Distribution of TT virus (TTV), TTV-like minivirus,and related viruses in humans and nonhuman primates

TT virus (TTV) and TTV-like minivirus (TLMV) are small DNA viruses with single-stranded, closed circular, antisense genomes infecting man. Despite their extreme sequence heterogeneity (>50%), a highly conserved region in the untranslated region (UTR) allows both viruses to be amplified by polymerase chain reaction (PCR). TTV/TLMV infection was detected in 88 of 100 human plasma samples; amplified sequences were differentiated into TTV and TLMV by analysis of melting profiles, showing that both viruses were similarly prevalent. PCR with UTR primers also detected frequent infection with TTV/TLMV-related viruses in a wide range of apes (chimpanzees, gorillas, orangutans, gibbons) and African monkey species (mangabeys, drills, mandrills). These findings support the hypothesis for the co-evolution of TTV-like viruses with their hosts over the period of primate speciation, potentially analogous to the evolution of primate herpesviruses.     

Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.      

Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities

An RNA-binding motif (RBM) gene family has been identified on the human Y chromosome that maps to the same deletion interval as the 'azoospermia factor' (AZF). We have identified the homologous gene family (Rbm) on the mouse Y with a view to investigating the proposal that this gene family plays a role in spermatogenesis. At least 25 and probably >50 copies of Rbm are present on the mouse Y chromosome short arm located between Sry and the centromere. As in the human, a role in spermatogenesis is indicated by a germ cell-specific pattern of expression in the testis, but there are distinct differences in the pattern of expression between the two species. Mice carrying the deletion Yd1, that maps to the proximal Y short arm, are female due to a position effect resulting in non-expression of Sry ; sex-reversing such mice with an Sry transgene produces males with a high incidence of abnormal sperm, making this the third deletion interval on the mouse Y that affects some aspect of spermatogenesis. Most of the copies of Rbm map to this deletion interval, and the Yd1males have markedly reduced Rbm expression, suggesting that RBM deficiency may be responsible for, or contribute to, the abnormal sperm development. In man, deletion of the functional copies of RBM is associated with meiotic arrest rather than sperm anomalies; however, the different effects of deletion are consistent with the differences in expression between the two species.      

ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome

It was shown recently that mutations of the ATRX gene give rise to a severe, X-linked form of syndromal mental retardation associated with alpha thalassaemia (ATR-X syndrome). In this study, we have characterised the full-length cDNA and predicted structure of the ATRX protein. Comparative analysis shows that it is an entirely new member of the SNF2 subgroup of a superfamily of proteins with similar ATPase and helicase domains. ATRX probably acts as a regulator of gene expression. Definition of its genomic structure enabled us to identify four novel splicing defects by screening 52 affected individuals. Correlation between these and previously identified mutations with variations in the ATR-X phenotype provides insights into the pathophysiology of this disease and the normal role of the ATRX protein in vivo.      

Molecular epidemiology of hepatitis C virus infection amongst intravenous drug users in rural communities

The prevalence of hepatitis C virus (HCV) infection amongst a group of intravenous drug users (IVDUs) resident in West Suffolk (East Anglia, England) was investigated and compared with the prevalence of infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV). In addition, both the level of HCV persistence, as defined by detection of viral RNA, and the HCV genotypes present in this population were determined. It was found that HCV antibodies were present in 59% of those tested; by comparison 22% had antibodies to HBV and 1% antibodies to HIV. HCV RNA was found in 44% of those with HCV antibody. HCV genotype 1 was the most prevalent within this population although both genotypes 2 and 3 were also represented. © 1995 Wiley-Liss, Inc.     

Safety and efficacy of the cystic fibrosis transmembrane conductance regulator potentiator icenticaftor (QBW251)

BackgroundThis is the first-in-human study of icenticaftor, an oral potentiator of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel. Restoration of CFTR activity has shown significant clinical benefits, but more studies are needed to address all CFTR mutations.MethodsSafety, pharmacodynamics/pharmacokinetics of icenticaftor were evaluated in a randomized, double-blind, placebo-controlled study in healthy volunteers. Efficacy was assessed in adult CF patients with ≥1 pre-specified CFTR Class III or IV mutation (150 and 450 mg bid), or homozygous for F508del mutation (450 mg bid). Primary efficacy endpoint was change from baseline in lung clearance index (LCI_2.5). Secondary endpoints included %predicted FEV₁ and sweat chloride level.ResultsClass IV mutations were present in 22 patients, Class III in 2 (both S549N), and 25 were homozygous for F508del. Icenticaftor was well-tolerated in healthy and CF subjects with no unexpected events or discontinuations in the CF groups. The most frequent study-drug related adverse events in CF patients were nausea (12.2%), headache (10.2%), and fatigue (6.1%). Icenticaftor 450 mg bid for 14 days showed significant improvements in all endpoints versus placebo in patients with Class III and IV mutations; mean %predicted FEV₁ increased by 6.46%, LCI_2.5 decreased by 1.13 points and sweat chloride decreased by 8.36 mmol/L. No significant efficacy was observed in patients homozygous for a single F508del.ConclusionsIcenticaftor was safe and well-tolerated in healthy volunteers and CF patients, and demonstrated clinically meaningful changes in lung function and sweat chloride level in CF patients with Class III and IV CFTR mutations.ClinicalTrials.gov: NCT02190604     

Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.

     

Pharmacokinetics and pharmacodynamics of prolonged oral etoposide in women with metastatic breast cancer

The pharmacokinetics and pharmacodynamics of prolonged oral etoposide chemotherapy were investigated in 15 women with metastatic breast cancer who received oral etoposide 100 mg as a single daily dose for up to 15 days. There was considerable interpatient variability in the day 1 pharmacokinetic parameters: area under the plasma concentration time curve (AUC) (0–24 h) 1.95±0.87 mg/ml per min (mean ± SD), apparent oral clearance 60.9±21.7 ml/min per 1.73 m², peak plasma concentration 5.6±2.5 g/ml, time to peak concentration 73±35 min and half-life 220±83 min. However, intrapatient variability in systemic exposure to etoposide was much less with repeated doses. The intrapatient coefficient of variation (CV) of AUC for day 8 relative to day 1 was 20% and for day 15 relative to day 1 was 15%, compared to the day 1 interpatient CV of 45%. Neutropenia was the principal toxicity. Day 1 pharmacokinetic parameters were related to the percentage decrease in absolute neutrophil count using the sigmoidal E_max equation. A good fit was found between day 1 AUC and neutrophil toxicity (R ²=0.77). All patients who had a day 1 AUC>2.0 mg/ml per min had WHO grade III or IV neutropenia. The predictive performance of the models for neutrophil toxicity was better for AUC (percentage mean predictive error 5%, percentage root mean square error 18.1%) than apparent oral clearance, peak plasma concentration, or daily dose (mg/m²). A limited sampling strategy was developed to predict AUC using a linear regression model incorporating a patient effect. Data sets were divided into training and test sets. The AUC could be estimated using a model utilizing plasma etoposide concentration at only two time points, 4 h and 6 h after oral dosing (R ²=98.9%). The equation AUC_pr=–0.376+0.631×C_4h+0.336×C_6h was validated on the test set with a relative mean predictive error of –0.88% and relative root mean square error of 6.4%. These results suggest monitoring of AUC to predict subsequent myelosuppression as a strategy for future trials with oral etoposide.Division of Haematology and Medical Oncology, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett St, Melbourne 3000, Australia     

Antioxidant effects of aminosalicylates and potential new drugs for inflammatory bowel disease: assessment in cell-free systems and inflamed human colorectal biopsies

BACKGROUND: The therapeutic efficacy of 5-aminosalicylic acid in inflammatory bowel disease may be related to its antioxidant properties. AIM: To compare in vitro the antioxidant effects of conventional drugs (5-aminosalicylic acid, corticosteroids, metronidazole), with new aminosalicylates (4-aminosalicylic acid, balsalazide) and other potential therapies (ascorbate, N-acetylcysteine, glutathione, verapamil). METHODS: Compounds were assessed for efficacy in reducing the in vitro production of reactive oxygen species by cell-free systems (using xanthine/xanthine oxidase, with or without myeloperoxidase) and by colorectal biopsies from patients with ulcerative colitis using luminol-amplified chemiluminescence. RESULTS: 5-aminosalicylic acid and balsalazide were more potent antioxidants than 4-aminosalicylic acid or N-acetyl-5-aminosalicylic acid in cell-free systems. 5-aminosalicylic acid (20 mM) and balsalazide (20 mM) inhibited rectal biopsy chemiluminescence by 93% and 100%, respectively, compared with only 59% inhibition by 4-aminosalicylic acid (20 mM). Hydrocortisone, metronidazole and verapamil had no significant effect on chemiluminescence in any system. Ascorbate (20 mM) inhibited chemiluminescence by 100% in cell-free systems and by 60% in rectal biopsies. N-acetyl cysteine (10 mM), and both oxidized and reduced glutathione (10 mM), completely inhibited chemiluminescence in cell-free systems, but not with rectal biopsies. CONCLUSIONS: The antioxidant effects of compounds varies between cell-free systems and inflamed colorectal biopsies. The effect of drugs on the chemiluminescence produced by these two assay systems is useful for screening potentially new antioxidant treatments for inflammatory bowel disease. Ascorbate seems worth further study as a novel therapy.     

STRESS FRACTURES OF THE FEMORAL SHAFT IN MILITARY RECRUITS

Stress fractures amongst military recruits are limited to the lower extremities; yet involvement of the shaft of the femur is unusual. Seven such cases in a series of 352 stress fractures are presented. The importance of early recognition and management is emphasized with a view to prevent bony disruption in an otherwise easily treatable condition.KEY WORDS: Fractures stress, Femoral fractures