Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation

Silane-coated silica particle solutions (ISolate(TM) and PureSperm)TM)) and iodixanol (OptiPrep(TM)) were compared to polyvinylpyrrolidone (PVP)-coated silica particles (Percoll(TM)) in their efficacy to recover spermatozoa by gradient centrifugation for use in assisted reproductive procedures. Efficacy was assessed in terms of percentages of sperm recovery, sperm vitality and motility, normal sperm morphology and normal sperm chromatin condensation. No significant difference was found in the recovery of spermatozoa for men with both normal sperm counts and oligozoospermia, between PVP-coated and silane-coated particle solutions. Iodixanol had significantly lower sperm recovery compared to the other products. Sperm vitality, progressive motility, normal morphology and normal chromatin condensation did not differ significantly between any of the sperm isolation products.      

Quantitative antimicrobial susceptibility test for Streptococcus pneumoniae using inoculum supplemented with whole defibrinated sheep blood.

The National Committee for Clinical Laboratory Standards recommends the use of lysed horse blood-supplemented Mueller-Hinton broth for determining the quantitative antimicrobial susceptibility of Streptococcus pneumoniae. This procedure may be difficult for laboratories using previously prepared or commercial MIC systems. Therefore, a study was undertaken to determine whether previously prepared microdilution trays containing Mueller-Hinton broth without blood could be used for determining the antimicrobial susceptibility of S. pneumoniae by adding whole defibrinated sheep blood to the bacterial suspension used to inoculate the trays. The presence of alpha-hemolysis was used as an indicator of bacterial growth. One hundred isolates of S. pneumoniae selected to represent a distribution of susceptibility patterns were tested by the National Committee for Clinical Laboratory Standards method and the sheep blood-supplemented-inoculum method. Greater than 94% agreement between the two methods was achieved. The sheep-blood-supplemented-inoculum procedure was highly reproducible and easy to perform and provides an acceptable alternative for determining the MICs for S. pneumoniae for laboratories using previously prepared or commercial microdilution systems.     

ACTH-elicited sodium appetite in sheep

Intramuscular injections of long-acting synthetic ACTH (45 U twice daily for 5 days) caused a large increase in the intake of 0.5 M NaCl in sheep. Mean Na intake of the sheep on the last 3 days of treatment approximated 50% of their total extracellular fluid Na. The mineral appetite was specific for NaCl. Intakes of 0.5 M KCl or 0.25 M CaCl2 were not significantly altered. The enhanced appetite for Na induced by ACTH appeared to precede any increase in urinary Na excretion. ACTH treatment was ineffective in adrenalectomized sheep. However, an infusion into adrenalectomized sheep of a combination of adrenal steroid hormones (including aldosterone, deoxycorticosterone, 11-deoxycortisol, cortisol, and corticosterone) that contrived blood levels similar to those, obtained with ACTH treatment in normal sheep did induce Na appetite. Thus, ACTH induces a specific, adrenal-steroid hormone-dependent Na appetite in sheep.     

Fos immunoreactivity in the lamina terminalis of adrenalectomized rats and effects of angiotension II type 1 receptor blockade or deoxycorticosterone

Neural activity, as measured immunohistochemically by the presence of Fos protein, was determined in the lamina terminalis, a thin strip of tissue forming the anterior wall of the third brain ventricle, after adrenalectomy. Several weeks after surgery, the adrenalectomized rats were maintained with access to water and a low sodium diet for five days. In addition, hypertonic (0.5M) NaCl solution was available for the entire five-day period (sodium available) or only during the first four days (sodium unavailable). The number of neurons expressing Fos, determined at the end of the fifth day, was increased in the adrenalectomized rats with or without NaCl solution to drink. Fos activity in the median preoptic nucleus was increased only in adrenalectomized rats without access to NaCl solution. Treatment of adrenalectomized rats with the sodium-retaining mineralocorticoid hormone, deoxycorticosterone, at the end of the fourth day, decreased Fos expression in the subfornical organ and the organum vasculosum of the lamina terminalis when NaCl solution was available but not when the NaCl solution was unavailable. In the adrenalectomized rats with NaCl solution available, mineralocorticoid treatment decreased both urinary sodium excretion and daily sodium intake. Brain nuclei in the lamina terminalis also became activated in intact rats made sodium deplete by treatment with the diuretic, furosemide. Relative to sodium-deplete intact rats, however, sodium-deplete adrenalectomized rats had a greater number of neurons expressing Fos in the organum vasculosum. Treatment of sodium-deplete rats, adrenalectomized or intact, with the angiotensin II-type 1 receptor antagonist, ZD7155, decreased sodium intake and Fos expression in the subfornical organ but not in the organum vasculosum of the lamina terminalis or median preoptic nucleus.In conclusion, the results demonstrated that activation of the brain nuclei located in the lamina terminalis of adrenalectomized rats was primarily related to sodium deficit and not to the absence of the mineralocorticoid hormones, although the adrenal hormones may have a role in limiting the activation of organum vasculosum of the lamina terminalis during sodium depletion. Furthermore, the results obtained with the administration of the angiotensin receptor antagonist are consistent with the proposal that sodium appetite of the sodium-deplete rat, adrenalectomized or intact, is mediated by circulating angiotensin II acting in the subfornical organ.     

Pregnancy: The effect of dose of mifepristone and gestation on the efficacy of medical abortion with mifepristone and misoprostol

Although it has been demonstrated that a combination of mifepristoneand a prostaglandin is an effective method of inducing abortionin early pregnancy, the optimum dose of the antigestogen isunknown. Women (n = 220) requesting abortion in early pregnancy(63 days amenorrhoea) were randomized to receive a single doseof either 600 or 200 mg mifepristone followed 48 h later bya single dose of 600 µg misoprostol by mouth. The percentageof women who had a complete abortion (93.6% confidence interval90.4–95.5%) was identical in the two groups. There wasno significant difference in the number of women who passedthe fetus within 4 h of receiving the prostaglandin (64 versus74%), the days of bleeding (14.6 ± 1.1 versus 15.3 ±0.9) nor in the onset of the next period (39.7 ± 1.3versus 36.7 ± 1.3) respectively between the groups receiving200 or 600 mg mifepristone. However, the complete abortion ratewas significantly higher in women 49 days compared to women50–63 days amenorrhoea (97.5 versus 89.1% respectively;P < 0.02). There was no difference in any of the other parametersat different weeks of gestation. We conclude: (i) that the recommendeddose of mifepristone could be reduced from 600 to 200 mg withoutloss of clinical efficacy, (ii) that the combination of mifepristoneand 600 µg misoprostol is a highly effective alternativeto vacuum aspiration for inducing abortion in women < 50days amenorrhoea and (iii) at gestation >56 days, this combinationmay result in too many incomplete abortions to be clinicallyacceptable.     

Identification of neural projections from the forebrain to the kidney, using the virus pseudorabies

These data demonstrate a chain of synaptically connected neurons extending to the rat kidney through several levels of the neuraxis from the forebrain, and the lamina terminalis -- an area known to be involved in the regulation of body fluid homeostasis. The Bartha strain of pseudorabies virus was injected into the kidney of male Sprague-Dawley rats, resulting in retrograde infections in spinal cord segments (T1-T8), and successive infection in five autonomic 'premotor' areas of the brain, the rostroventrolateral medulla, rostroventromedial medulla, raphe nuclei, A5 region of the pons, and the paraventricular nucleus of the hypothalamus, as well as the nucleus of the solitary tract, locus coeruleus, and subcoeruleus nuclei. Higher order labelling was found in regions of the forebrain, including the organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, medial preoptic area, bed nucleus of the stria terminalis, anteroventral periventricular nucleus, lateral preoptic area, suprachiasmatic nucleus, retrochiasmatic nucleus, primary motor cortex, and visceral cortex. This polysynaptic pathway to the kidney may form the substrate underlying the impact of forebrain structures on renal function.     

Selective expansion of specific T cell receptors in the inflamed colon of Crohn's disease.

To identify disease-specific T cell changes that occur in Crohn's disease (CD), the T cell receptor BV repertoires of lamina propria lymphocytes (LPL) isolated from both the inflamed and "disease-inactive" colons of seven CD patients were compared by the quantitative PCR and DNA sequence analysis. It was observed that the BV repertoires of LPL isolated from the disease-active and disease-inactive parts of the colon from the same individual were very different. Furthermore, nearly all of the differences occurred in CD4+ LPL, with very few differences in the CD8+ population of LPL. Although the pattern of BV segments that was increased in disease-active tissue relative to disease-inactive tissue was different for all seven CD patients, there were several BV segments that increased uniformly in the disease-active tissue of all seven individuals. CDR3 length analysis and DNA sequencing of these BV segments revealed that in six of the seven CD patients there was a striking degree of oligoclonality that was absent from disease-inactive tissue of the same individual. These observations suggest that at least some of the inflammation in CD is the result of responses by CD4+ T cells to specific antigens. The isolation of such inflammation-specific CD4+ T cells may make it possible to identify the antigens that are responsible for the inflammatory process in CD and provide a better understanding of its pathogenesis.     

Fatal meningoencephalitis due to Bacillus anthracis

Abstract: We report the first case of fatal anthrax meningoencephalitis in Hong Kong over the past 60 years. A 13 year-old boy presented with right lower quadrant pain, diarrhoea and progressive headache. Lumbar puncture yielded gram positive bacilli initially thought to be Bacillus cereus, a contaminant. He was treated with ampicillin and cefotaxime, but died 3 days after hospitalization. The organism isolated from blood and cerebrospinal fluid was later identified as Bacillus anthracis.