was performed in order to obtain the thiopurine methyltransferase (TPMT) activity frequency distribution histogram in a Spanish population. A total of 3640 Spanish clinical laboratory samples were evaluated, which included 1249 patients with Crohn's disease, 589 with ulcerative colitis, 348 with multiple sclerosis (MS), 487 with several autoimmune diseases different from the above-mentioned diseases and 967 a donor group. We have measured the TPMT activity in red blood cells (RBCs) by a radiochemical method, using S-adenosyl-L-[methyl-3H]methionine as methyl donor. The different groups present in their entirety a normal distribution histogram and a wide range of TPMT activity from 0 to 41 U/ml RBCs. The differences found between the Spanish population TPMT activity frequency distribution histogram and the pattern previously described in a North American population were not due to azathioprine treatment or gender. The effect of autoimmune diseases on TPMT activity was evaluated: the enzymatic activity was similar in the donor group (19.9 +/- 6.3 U/ml RBCs) and in the patients with Crohn's disease (20.0 +/- 5.8 U/ml RBCs) and ulcerative colitis (19.7 +/- 6.1 U/ml RBCs); however, it decreased significantly (p<0.0001) in MS patients (17.1 +/- 6.1 U/ml RBCs) with respect to the donor group. In conclusion, our results show that the Spanish population TPMT distribution is closer to that of the Jewish population of Israel than to North American populations, and that in MS the enzymatic activity of TPMT decreases significantly. This observation may take into account the usage of azathioprine as therapeutic agent in Spanish MS patients.     

Brain biopsy in the diagnosis of cerebral mycosis fungoides

A case of cerebral mycosis fungoides co-existing with progressive multifocal leucoencephalopathy presented with dementia. Brain biopsy established the diagnosis of mycosis fungoides after cerebrospinal fluid examinations and computerised tomographic scanning of the brain produced non-specific abnormalities.     

LRTOMT: a new tone in understanding the symphony of non-syndromic deafness

Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans
Ahmed et al. (2008)
Nature Genetics 40: 1335–1340.     

The effectiveness of a treatment protocol for male lower urinary tract symptoms in general practice: a practical randomised controlled trial

BACKGROUND: Randomised controlled trials have shown the efficacy of several treatment modalities for lower urinary tract symptoms (LUTS) in selected populations. The effectiveness in daily practice has hardly been investigated, especially in primary care and is dependent on choices between all possible treatment options and best investigated in a comprehensive study, including all treatment modalities (watchful waiting, alpha-blockers, 5-alpha-reductase inhibitors, and surgery). AIM: Assessment of the effectiveness of a comprehensive treatment protocol for LUTS in primary care. DESIGN OF STUDY: Randomised controlled trial. SETTING: Fourteen general practices in the Netherlands. METHOD: Intervention: treatment protocol based on a formalised expert opinion. Control condition: usual care. Study population: 208 subjects with moderate to severe LUTS (IPSS > or =8, median = 13). OUTCOME MEASURES: symptom severity (IPSS [International Prostate Symptom Score]), bother score (Dan-PSS [Danish Prostate Symptom Score]), and maximum urinary flow (Q(max)); incidence of acute urinary retention and urinary tract infections. RESULTS: In the intervention group markedly more subjects used an alpha-blocker at end of follow-up than in the usual care group (24% versus 6%). No significant differences were found between intervention and control group in IPSS, Q(max) or Dan-PSS. CONCLUSION: alpha-blockers and watchful waiting are the most frequent treatment modalities for LUTS in primary care. Our study showed no evidence that a protocol using well-defined indications for all possible treatment modalities based on a formalised expert opinion procedure has added value. Based on our results, we cannot recommend a broadening of the indication for alpha-blockers, which, however, seems to be the current trend.     

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Differences in the global methylation pattern, ie hyper‐ as well as hypo‐methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5‐ cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam‐2 before and after demethylation using 5‐azacytidine. Exposure to 5‐azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell‐specific marker VASA, showed increased expression. Following treatment with 5‐azacytidine, TCam‐2 cells were analysed using a high‐throughput methylation screen for changes in the methylation sites of 14 000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

p.R254Q mutation in the aquaporin‐2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin‐induced phosphorylation

Vasopressin regulates human water homeostasis by re‐distributing homotetrameric aquaporin‐2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP‐dependent protein kinase A (PKA) is thought to be essential. Dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by AQP2 gene mutations. Here, we investigated a reported patient case of dominant NDI caused by a novel p.R254Q mutation. Expressed in oocytes, AQP2‐p.R254Q appeared to be a functional water channel, but was impaired in its transport to the cell surface to the same degree as AQP2‐p.S256A, which mimics non‐phosphorylated AQP2. In polarized MDCK cells, AQP2‐p.R254Q was retained and was distributed similarly to that of unstimulated wt‐AQP2 or AQP2‐p.S256A. Upon co‐expression, AQP2‐p.R254Q interacted with, and retained wt‐AQP2 in intracellular vesicles. In contrast to wild‐type AQP2, forskolin did not increase AQP2‐p.R254Q phosphorylation at S256 or its translocation to the apical membrane. Mimicking constitutive phosphorylation in AQP2‐p.R254Q with the p.S256D mutation, however, rescued its apical membrane expression. These date indicate that a lack of S256 phosphorylation is the sole cause of dominant NDI here, and thereby, p.R254Q is a loss of function instead of a gain of function mutation in dominant NDI. © 2009 Wiley‐Liss, Inc.     

Targeting antigens to an invariant epitope of the MHC Class II DR molecule potentiates the immune response to subunit vaccines

Recombinant subunit and peptidic vaccines in general present a reduced immunogenicity in vaccinated individuals with respect to the whole pathogen from which they derived. The generation of strong immune responses to these vaccines requires the use of potent adjuvants, high antigen doses and repetitive vaccinations. In this report, we document the enhanced antibody response obtained against two recombinant subunit vaccines by means of targeting to antigen-presenting cells by a recombinant single chain antibody. This antibody, named APCH1, recognizes an epitope of MHC Class II DR molecule preserved in different animal species, including humans. We showed that vaccinal antigens translationally fused to APCH1 antibody and produced by recombinant baculoviruses in insect larvae (Trichoplusia ni), elicited an increased antibody response in comparison with the same antigens alone or fused to a carrier molecule. These results suggest that targeting of antigens to this invariant MHC Class II epitope has immunopotentiating effects that could circumvent the reduced potency of peptidic or subunit vaccines, opening the possibility of widespread application of APCH1 as a new adjuvant antibody of general use.