Determination of homozygosity or heterozygosity for the G2m(n) allotype by a monoclonal, precipitating antibody

Monoclonal antibody SH 21 was found to be a good anti-IgG2m(n) reagent both for the conventional typing (HA inhibition) and for precipitation in polyethylene glycol containing gel. Antibody SH 21, together with monoclonal antibody HP 6014 (anti-IgG2), was used for the discrimination between G2m(+n) homozygotes and heterozygotes in a double diffusion assay, where the two antibodies and a serum sample were placed in corners of a triangle. In the case of a homozygote SH 21 was able to stop the diffusion of IgG2 to a precipitation line, but some IgG2 of a heterozygote was able to diffuse beyond the precipitate, forming a crossed precipitation pattern. The validity of the typing was checked with serum samples from 54 families. All the 33 'obligatory heterozygotes' (G2m(+n) parent(s) of a G2m(-n) child, or the other way round) exhibited the crossed precipitate. The frequency of the G2mn allele in a Finnish population of 297 unrelated adults was 0.409.     

The distribution of small and intermediate conductance calcium-activated potassium channels in the rat sensory nervous system

Small (SK) and intermediate (IK) conductance calcium-activated potassium channels are candidate ion channels for the regulation of excitability in nociceptive neurones. We have used unique peptide-directed antisera to describe the immunocytochemical distribution of the known isoforms of these ion channels in dorsal root ganglia (DRG) and spinal cord of the rat. These investigations sought to characterize further the phenotype and hence possible functions of nociceptive neurone subpopulations in the rat. In addition, using Western blotting, we sought to determine the level of protein expression of SK and IK channels in sensory nervous tissues following induction of inflammation (Freund's Complete Adjuvant (FCA) arthritis model) or nerve injury (chronic constriction injury model). We show that SK1, SK2, SK3 and IK1 are all expressed in DRG and spinal cord. Morphometric analysis revealed that SK1, SK2 and IK1 were preferentially localized to neurones having cell bodies <1000 microm2 (putative nociceptors) in DRG. Dual labeling immunocytochemistry showed that these ion channels co-localize with both CGRP and IB4, known markers of nociceptor sub-populations. SK2 was localized almost exclusively in the superficial laminae of the spinal cord dorsal horn, the region in which many sensory afferents terminate; the distribution of SK1 and IK1 was more widespread in spinal cord, although some preferential labeling within the dorsal horn was observed in the case of IK1. Here we show evidence for a distinctive pattern of expression for certain members of the calcium-activated potassium channel family in the rat DRG.     

The challenges of implementing quality improvement in small rural hospitals

In the five years since launching its QI initiative, Mason General Hospital has dealt with many of the challenges that commonly arise in healthcare organizations during TQM implementation, including involving physicians and training staff. It also had to address a host of issues common to small rural organizations. At first glance, one might suspect that the move toward QI would be easier in a small organization because there are fewer leaders to orient, fewer people involved in work processes, and fewer levels of management. In fact, the small organization presents special challenges, including limited staff and financial resources, high turnover rates in key medical staff leadership positions, and small sample sizes. The strategies used by Mason General Hospital to respond to these and other challenges provide a model for small rural healthcare organizations.     

The role of PGE2 in the sensitization of mechanoreceptors in normal and inflamed ankle joints of the rat

Summary The role of PGE₂ in the sensitization of highthreshold tarsal joint mechanoreceptors (putative nociceptors) has been investigated in 11 arthritic and 16 normal rats. Injections of a low dose of Freund's complete adjuvant at multiple sites into the tissues surrounding the ankle joint induced a chronic unilateral monoarthritis in the injected limb. Measurements of both spontaneous activity and responses of tarsal joint mechanoreceptors to repeated graded mechanical stimuli were made. All of the mechanoreceptors examined had afferent fibres with conduction velocities in the C or A- range. Using this new model of joint inflammation we have shown that lysine acetylsalicylate reduces the mechanical sensitivity of these joint mechanoreceptors and reduces the spontaneous activity in afferent nerve fibres. Prostaglandin E₂ is unable to restore either the spontaneous activity in the afferent axon or the mechanical sensitivity of tarsal joint mechanoreceptors after lysine acetylsalicylate in the arthritic rat. Similarly, PGE₂ does not sensitize or excite tarsal joint mechanoreceptors in the normal rat. In the normal rat, however, PGE₂ potentiates the excitatory action of bradykinin and enhances the sensitizing effect of bradykinin on the responses of joint mechanoreceptors to mechanical stimulation when both substances are injected simultaneously. These results indicate that PGE₂ is not important in the sensitization of these joint mechanoreceptors in this model of chronic joint inflammation but that in other circumstances PGE₂ may be able to contribute to a sensitization of joint mechanoreceptors by enhancing the action of bradykinin.     

Memory impairment in out-of-hospital cardiac arrest survivors is associated with global reduction in brain volume, not focal hippocampal injury

BACKGROUND AND PURPOSE: More than 30% of out-of-hospital cardiac arrest (OHCA) survivors suffer significant memory impairment. The hippocampus may be vulnerable to hypoxic injury during cardiac arrest. The purpose of this study was to determine whether selective hippocampal injury is the substrate for this memory impairment. METHODS: Seventeen OHCA survivors and 12 patients with uncomplicated myocardial infarction were studied. OHCA survivors were divided into those with impaired and intact memory. Memory was assessed by use of the Rivermead Behavioural Memory Test and Doors and People Test. MRI was used to determine intracranial, whole-brain, amygdala-hippocampal complex, and temporal lobe volumes. Brain structure was also examined by statistical parametric mapping. RESULTS: Left amygdala-hippocampal volume was reduced in memory-impaired OHCA victims compared with control subjects (mean 3. 93 cm(3) and 95% CI 3.50 to 4.36 cm(3) versus mean 4.65 cm(3) and 95% CI 4.37 to 4.93 cm(3); P=0.002). Left temporal lobe and whole-brain volumes were also reduced. There were no differences in amygdala-hippocampal volume indexed against ipsilateral temporal lobe volume. Significant correlations were observed between total brain volume and Rivermead Behavioural Memory Test (r=0.56, P<0.05) and Doors and People Test (r=0.67, P<0.01) scores in OHCA survivors. Both recall and recognition were compromised in memory-impaired subjects. Statistical parametric mapping did not detect focal brain abnormalities in these subjects. Global cerebral atrophy was confirmed by qualitative assessment. CONCLUSIONS: Memory impairment in OHCA survivors is associated with global cerebral atrophy, not selective hippocampal damage. Rehabilitation protocols need to account for the global nature of the brain injury.     

Dietary influences on bone mass and bone metabolism: further evidence of a positive link between fruit and vegetable consumption and bone health?

BACKGROUND: The role of nutritional influences on bone health remains largely undefined because most studies have focused attention on calcium intake. OBJECTIVE: We reported previously that intakes of nutrients found in abundance in fruit and vegetables are positively associated with bone health. We examined this finding further by considering axial and peripheral bone mass and markers of bone metabolism. DESIGN: This was a cross-sectional study of 62 healthy women aged 45-55 y. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry at the lumbar spine and femoral neck and by peripheral quantitative computed tomography at the ultradistal radial total, trabecular, and cortical sites. Bone resorption was calculated by measuring urinary excretion of pyridinoline and deoxypyridinoline and bone formation by measuring serum osteocalcin. Nutrient intakes were assessed by using a validated food-frequency questionnaire; other lifestyle factors were assessed by additional questions. RESULTS: After present energy intake was controlled for, higher intakes of magnesium, potassium, and alcohol were associated with higher total bone mass by Pearson correlation (P < 0.05 to P < 0.005). Femoral neck BMD was higher in women who had consumed high amounts of fruit in their childhood than in women who had consumed medium or low amounts (P < 0.01). In a regression analysis with age, weight, height, menstrual status, and dietary intake entered into the model, magnesium intake accounted for 12.3% of the variation in pyridinoline excretion and 12% of the variation in deoxypyridinoline excretion. Alcohol and potassium intakes accounted for 18.1% of the variation in total forearm bone mass. CONCLUSION: The BMD results confirm our previous work (but at peripheral bone mass sites), and our findings associating bone resorption with dietary factors provide further evidence of a positive link between fruit and vegetable consumption and bone health.     

Increased hepatobiliary clearance of unconjugated thyroxine determines DMP 904-induced alterations in thyroid hormone homeostasis in rats.

4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective antagonist of corticotropin releasing factor receptor-1 (CRF1 receptor) with an efficacious anxiolytic profile in preclinical animal models. In subchronic toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia, and a low incidence of follicular cell adenoma. The current investigations were designed to determine the mode of action by which DMP 904 disrupts thyroid homeostasis in male rats. Five-day treatment with DMP 904 (300 mg/kg/day) dramatically lowered serum thyroxine (T4) to levels below detectable limits (< 1 microg/dl) by 72 h, with concurrent decreases in triiodothyronine (T3, about a 70% decrease) and increases in thyroid stimulating hormone (TSH; about a three-fold increase). DMP 904 increased [125I]T4 total body clearance (Cl tb) (38.21 +/- 10.45 ml/h) compared to control (5.61 +/- 0.59 ml/h) and phenobarbital-treated rats (7.92 +/- 1.62 ml/h). This increase in Cl(tb) was associated with a significant increase in biliary clearance (Cl bile) of unconjugated [125I]T4 (nearly 80-times control rates) and increased liver:blood ratios of T4, suggestive of enhanced hepatic uptake of T4. A single dose of DMP 904 (200 mg/kg) increased mRNA levels of hepatic cytochrome P450s (CYP 3A1 and CYP 2B1) and UDP-glucuronosyltransferases (UGT 1A1 and UGT 1A2). DMP 904 also induced mRNAs of the canalicular transporter, multi-drug resistance protein-2 (Mrp2) and sinusoidal transporters, organic anion transporting proteins (Oatp1 and Oatp2) within 24 h. Western blot analysis confirmed DMP 904 related increases in Oatp2 protein expression. Collectively, these data suggest that DMP 904 is an agonist of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and that the decreased serum levels of T4 and T3 resulted from increased hepatobiliary clearance. However, DMP 904 is distinguished from other compounds associated with similar effects on thyroid hormone homeostasis because its effects were primarily related to increased biliary excretion of unconjugated T4.     

Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer.

PURPOSE: The relationships between mismatch repair (MMR) protein expression, microsatellite instability (MSI), family history, and germline MMR gene mutation status have not been studied on a population basis. METHODS: We studied 131 unselected patients with colorectal cancer diagnosed younger than age 45 years. For the 105 available tumors, MLH1, MSH2, MSH6, and PMS2 protein expression using immunohistochemistry (IHC) and MSI were measured. Germline DNA was screened for hMLH1, hMSH2, hMSH6, and hPMS2 mutations for the following patients: all from families fulfilling the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer (HNPCC); all with tumors that were high MSI, low MSI, or that lacked expression of any MMR protein; and a random sample of 23 with MS-stable tumors expressing all MMR proteins. RESULTS: Germline mutations were found in 18 patients (nine hMLH1, four hMSH2, four hMSH6, and one hPMS2); all tumors exhibited loss of MMR protein expression, all but one were high MSI or low MSI, and nine were from a family fulfilling Amsterdam Criteria. Sensitivities of IHC testing, MSI (high or low), and Amsterdam Criteria for MMR gene mutation were 100%, 94%, and 50%, respectively. Corresponding positive predictive values were 69%, 50%, and 75%. CONCLUSIONS: Tumor IHC analysis of four MMR proteins and MSI testing provide a highly sensitive strategy for identifying MMR gene mutation-carrying, early-onset colorectal cancer patients, half of whom would have been missed using Amsterdam Criteria alone. Tumor-based approaches for triaging early-onset colorectal cancer patients for MMR gene mutation testing, irrespective of family history, appear to be an efficient screening strategy for HNPCC.