Bioisosteres of ethyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo [1,5-a][1,4]diazepine-3-carboxylate (HZ-166) as novel alpha 2,3 selective potentiators of GABAA receptors: Improved bioavailability enhances anticonvulsant efficacy.

HZ-166 has previously been characterized as an α2,3-selective GABAA receptor modulator with anticonvulsant, anxiolytic, and anti-nociceptive properties but reduced motor effects. We discovered a series of ester bioisosteres with reduced metabolic liabilities, leading to improved efficacy as anxiolytic-like compounds in rats. In the present study, we evaluated the anticonvulsant effects KRM-II-81 across several rodent models. In some models we also evaluated key structural analogs. KRM-II-81 suppressed hyper-excitation in a network of cultured cortical neurons without affecting the basal neuronal activity. KRM-II-81 was active against electroshock-induced convulsions in mice, pentylenetetrazole (PTZ)-induced convulsions in rats, elevations in PTZ-seizure thresholds, and amygdala-kindled seizures in rats with efficacies greater than that of diazepam. KRM-II-81 was also active in the 6 Hz seizure model in mice. Structural analogs of KRM-II-81 but not the ester, HZ-166, were active in all models in which they were evaluated. We further evaluated KRM-II-81 in human cortical epileptic tissue where it was found to significantly-attenuate picrotoxin- and AP-4-induced increases in firing rate across an electrode array. These molecules generally had a wider margin of separation in potencies to produce anticonvulsant effects vs. motor impairment on an inverted screen test than did diazepam. Ester bioisosters of HZ-166 are thus presented as novel agents for the potential treatment of epilepsy acting via selective positive allosteric amplification of GABAA signaling through α2/α3-containing GABA receptors. The in vivo data from the present study can serve as a guide to dosing parameters that predict engagement of central GABAA receptors.

An antidepressant-related pharmacological signature for positive allosteric modulators of α2/3-containing GABAA receptors.

Data from transgenic animals and novel pharmacological agents has realigned scientific scrutiny on the therapeutic potential of positive allosteric modulators (PAMs) of α2/3-containing GABAA receptors. Evidence for analgesic, anticonvulsant, and anxiolytic activity of α2/3-selective PAMs has been presented along with the clinical potential for a milder motor-impacting profile compared to non-selective GABAA receptor PAMs. A new series of α2/3-selective PAMs was recently introduced which has anxiolytic and anticonvulsant activity in rodent models. These molecules also produce efficacy against pain in multiple animal models. Additionally, co-morbid states of depression are prevalent among patients with pain and patients with anxiety. Compounds were shown to be selective for α2 and α3 constructs over α1 (except KRM-II-82), α4, α5, and α6 proteins in electrophysiological assays in transfected HEK-293T cells. Utilizing the forced-swim assay in mice that detects conventional and novel antidepressant drugs, we demonstrate for the first time that α2/3-selective PAMs are active in the forced-swim assay at anxiolytic-producing doses. In contrast, activity in a related model, the tail-suspension test, was not observed. Diazepam was not active in the forced-swim assay when given alone but produced an antidepressant-like effect in mice when given in conjunction with the α1-preferring antagonist, ß-CCT, that attenuated the motor-impairing effects of diazepam. We conclude that these α2/3-selective PAMs deserve further scrutiny for their potential treatment of major depressive disorder. If effective, such a mechanism could add a beneficial antidepressant component to the anxiolytic, analgesic, and anticonvulsant spectrum of effects of these compounds.

PDE11A negatively regulates lithium responsivity.

Lithium responsivity in patients with bipolar disorder has been genetically associated with Phosphodiesterase 11A (PDE11A), and lithium decreases PDE11A mRNA in induced pluripotent stem cell-derived hippocampal neurons originating from lithium-responsive patients. PDE11 is an enzyme uniquely enriched in the hippocampus that breaks down cyclic AMP and cyclic GMP. Here we determined whether decreasing PDE11A expression is sufficient to increase lithium responsivity in mice. In dorsal hippocampus and ventral hippocampus (VHIPP), lithium-responsive C57BL/6J and 129S6/SvEvTac mice show decreased PDE11A4 protein expression relative to lithium-unresponsive BALB/cJ mice. In VHIPP, C57BL/6J mice also show differences in PDE11A4 compartmentalization relative to BALB/cJ mice. In contrast, neither PDE2A nor PDE10A expression differ among the strains. The compartment-specific differences in PDE11A4 protein expression are explained by a coding single-nucleotide polymorphism (SNP) at amino acid 499, which falls within the GAF-B homodimerization domain. Relative to the BALB/cJ 499T, the C57BL/6J 499A decreases PDE11A4 homodimerization, which removes PDE11A4 from the membrane. Consistent with the observation that lower PDE11A4 expression correlates with better lithium responsiveness, we found that Pde11a knockout mice (KO) given 0.4% lithium chow for 3+ weeks exhibit greater lithium responsivity relative to wild-type (WT) littermates in tail suspension, an antidepressant-predictive assay, and amphetamine hyperlocomotion, an anti-manic predictive assay. Reduced PDE11A4 expression may represent a lithium-sensitive pathophysiology, because both C57BL/6J and Pde11a KO mice show increased expression of the pro-inflammatory cytokine interleukin-6 (IL-6) relative to BALB/cJ and PDE11A WT mice, respectively. Our finding that PDE11A4 negatively regulates lithium responsivity in mice suggests that the PDE11A SNPs identified in patients may be functionally relevant.

Contributions of different kainate receptor subunits to the properties of recombinant homomeric and heteromeric receptors.

The tetrameric kainate receptors can be assembled from a combination of five different subunit subtypes. While GluK1-3 subunits can form homomeric receptors, GluK4 and GluK5 require a heteromeric partner to assemble, traffic to the membrane surface, and produce a functional channel. Previous studies have shown that incorporation of a GluK4 or GluK5 subunit changes both receptor pharmacology and channel kinetics. We directly compared the functional characteristics of recombinant receptors containing either GluK4 or GluK5 in combination with the GluK1 or GluK2 subunit. In addition, we took advantage of mutations within the agonist binding sites of GluK1, GluK2, or GluK5 to isolate the response of the wild-type partner within the heteromeric receptor. Our results suggest that GluK1 and GluK2 differ primarily in their pharmacological properties, but that GluK4 and GluK5 have distinct functional characteristics. In particular, while binding of agonist to only the GluK5 subunit appears to activate the channel to a non-desensitizing state, binding to GluK4 does produce some desensitization. This suggests that GluK4 and GluK5 differ fundamentally in their contribution to receptor desensitization. In addition, mutation of the agonist binding site of GluK5 results in a heteromeric receptor with a glutamate sensitivity similar to homomeric GluK1 or GluK2 receptors, but which requires higher agonist concentrations to produce desensitization. This suggests that onset of desensitization in heteromeric receptors is determined more by the number of subunits bound to agonist than by the identity of those subunits. The distinct, concentration-dependent properties observed with heteromeric receptors in response to glutamate or kainate are consistent with a model in which either subunit can activate the channel, but in which occupancy of both subunits within a dimer is needed to allow desensitization of GluK2/K5 receptors.

Demographic and phenotypic features of 70 families segregating Barrett's oesophagus and oesophageal adenocarcinoma.

BACKGROUND: Based on reported familial patterns, inheritance of a predisposition of developing Barrett's oesesophagus (BO) and oesophageal adenocarcinoma (OAC) likely follows an autosomal dominant model of most inherited cancer syndromes. oesophagus (BO) and oesophageal adenocarcinoma (OAC) likely follows an autosomal dominant model of most inherited cancer syndromes. AIMS: We analysed the phenotypic features of 70 familial BO/OAC families accrued for the purpose of initiating a linkage study to search for genes that contribute to susceptibility for BO/OAC. METHODS: Families with young or familial BO/OAC were recruited from participating institutions and self-referral from advertisement. RESULTS: A total of 70 families (173 affected and 784 unaffected individuals) were recruited into this study. Mean ages of diagnosis of BO and OAC among males were 50.6 and 57.4 years, respectively; among females, 52.1 and 63.5 years, respectively. The standardised incidence ratio (SIR) of cancers other than OAC or oesophagogastric junctional adenocarcinoma (OGJAC), among probands was 0.71. Seventy one percent of the pedigrees have "typical" structures with less than three affected individuals. Power calculations under realistic model assumptions suggest that if genetic heterogeneity is absent or limited, then DNA collection from members of these pedigrees could enable the identification of a novel candidate susceptibility gene for BO/OAC in a genome scan. CONCLUSIONS: This is the largest series of families with BO/OAC yet reported, features of which are consistent with inherited germline predisposition. Further, the SIR of cancers other than OAC/OGJAC was 0.71 among 70 probands, indicating these individuals were not more likely to develop non-OAC cancers.

Improvements in GORE-TEX vascular graft performance by Carmeda BioActive surface heparin immobilization.

OBJECTIVES: a performance improvement in small-diameter bypass grafts remains a clinical objective. The purpose of the present investigation was to evaluate the potential of enhancing the thromboresistance of ePTFE grafts using a bioactive heparinized graft luminal surface in a canine model. MATERIAL AND METHODS: this study investigated the utility of heparin immobilization onto expanded polytetrafluoroethylene using Carmeda BioActive Surface technology (CBAS-ePTFE) as a means of improving vascular graft thromboresistance. Graft luminal surfaces were covered uniformly with the stably bound, end-point immobilized heparin. RESULTS: acute canine (5 greyhounds) interposition experiments comparing CBAS-ePTFE grafts to control ePTFE grafts showed that CBAS-ePTFE grafts remained patent and had significantly greater thrombus-free luminal surface (p<0.05). In a chronic canine (16 greyhounds) interposition experiment, significantly improved patency (p<0.05) was observed with CBAS-ePTFE grafts compared to controls. Long-term in vivo heparin bioactivity was demonstrated on CBAS-ePTFE grafts explanted between 1 and 12 weeks. On all CBAS-ePTFE grafts, heparin activity levels ranged from 15-25pmol/cm(2) and did not differ significantly (p>0.05). DISCUSSION: these results support the conclusion that a stable, CBAS-ePTFE surface provides improved thromboresistance and improved patency in canine interposition models. Maintenance of heparin catalytic activity on the graft surface in vivo likely contributes to this outcome and holds promise for the utility of this graft surface for clinical applications.

SPARC modulates cell growth, attachment and migration of U87 glioma cells on brain extracellular matrix proteins.

We have identified secreted protein acidic and rich in cysteine (SPARC) as a potential glioma invasion-promoting gene. To determine whether SPARC alters the growth, attachment, or migration of gliomas, we have used U87T2 and doxycycline-regulatable SPARC-transfected clones to examine the effects of SPARC on (1) cell growth, (2) cell cycle progression, (3) cell attachment, and (4) cell migration, using growth curves, flow cytometry, attachment, and migration analyses on different brain ECMs, including collagen IV, laminin, fibronectin, vitronectin, hyaluronic acid, and tenascin. Our data indicate that SPARC delays tumor cell growth in the log phase of the growth curve. The clones secreted different levels of SPARC. The clone secreting the lowest level of SPARC was associated with a higher percentage of cells in G2M, whereas the clones secreting the higher levels of SPARC were associated with a greater percentage of cells in G0/G1. In comparison to the parental U87T2 clone, the SPARC-transfected clones demonstrated increased attachment to collagen, laminin, hyaluronic acid, and tenascin, but not to vitronectin or fibronectin. SPARC-transfected clones also demonstrated altered migration on the different extracellular matrix proteins. The modulation of migration, either positive or negative, was associated with changes in the level of secreted SPARC. These data suggest that SPARC may modulate glioma proliferation and invasion by modulating both the growth and migration of glioma cells.

Correlation between magnetic resonance spectroscopy imaging and image-guided biopsies: semiquantitative and qualitative histopathological analyses of patients with untreated glioma.

OBJECTIVE: Since intratumoral heterogeneity of gliomas is not adequately reflected in conventional magnetic resonance imaging (MRI), we sought to determine a correlation between different proton magnetic resonance spectroscopic imaging ((1)H MRSI) metabolic ratios and the degree of tumor infiltration in diffusely infiltrating gliomas. In this report, we describe the microscopic anatomy of gliomas on imaging. METHODS: Image-guided biopsies with semiquantitative and qualitative histopathological analyses from a series of 31 untreated patients with low- and high-grade gliomas were correlated with multivoxel (1)H MRSI referenced to the same spatial coordinates. RESULTS: This series yielded 247 tissue samples and 307 observations. Choline-containing compounds using contralateral creatine and choline for normalization or ipsilateral N-acetylaspartate appear to correlate best with the degree of tumor infiltration. Similar correlations were present within each grade after stratification. Despite the interpatient overlap of metabolic ratios between normal tissue and mild tumor infiltration, preliminary analyses revealed that (1)H MRSI appears more accurate than conventional MRI in defining the tumor boundary and quantifying the degree of tumor infiltration. CONCLUSION: This is the first study showing histopathological validation of tumor boundaries using (1)H MRSI. These results support the conclusion that (1)H MRSI accurately reflects the extent of the disease in patients with gliomas. This has important diagnostic and therapeutic implications for more accurately assessing the burden of disease as well as for planning and assessing response to therapy.

The expression of the urokinase plasminogen activator system in metastatic murine osteosarcoma: an in vivo mouse model.

The role of urokinase plasminogen activator (uPA) in osteosarcoma is poorly understood. We examined the importance of uPA, its receptor, uPAR, and its inhibitor, PAI-1, in our in vivo model of metastatic osteosarcoma. Rodent osteosarcoma cells (UMR 106-01) were inoculated into the tibia of athymic mice. Animals were sacrificed and autopsied at 4 days to 5 weeks after inoculation. Tibiae and lungs were excised, fixed, and examined histologically and by in situ hybridization. Osteosarcoma development was associated with tibial swelling and lameness, and radiographic changes included osteolysis and new bone formation. Lung metastases developed spontaneously. In the tibial tumors, uPAR mRNA was expressed early (4 days), whereas uPA and PAI-1 mRNA increased as the tumor invaded the surrounding tissue (3 weeks). There was also an increase in the mRNA expression of the osteoblast-related genes, alpha1(I) procollagen and osteopontin, but not matrix Gla protein. Lung metastases also expressed mRNA for the uPA system and the bone-related proteins. We have produced a model of metastatic osteosarcoma, which typifies the characteristics of the human tumor. Our results suggest that the uPA system plays a role in the local aggressiveness and metastasis of osteosarcoma and, in particular, indicates a possible therapeutic role for uPAR antagonists in the treatment of osteosarcoma.

Bisphosphonates regulate cell growth and gene expression in the UMR 106-01 clonal rat osteosarcoma cell line.

Local growth of osteosarcoma involves destruction of host bone by proteolytic mechanisms and/or host osteoclast activation. Osteoclast formation and activity are regulated by osteoblast-derived factors such as the osteoclast differentiating factor, receptor activator of NF-kappaB ligand (RANKL) and the inhibitor osteoprotegerin (OPG). We have investigated the in vitro effects of bisphosphonates on a clonal rat osteosarcoma cell line. The aminobisphosphonate pamidronate was added to UMR 106-01 cell cultures (10(-8)M to 10(-4)M up to 5 days). The non-aminobisphosphonate clodronate was administered for the same time periods (10(-6)M to 10(-2)M). Cell proliferation, apoptosis and mRNA expression was assessed. Both agents inhibited cell proliferation in a time- and dose-dependent manner. ELISA analysis demonstrated an increase in DNA fragmentation although there was no significant dose-related difference between the doses studied. Bisphosphonate-treated cultures had a greater subpopulation of cells exhibiting morphological changes of apoptosis. Expression of mRNA for osteopontin and RANKL was down-regulated by both agents, while the expression of mRNA for alkaline phosphatase, pro-alpha1(I) collagen and OPG was not altered. Out in vitro work suggests the bisphosphonates not only have direct effects on osteosarcoma cell growth and apoptosis, but also, by altering the relative expression of osteoclast-regulating factors, they may inhibit the activity of osteoclasts and their recruitment.

Child Sexual Behavior Inventory: normative, psychiatric, and sexual abuse comparisons.

A normative sample of 1,114 children was contrasted with a sample of 620 sexually abused children and 577 psychiatric outpatients on the Child Sexual Behavior Inventory (CSBI), a 38-item behavior checklist assessing sexual behavior in children 2 to 12 years old. The CSBI total score and each individual item differed significantly between the three groups after controlling for age, sex, maternal education, and family income. Sexually abused children exhibited a greater frequency of sexual behaviors than either the normative or psychiatric outpatient samples. Test-retest reliability and interitem correlation were satisfactory. Sexual behavior problems were related to other generic behavior problems. This contributed to the reduced discrimination between psychiatric outpatients and sexually abused children when compared to the normative/sexually abused discrimination.

Pharmacokinetic-pharmacodynamic relationships of the bispecific antibody MDX-H210 when administered in combination with interferon gamma: a multiple-dose phase-I study in patients with advanced cancer which overexpresses HER-2/neu.

INTRODUCTION: MDX-H210 is a Fab'xFab' bispecific antibody (BsAb) constructed chemically by crosslinking Fab' mAb 520C9 (anti-HER-2/neu) and Fab' mAbH22 (anti-CD64). STUDY DESIGN AND OBJECTIVES: This was a dose escalation study of intravenous MDX-H210 (1-70 mg/m(2)), preceded 24 h beforehand by subcutaneous IFNgamma (50 microg/m(2) to up-regulate FcgammaRI) administered three times a week for 3 weeks. We investigated the pharmacokinetic-pharmacodynamic relationships between MDX-H210 C(max) and AUC and (i) MDX-H210 binding to peripheral blood monocytes and neutrophils, (ii) the peak plasma G-CSF, IL-6, IL-8 and TNFalpha concentrations, and (iii) the observed clinical toxicity. RESULTS: 23 patients (19F:4M; median age 51.5; range 25-72 y) with advanced HER-2/neu positive cancers (19 breast, three prostate and one lung) were studied. Plasma MDX-H210 concentrations over time, circulating numbers of monocytes and neutrophils, percent saturation of monocyte and neutrophil FcgammaRI, and plasma concentrations over time of G-CSF, IL-6, IL-8 and TNFalpha were measured and clinical toxicity monitored. The E(max) pharmacodynamic model best fitted the relationship of MDX-H210 C(max) and the maximum percent saturation of both monocytes (E(max)=74.6; EC(50)=0.9 microg/ml) and neutrophils (E(max)=66.2; EC(50)=2.3 microg/ml) on the first day of treatment. On the last day of treatment, day 19, these parameters were E(max)=57.0% and EC(50)=0.46 microg/ml for monocytes and E(max)=61.9% and EC(50)=0.26 microg/ml for neutrophils. No positive relationship was defined between the log MDX-H210 C(max) and the log peak plasma IL-6, G-CSF, TNF or IL-8 concentrations on day 1. On day 19 these plasma cytokine concentrations were undetectable post MDX-H210 therapy. There was no consistent relationship between MDX-H210 C(max) and the observed clinical toxicities. CONCLUSIONS: These data suggest that MDX-H210 C(max) and AUC could be related by the E(max) model to maximum percent FcgammaRI saturation on circulating monocytes and neutrophils in the patients studied. After day 1, the post MDX-H210 therapy cytokine response attenuated over time, consistent with desensitization. We did not find a relationship between log MDX-H210 C(max) and peak plasma cytokine concentrations or clinical toxicities.

Bispecific antibody-targeted phagocytosis of HER-2/neu expressing tumor cells by myeloid cells activated in vivo.

Studies from our laboratory and others have established that both mononuclear phagocytes and neutrophils mediate very efficient cytotoxicity when targeted through Fc receptors using a suitable monoclonal or bispecific antibody (BsAb). Cross-linking an Fc receptor for IgG (FcgammaR) triggers multiple anti-tumor activities including superoxide generation, cytokine and enzyme release, phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). In this report, using unfractionated leukocytes and two color flow cytometric analysis, we describe the phagocytic capacity of peripheral blood polymorphonuclear cells (PMN) and monocytes isolated from patients enrolled in a phase I clinical trial of MDX-H210 given in combination with IFNgamma. MDX-H210 is a BsAb targeting the myeloid trigger molecule FcgammaRI and the HER-2/neu proto-oncogene product overexpressed on a variety of adenocarcinomas. In this trial, cohorts of patients received escalating doses of MDX-H210 3 times per week for 3 weeks. Interferon-gamma (IFNgamma) was given 24 h prior to each BsAb infusion. Our results demonstrate that monocytes from these patients were inherently capable of phagocytosing the HER-2/neu positive SK-BR-3 cell line and that addition of MDX-H210 into the assay significantly enhanced the number of targets phagocytosed. Two days after administration of an immunologically active dose of MDX-H210 (10 mg/m2), monocytes from these patients were able to phagocytose greater amounts of target cell material, indicating that these cells remained armed with functionally sufficient BsAb for at least 48 h. PMN from these patients very efficiently mediated phagocytosis through FcgammaRI after being treated with IFNgamma, but not before. We conclude that phagocytosis is not only an efficient mechanism of myeloid cell-mediated cytotoxicity, but may also be a mechanism by which antigens from phagocytosed cells can enter a professional antigen presenting cell for processing and presentation.

Nicotinic receptors on hippocampal cultures can increase synaptic glutamate currents while decreasing the NMDA-receptor component.

Activation of presynaptic nicotinic acetylcholine receptors (nAChRs) can enhance the release of glutamate from synapses in hippocampal slices and cultures. In hippocampal cultures making autaptic connections, rapid application of a high concentration of nicotine activated presynaptic, postsynaptic, and somatic nAChRs, which consequently enhanced the amplitude of evoked excitatory postsynaptic currents (eEPSCs) mediated by glutamate receptors. The increased eEPSC amplitudes arose from enhanced glutamate release caused by presynaptic nAChRs (Radcliffe and Dani, 1998, Journal of Neuroscience 18, 7075). The same whole-cell nicotine applications that enhanced non-NMDA eEPSCs often decreased the NMDA-receptor component of the eEPSCs. Furthermore, whole-cell activation of nAChRs by nicotine selectively reduced the amplitude of the whole-cell NMDA-receptor currents without affecting the non-NMDA receptor currents. The inhibition by nicotine was prevented by the alpha7-specific antagonist, methyllycaconitine, and required the presence of extracellular Ca(2+). The calmodulin antagonist fluphenazine prevented inhibition of the NMDA-receptor current by nAChR activity, suggesting that a Ca(2+)-calmodulin-dependent process mediated the effect of nicotine. Our results indicate that activation of nAChRs can modulate glutamatergic synapses in several ways. Presynaptic nAChR activity enhances synaptic transmission by increasing transmitter release. Additionally, somatic or postsynaptic nAChRs can initiate a Ca(2+) signal that can act via calmodulin to reduce the responsiveness of NMDA receptors.

Urokinase plasminogen activator system gene expression is increased in human breast carcinoma and its bone metastases--a comparison of normal breast tissue, non-invasive and invasive carcinoma and osseous metastases.

The urokinase plasminogen activator (uPA) system has been widely associated with the development of breast carcinoma. However, the role of the urokinase pathway in the development of osseous breast cancer metastases has been largely overlooked. We studied the expression of uPA, urokinase plasminogen activator receptor (uPAR)- and plasminogen activator inhibitor type-1 (PAI-1) in human breast carcinomas and their bone metastases, using in situ hybridisation. Studies were performed using paraffin-embedded tissue from 13 ductal carcinomas, 23 invasive ductal carcinomas, five normal breasts and 25 bone metastases. The majority of the tumours examined expressed low to moderate levels of uPA mRNA and low to high levels of uPAR and PAI-1 mRNA, which was predominantly localised to the epithelial tumour cells. There was slight over-expression of uPA and PAI-1 mRNA and a marked increase in uPAR mRNA expression in the malignant tumours compared with benign tissue. Overall, uPAR and PAI-1 mRNA expression was found to be more variable than uPA mRNA, suggesting a possible role of the receptor and inhibitor in the regulation of uPA activity. Increased alpha1(I) procollagen (COL) and osteopontin (OPN) mRNA expression was detected, primarily in the stromal cells, in malignant tumours compared with the benign tissue. The increased expression of the components of the uPA system on the epithelial tumour cells may account for the activation of the proteolytic cascade that occurs during breast cancer metastasis to bone. Furthermore, the over-expression of COL and OPN suggests a possible interaction between these matrix proteins and the uPA system.

Characteristics and outcome of children with carbon monoxide poisoning with and without smoke exposure referred for hyperbaric oxygen therapy.

OBJECTIVES: To describe the clinical characteristics and outcome of children with carbon monoxide (CO) poisoning with and without smoke exposure referred for hyperbaric oxygen therapy (HBOT), and to determine the association between any of these characteristics and death. SETTING: Regional hyperbaric referral center. PATIENTS: The medical records of 150 children with CO poisoning (COP) who were treated with HBOT between August 92 and September 95 were reviewed. MEASUREMENTS/MAIN RESULTS: COP was defined as a history of probable exposure to CO, with either a carboxyhemoglobin level (COHb) > 25, or COHb < 25 with neurological, respiratory, or cardiac compromise. Major cutaneous burns were described as second degree burns over greater than 20% of the patient's total body surface area (TBSA), or third degree burns over greater than 10% of the patient's TBSA. Children extracted from a closed-space fire who had airway soot, singed facial hair/facial burns, or respiratory distress were defined as having smoke inhalation and carbon monoxide poisoning (CO/SI). CO/SI occurred in 40.1% of patients. Compared to children with COP alone, those with CO/SI were significantly more likely to have a depressed mental status upon arrival to an ED (76.3 % vs 13.6 %, P < 0.001), lower mean initial GCS (6.7 vs 14.7, P < 0.001), lower mean initial pH (7.2 vs 7.4, P < 0.001), respiratory arrest at the scene (68.5% vs 0%, P < 0.001), and cardiac arrest at the scene (25.9% vs 0%, P < 0.001). Children with CO/SI were significantly more likely to have a poor outcome (death) than children with COP alone (22.6% vs. 0%, P < 0.001). Comparing children with CO/SI who died versus survivors, there were significant differences in mean initial COHb (38.3 vs 24.3, P = 0.03), mean initial temperature upon arrival in an ED (94.9 degrees F vs 98.2 degrees, P < 0.006), respiratory arrest at the scene (92% vs 59.6%, P = 0.04), and cardiac arrest at the scene (66.7% vs 13.5%, P < 0.001). Sixty percent of children died who had a combination of risk factors of smoke inhalation, low temperature, high COHb level, and respiratory and cardiac arrest in the field. CONCLUSIONS: These preliminary data suggest that children with COP alone who are treated with HBOT are at low risk for dying regardless of initial COHb level. Children with CO/SI have a significantly higher risk of dying than those children with COP alone. A combination of smoke inhalation, low temperature, high COHb level, respiratory arrest, and cardiac arrest is highly associated with death. Prospective studies are needed to confirm and further define these associations.

Loreclezole inhibition of recombinant alpha1beta1gamma2L GABA(A) receptor single channel currents.

Loreclezole had two different effects on GABA(A) receptor (GABAR) currents. When applied to GABARs that contained a beta2 or beta3 subunit subtype, but not a beta1 subtype, loreclezole potentiated the peak current evoked by sub-maximal concentrations of GABA. Loreclezole also increased the rate and degree of apparent desensitization of GABAR whole-cell currents, an effect that was independent of the beta subunit subtype, suggesting that potentiation and inhibition of GABAR current by loreclezole occurred through separate sites. We used patch-clamp recording from outside-out and inside-out patches from L929 fibroblasts transiently transfected with rat GABAR subunits to examine the properties of inhibition of alpha1beta1gamma2L single channel currents by loreclezole. Loreclezole decreased the mean open time of the channel by decreasing the average durations of the open states. Loreclezole also increased the occurrence of a closed component with an average duration near 20 ms. Inhibition by loreclezole was not voltage-dependent. Loreclezole was equally effective when applied to the intracellular side of the receptor, suggesting that its binding site was readily accessible from both sides of the membrane. Pre-application of loreclezole effectively inhibited the GABAR current in macropatches, indicating that binding did not require an open channel. These findings were consistent with a mechanism of allosteric modulation at a site formed by the membrane spanning regions of the receptor.

A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens.

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.

Effects of a replication of a multicomponent model for preventing adolescent pregnancy in three Kansas communities.

CONTEXT: A significant amount of attention has been devoted to the complex issue of teenage pregnancy and to programs for reducing pregnancy among adolescents. Careful evaluations of such programs are needed to ascertain what strategies will be most effective at reducing teenage pregnancy. METHODS: A pretest-posttest comparison group design was used to analyze the effects of a comprehensive multicomponent school and community intervention on estimated pregnancy rates and birthrates among young people in three Kansas communities: Geary County, Franklin County and selected neighborhoods of Wichita. RESULTS: There were high levels of program activity in all three communities during the intervention period, including teacher training and sexuality education for students. Survey respondents rated highly such project interventions as the extension of school-linked clinic hours to accommodate student schedules and support groups established in middle schools. Between 1994 and 1997, the proportions of adolescents reporting that they had ever had sex decreased significantly among all ninth and 10th graders in Geary County, from 51% to 38% among females and from 63% to 43% among males. In Franklin County, more males in grades 11 and 12 reported using condoms in 1996 (55%) than had done so in 1994 (39%). Age at first intercourse remained relatively stable in Franklin and Geary counties during the intervention period. The estimated pregnancy rate among adolescents aged 14-17 decreased between 1994 and 1997 in Geary Country, while it increased in comparison areas. The estimated pregnancy rates among 14-17-year-olds decreased in both Franklin County and its comparison communities. The birthrate declined both in one target area of Wichita and in its comparison area from 1991-1993 to 1994-1996. Over the same time period, the birthrate increased in a second target area of Wichita, while it decreased in the comparison community. CONCLUSIONS: This evaluation of a comprehensive multicomponent program for adolescent pregnancy prevention contributes to our understanding of this model and its replicability in diverse communities. Ongoing program evaluation is important for developing initiatives and for refining strategies so they respond to local conditions.

PIP: This paper evaluates a multi-component program for reducing pregnancy among adolescents in the US. The study employed a pretest-posttest comparison group design to analyze the effects of a comprehensive multi-component school and community intervention on estimated pregnancy rates and birthrates among young people in three Kansas communities: Geary County, Franklin County and selected neighborhoods of Wichita. Results revealed high levels of program activity in all three communities during the intervention period, including teacher training and sexuality education for students. From 1994-97, the proportion of adolescents who reported that they had experienced sex decreased significantly among all 9th and 10th graders in Geary County. Condom use among males in grades 11 and 12 in Franklin County increased from 39% in 1994 to 55% in 1996. In Franklin County and its comparison areas, the estimated pregnancy rates decreased among adolescents aged 14-17 years. The birthrate declined both in one target area of Wichita and in its comparison area from 1991-93 and 1994-96. In general, this research contributed to an understanding on the impact of multi-component school- and community-based interventions on adolescent pregnancy rates.