Genotypic variation and stability of four variable-number tandem repeats and their suitability for discriminating strains of Mycobacterium leprae

It has not been possible to distinguish different strains of Mycobacterium leprae according to their genetic sequence. However, the genome contains several variable-number tandem repeats (VNTR), which have been used effectively in strain typing of other bacteria. To determine their suitability for differentiating M. leprae, we developed PCR systems to amplify 5 different VNTR loci and examined a battery of 12 M. leprae strains derived from patients in different regions of the United States, Brazil, Mexico, and the Philippines, as well as from wild armadillos and a sooty mangabey monkey. We found diversity at four VNTR (D = 0.74), but one system (C(16)G(8)) failed to yield reproducible results. Alleles for the GAA VNTR varied in length from 10 to 16 copies, those for AT(17) varied in length from 10 to 15 copies, those for GTA varied in length from 9 to 12 copies, and those for TA(18) varied in length from 13 to 20 copies. Relatively little variation was seen with interspecies transfer of bacilli or during short-term passage of strains in nude mice or armadillos. The TA(18) locus was more polymorphic than other VNTR, and genotypic variation was more common after long-term expansion in armadillos. Most strain genotypes remained fairly stable in passage, but strain Thai-53 showed remarkable variability. Statistical cluster analysis segregated strains and passage samples appropriately but did not reveal any particular genotype associable with different regions or hosts of origin. VNTR polymorphisms can be used effectively to discriminate M. leprae strains. Inclusion of additional loci and other elements will likely lead to a robust typing system that can be used in community-based epidemiological studies and select clinical applications.     

Pain relief following Arthroscopy - a comparative study of Intra-articular Bupivacaine,Morphine and Neostigmine

Pain after arthroscopy is quite distressing. Intra-articular bupivacaine produces transient analgesia and reports of analgesia using intra-articular opioids have produced conflicting results. Recently, spinal administration of neostigmine was shown to produce dose-dependant analgesia. However, this was limited by adverse effects. The purpose of this study was to compare the effects on intra-articular neostigmine, bupivacaine and morphine. 75 patients were randomized to receive intra-articular saline, bupivacaine, morphine, neostigmine and bupivacaine-neostigmine after arthroscopic surgery under spinal anaesthesia. Visual analog pain scores (VAS), duration of analgesia as defined as time for first demand for parenteral opioids and the total subsequent consumption of morphine was evaluated. Intra-articular bupivacaine resulted in significant VAS reduction at one and four hours as compared to those receiving intra-articular saline and morphine. Analgesia lasted longer after 500ugm intra-articular neostigmine as compared with bupivacaine, morphine or saline. The need for supplementary analgesia was lowest in the neostigmine group as compared to the other groups. No significant difference was found if bupivacaine was added to neostigmine. Among all the groups, no significant side-effects were observed.Key Words: Arthroscopy, Bupivacaine, Morphine, Neostigmine     

Toxicity and Cerebrospinal Fluid Levels of Carboplatin Chronically Infused into the Brainstem of a Primate

PURPOSE: Carboplatin was infused into the brainstem of cynomolgus monkeys to investigate neurotoxicity and systemic exposures following chronic local delivery. METHODS: Infusions at 0.42 microl/h were intended to deliver 0.025 (n = 2), 0.075 (n = 3), 0.25 (n = 5), and 0.75 (n = 3) mg/kg by day 30. Laboratory tests, radiographic measurements, and clinical observations were used to monitor toxicity. Blood and cerebrospinal fluid (CSF) were sampled for platinum. RESULTS: Lethargy and ataxia were observed after week 4 in the monkeys given 0.075 mg/kg, and week 2 in the monkeys given 0.25 mg/kg when the infused doses were approximately 250 and 400 microg, respectively. Rapidly progressive neurotoxicity with the 0.75 mg/kg dose required termination of the infusions at days 4-10. Hematology and chemistry values were unremarkable in all groups. Blood levels of platinum remained undetectable in 0.025 and 0.075 mg/kg dose groups. Levels in the 0.25 mg/kg group were 3.1 +/- 0.6 microg/l at 2 weeks and 5.2 +/- 0.8 microg/l at 1 month. The CSF platinum levels varied. Animals in the 0.25 mg/kg group had higher CSF levels at 2 weeks (avg. 65 microg/l, range 36-89) compared to their 1 month value (avg. 60 microg/l, range 7-170), despite the constant infusion. CONCLUSION: Carboplatin can be chronically infused into monkey brainstems. Neurotoxicity is the predominant side effect and is dose-dependent. Pharmacokinetics of local and systemic delivery are different for carboplatin. Further studies are needed to monitor toxicity at higher flow rates and to investigate drug binding to abnormal central nervous system (CNS) tissues.     

Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.     

Specialisation and breast cancer survival in the screening era

It is recommended that specialist surgeons treat all breast cancer, although the limited evidence to support this is based on treatment patterns prior to the introduction of screening. Whether a specialist survival advantage exists in the post-screening era is uncertain, as referral and treatment patterns may have changed, in addition to the effect of screening on the natural history of breast cancer. Our aim was to determine the impact of screening on the caseload and case-mix of specialist surgeons, to determine if the survival advantage associated with specialist care is maintained with longer follow-up and persists after the introduction of screening. Using the West of Scotland Cancer Registry, all 7197 women treated for breast cancer in a 15-year time period (1980-1994) in a geographically defined cohort were followed up for an average of 9 years, and pathological stage and socioeconomic status were linked with mortality data. We show that the caseload of specialists has increased substantially (from 11 to 59% of the total workload) and that smaller cancers have been selectively referred. However, even after allowing for pathological stage, socioeconomic status and method of detection, specialist treatment was associated with a significantly lower risk of dying (prescreening: relative risk of dying=0.83, 95% CI=0.75-0.92; post-screening: relative risk of dying=0.89, 95% CI=0.78-1.00). We conclude that this survival benefit is most consistent with effective surgical management rather than selective referral, the influx of screen-detected cancers or adjuvant therapies.     

Hemifacial microsomia in two patients further supporting chromosomal mosaicism as a causative factor

We report two cases with hemifacial microsomia with body asymmetry associated with mosaic trisomies. The child with mosaic trisomy 9 had skin pigmentary changes. In the boy with mosaic trisomy 22, the extra chromosome 22 originated from a maternal meiosis I error.