Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level.

Progress in understanding the basis of resistance to isoniazid (INH) and rifampin (RMP) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. Consecutive isolates (n = 95) of Mycobacterium tuberculosis, from a Spanish reference laboratory investigating outbreaks of multidrug-resistant tuberculosis, were coded and sent to two external laboratories for genotypic analysis of INH and RMP resistance by PCR-single-strand conformation polymorphism (SSCP) analysis of specific regions of four genes: part of the coding sequence of katG and the promoter regions of inhA and ahpC for INH and the RMP resistance region of rpoB. After correction for the presence of outbreak strains and multiple isolates from single patients, RMP resistance was detected successfully by PCR-SSCP in > 96% of the RMP-resistant strains. PCR-SSCP had a sensitivity of 87% for INH resistance detection, and mutations in katG, inhA, katG-inhA, ahpC, and katG-ahpC were identified in 36.8, 31.6, 2.6, 13.2, and 2.6%, respectively, of the unique strains. Specificity was 100%. Molecular detection of resistance to the two main antituberculous drugs, INH and RMP, can be accomplished accurately by using a strategy which limits analysis to four genetic regions. This may allow the expedient analysis of drug resistance by reference laboratories.     

Interactions of chrysotile asbestos fibres with the complement system.

Type A chrysotile fibres (white asbestos) were tested in vitro for activation of the complement system. Fibres were incubated in normal human serum (NHS), factor B-depleted human serum, and normal and C4-deficient guinea-pig sera; the supernates were assayed for the remaining complement activity. Activation of the alternative pathway (AP) was shown in three ways. First, quantitative measurement of factor B; second, kinetic analysis of rabbit red blood cell lysis in whole alternative pathway (AP) and factor B lytic assays; third, qualitative measurement of C3 and factor B conversion by crossed immunoelectrophoresis. No C3 convertase activity could be demonstrated on the fibres but other possible mechanisms of AP activation are discussed. Magnesium itself is not responsible for this activation because acid-treated fibres retain this property. The early classical pathway is not involved as shown by normal whole complement activity of a factor B-depleted human serum and the absence of decrease of C4 functional activity. Knowing that complement proteins are present in pulmonary alveoli, mainly provided by cell synthesis, we suggest that complement activation in vivo may be relevant to the genesis of the chronic inflammation and fibrosis in the lung.     

A unique rat model of bile ductular hyperplasia in which liver is almost totally replaced with well-differentiated bile ductules.

A novel rat model was developed in which furan combined in a unique synergistic manner with bile duct ligation to induce replacement of most of liver with well-differentiated hyperplastic bile ductules. Multiple tissue sections of liver from Fischer 344 male rats first subjected to a bile duct ligation and 1 week later given furan by gavage at 45 mg/kg body weight, once a day, five times weekly for 5 to 6 weeks, exhibited a mean percent of bile ductule tissue per total liver section area of 72.6 +/- 16.3% compared to control values of 20.0 +/- 4.2% for bile duct-ligated rats that received corn oil by gavage instead of furan and 11.9 +/- 3.1% for rats that were given a sham operation followed by furan. This dramatic difference was also reflected by the very high mean gamma-glutamyl transpeptidase specific activity of liver homogenates from the bile duct-ligated/furan-treated rats, which was approximately 8 x 10(3) nmoles p-nitroaniline/mg protein/hour versus values of approximately 2 x 10(3) for bile duct-ligated/corn oil control, approximately 1 x 10(3) for sham-operated/furan-treated control, and 44.9 for untreated rat. The data presented support a potentially powerful experimental model for investigating bile ductular cell functions, differentiation, and proliferation.     

Subarachnoid hemorrhage in the African-American population: a cooperative study.

The clinical outcome of patients following subarachnoid hemorrhage is complicated by delayed cerebral ischemia and contributing factors such as hypertension. To observe the impact of hypertension and delayed cerebral ischemia on the outcome of a predominantly African-American cohort following subarachnoid hemorrhage, both retrospective (n = 42) and prospective (n = 21) studies were conducted. In the total pool (n = 63), the mean age was 49.7 years (range: 17 to 80) with a preponderance of female patients (70%). Aneurysm formation was significant in the region of the posterior communicating artery. Of the patients reviewed, 73.8% had preexisting hypertension and 45.9% developed delayed cerebral ischemia. Approximately 89% of the patients who suffered from delayed cerebral ischemia had hypertension. Results failed to display any significant beneficial association between the use of the calcium channel blocker nimodipine and delayed cerebral ischemia. Use of the antifibrinolytic drug aminocaproic acid demonstrated a worse patient outcome. It is not recommended that aminocaproic acid be used in this population. Subsequently, due to the proportional occurrence of delayed cerebral ischemia in hypertensive patients following subarachnoid hemorrhage, it is suggested that prophylactic surgical management of unruptured intracranial aneurysms be considered in hypertensive patients. Further study is needed to discern the association between hypertension, delayed cerebral ischemia, and stroke in patients following subarachnoid hemorrhage.     

X inactivation as a mechanism of selection against lethal alleles: further investigation of incontinentia pigmenti and X linked lymphoproliferative disease.

Thirty-one females with incontinentia pigmenti (IP), 42 controls, and 11 females from four families segregating for X linked lymphoproliferative disease (XLP) were studied for evidence of skewed X inactivation by analysis of methylation at sites in the HPRT, PGK, and M27 beta (DXS255) regions of the X chromosome. Extensive skewing of X inactivation was present in blood from 4/42 (9.5%) control females and 11/31 (35%) of those with IP. This frequency of skewed inactivation was seen in both familial and sporadic cases of IP. Analysis of inactivation in mother/daughter pairs, both affected and control subjects, showed no familial consistency of pattern, arguing against specific mutations being associated with particular patterns of inactivation. In the only informative family where both mother and daughter were affected by IP and showed skewed inactivation, the IP mutation was on the active X chromosome. This argues against cell selection during early embryogenesis being the explanation for the skewed inactivation observed. These data confirm that skewed inactivation of one X is observed in lymphocytes from a significant minority of normal females, and is seen with raised frequency in IP heterozygotes. It is not, however, a universally observed phenomenon, and the relationship of X inactivity to the IP mutation appears to be complex. In the case of XLP, though skewed X inactivation patterns are seen in most disease carriers, the frequency with which this phenomenon occurs in normal females renders it an unreliable diagnostic marker for XLP carriers.     

Expression of interleukin-6 in the cornea in response to infection with different strains of Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa causing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither. In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains of P. aeruginosa. The number of IL-6-positive cells was determined by image analysis. IL-6 protein levels were measured by an enzyme-linked immunosorbent assay. BALB/c mice were challenged by use of the wounded-cornea model with P. aeruginosa 6294, 6206, or Paer1 (2 x 10(6) CFU). At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA. IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206. At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA. In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge. Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates of P. aeruginosa. The data obtained for mRNA expression in the cornea for all three strains of P. aeruginosa correlated well with IL-6 protein analysis of whole-eye homogenates. Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.     

CD4+ helper T cell depression in autism

CD4+ (helper) T cells are a heterogenous population of lymphocytes including at least two distinct subpopulations. To investigate the possibility that immune abnormalities in some subjects with autism may involve abnormal distributions of CD4+ and/or CD8+ cells, (suppressor) T cells, peripheral blood lymphocytes of 25 autistic subjects were characterized with monoclonal antibodies and flow cytometry. The autistic subjects had a significantly lower percentage and number of CD4+ cells, a lower number of T cells (CD2+ cells) and B cells (CD20+ cells), and a lower percentage and number of total lymphocytes than siblings and normal subjects. The level of blood values for female subjects appeared lower than those for males as compared to normal subjects of the same sex. These results suggest that a decrease in CD4+ cells is associated with autism.     

Steady-state visual evoked potentials reveal frontally-mediated working memory activity in humans

Steady-state visual evoked potentials (SSVEPs) reflect power changes at the stimulus driving frequency and have been used to assess brain activity reflecting cognitive processing. Only one study has demonstrated SSVEP modulation associated with working memory (WM), and none have compared the spatial localization of SSVEP modulations during WM performance with other brain imaging methods. Here we examined WM-related activity recorded with dense-array SSVEPs, analyzed using low resolution electromagnetic tomography, and compared the results to our previous findings using functional magnetic resonance imaging (fMRI). WM was associated with increased SSVEP activity over the right dorsolateral prefrontal cortex, paralleling our previous fMRI findings. Frontal WM-related SSVEP power correlated selectively with task performance. These results demonstrate the utility of SSVEPs for studying representational aspects of cognition.     

Semiautomated Quantitation of Carotid Artery Stenosis in Gadolinium-Bolus Magnetic Resonance Angiography

Magnetic resonance angiography (MRA) has become the standard method for evaluation of carotid occlusive disease. Fast imaging methods combined with bolus intravenous injection of gadolinium contrast have improved the quality of these images. Nevertheless, the gold standard for evaluation was based on projection arterial angiography. The properties of these images are rather different. Whereas most previous evaluations of MRA have used visual assessment of images, we evaluate an algorithm in which a computer algorithm plays the primary role in defining arterial lumen margins, hence, disease. The accuracy of this semiautomated algorithm is shown to compare favorably with gold-standard arteriography in a series of 50 patients.     

Intracellular Abeta is increased by okadaic acid exposure in transfected neuronal and non-neuronal cell lines.

Intracellular Abeta was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Abeta immunostaining was enhanced, as demonstrated by two independent anti-Abeta antibodies. The confocal microscopic study revealed that the immunoreactivity of Abeta was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Abeta sandwich ELISA, showed significantly increased intracellular Abeta. False positive detection of Abeta by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Abeta, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Abeta.