Catching and killing of airborne SARS-CoV-2 to control spread of COVID-19 by a heated air disinfection system.

Airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via air-conditioning systems poses a significant threat for the continued escalation of the current coronavirus disease (COVID-19) pandemic. Considering that SARS-CoV-2 cannot tolerate temperatures above 70 °C, here we designed and fabricated efficient filters based on heated nickel (Ni) foam to catch and kill SARS-CoV-2. Virus test results revealed that 99.8% of the aerosolized SARS-CoV-2 was caught and killed by a single pass through a novel Ni-foam-based filter when heated up to 200 °C. In addition, the same filter was also used to catch and kill 99.9% of Bacillus anthracis, an airborne spore. This study paves the way for preventing transmission of SARS-CoV-2 and other highly infectious airborne agents in closed environments.

Analysis of the sporicidal activity of chlorine dioxide disinfectant against Bacillus anthracis (Sterne strain).

Routine surface decontamination is an essential hospital and laboratory procedure, but the list of effective, noncorrosive disinfectants that kill spores is limited. We investigated the sporicidal potential of an aqueous chlorine dioxide solution and encountered some unanticipated problems. Quantitative bacteriological culture methods were used to determine the log(10) reduction of Bacillus anthracis (Sterne strain) spores following 3min exposure to various concentrations of aqueous chlorine dioxide solutions at room temperature in sealed tubes, as well as spraying onto plastic and stainless steel surfaces in a biological safety cabinet. Serial 10-fold dilutions of the treated spores were then plated on 5% sheep blood agar plates, and the survivor colonies were enumerated. Disinfection of spore suspensions with aqueous chlorine dioxide solution in sealed microfuge tubes was highly effective, reducing the viable spore counts by 8log(10) in only 3min. By contrast, the process of spraying or spreading the disinfectant onto surfaces resulted in only a 1log(10) kill because the chlorine dioxide gas was rapidly vaporised from the solutions. Full potency of the sprayed aqueous chlorine dioxide solution was restored by preparing the chlorine dioxide solution in 5% bleach (0.3% sodium hypochlorite). The volatility of chlorine dioxide can cause treatment failures that constitute a serious hazard for unsuspecting users. Supplementation of the chlorine dioxide solution with 5% bleach (0.3% sodium hypochlorite) restored full potency and increased stability for one week.

Interferon protects mice against inhalation anthrax.

Interferons (IFNs) play a role in innate immunity during many viral, bacterial, and protozoal infections. With the increasing threat of bioterrorist attacks with Bacillus anthracis, its high lethality, and the limited effectiveness of antibiotics, alternative treatments are being studied. Antibodies to protective antigen (PA) are promising, as is IFN. During many bacterial infections, production of and protection by IFNs has been reported, including B. anthracis in vitro. In vivo, we find that (1) the type I IFN inducer, Poly-ICLC, strongly and rapidly protects mice; (2) the protection is IFN-mediated since recombinant murine IFN-beta can protect, and protection by Poly-ICLC is abrogated in IFN type I receptor knockout mice. The greatest protection by Poly-ICLC was conferred by intranasal treatment. A delay in death was observed with the intramuscular route alone, but was not significant. Together, the results suggest the IFN defense could protect mice, up to 60%, against lethal inhalational anthrax, and thus have important medical implications for therapy of human anthrax.

Proteomic analyses of murine macrophages treated with Bacillus anthracis lethal toxin.

Bacillus anthracis is the etiological agent of anthrax and the bacterium produces a tripartite anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA represents the binding domain of the toxin and acts in concert with either LF, a metalloprotease, or EF, an adenylate cyclase, to form lethal toxin (LeTx) or edema toxin (EdTx), respectively. We analyzed the proteomics response of two murine macrophage cell lines (J774.1A and RAW264.7) following B. anthracis LeTx treatment to detect unique host proteins involved in anthrax infection using difference in-gel electrophoresis (DIGE) followed by nanoLC-MS for identification of the proteins. The comparative proteomics approach identified a set of proteins in each cell line that was consistently upregulated when the two macrophage cell lines were treated with LeTx. The upregulated proteins include those involved in energy metabolism, cytoskeleton structure and stress response. A subset of five proteins (ATP synthase beta subunit, beta-actin, Hsp70, vimentin, and Hsp60 homolog) was identified that were commonly upregulated in both cell lines. The proteomic data suggest the involvement of reactive oxygen species (ROS) in cell lysis as seen by the upregulation of proteins that lead to the production of ROS in both the cell lines used in our study. However, proteins that afford protection against ROS may play an important role in the survival of the macrophage to LeTx infection as shown by the differences in proteomic responses of the two cell lines to the action of LeTx. These identified proteins may have the potential to be used as biomarkers for diagnostics and therapeutics.

Evaluation of the protective effects of quinacrine against Bacillus anthracis Ames.

Bacillus anthracis has gained notoriety as a dangerous biological weapon because of its virulence and ability to produce highly resistant spores. In addition, the ability of this organism to produce plasmid-encoded edema toxin (EdTx) and lethal toxin (LeTx) plays a pivotal role in the pathogenesis of anthrax. In this study, the efficacy of quinacrine was evaluated against the effects of anthrax toxins in vitro and its ability to provide protection against challenge with B. anthracis Ames strain spores in an intranasal mouse and guinea pig model. Quinacrine protected murine macrophages in vitro against cytotoxicity and cAMP production induced by LeTx and EdTx, respectively, at concentrations of 40-80 microM, most likely by preventing acidification of the endosomes. However, animals dosed with human equivalent doses of quinacrine were not protected against respiratory spore challenge. The failure of quinacrine to provide protection against inhalation anthrax was attributed to our inability to attain inhibitory concentrations of the drug in the serum or tissues. After daily administration of 43.3 mg quinacrine to guinea pigs (300 g), serum levels after 96 h were only 9.9 microM, a concentration not sufficient to protect macrophages in vitro. Administration of high doses of quinacrine (86.6 mg/kg) was toxic to the animals. These results illustrate some of the difficulties in developing protective therapeutic strategies against inhalation anthrax even when antitoxic drugs appear effective in vitro.

Molecular characterization of a glucose-inhibited division gene, gidA, that regulates cytotoxic enterotoxin of Aeromonas hydrophila.

By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

Early cell signaling by the cytotoxic enterotoxin of Aeromonas hydrophila in macrophages.

A cytotoxic enterotoxin (Act) of Aeromonas hydrophila is an important virulence factor with hemolytic, cytotoxic and enterotoxic activities. In this report, we demonstrated Act rapidly mobilized calcium from intracellular stores and evoked influx of calcium from the extracellular milieu in macrophages. A direct role of calcium in Act-induced prostaglandin (e.g. PGE(2)) and tumor necrosis factor alpha (TNF alpha) production was demonstrated in macrophages using a cell-permeable calcium chelator BAPTA-AM, which also down-regulated activation of transcription factor NF-kappa B. We showed that Act's capacity to increase PGE(2) and TNF alpha production could be blocked by inhibitors of tyrosine kinases and protein kinase A. In addition, Act caused up-regulation of the DNA repair enzyme redox factor-1 (Ref-1), which potentially could promote DNA binding of the transcription factors allowing modulation of various genes involved in the inflammatory response. Taken together, a link between Act-induced calcium release, regulation of downstream kinase cascades and Ref-1, and activation of NF-kappa B leading to PGE(2) and TNF alpha production was established. Since Act also caused extensive tissue damage, we showed that Act increased reactive oxygen species, and the antioxidant N-acetyl cysteine, blocked Act-induced PGE(2) and TNF alpha production, as well as NF-kappa B nuclear translocation in macrophages. We have demonstrated for the first time early cell signaling initiated in eukaryotic cells by Act, which leads to various biological effects associated with this toxin.

Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system that causes motor, sensory, and cognitive deficits. The present study characterized demyelinated lesions in the cerebral cortex of MS patients. One hundred twelve cortical lesions were identified in 110 tissue blocks from 50 MS patients. Three patterns of cortical demyelination were identified: Type I lesions were contiguous with subcortical white matter lesions; Type II lesions were small, confined to the cortex, and often perivascular; Type III lesions extended from the pial surface to cortical layer 3 or 4. Inflammation and neuronal pathology were studied in tissue from 8 and 7 patients, respectively. Compared to white matter lesions, cortical lesions contained 13 times fewer CD3-positive lymphocytes (195 vs 2,596/mm3 of tissue) and 6 times fewer CD68-positive microglia/macrophages (11,948 vs 67,956/mm3 of tissue). Transected neurites (both axons and dendrites) occurred at a density of 4,119/mm3 in active cortical lesions, 1,107/mm3 in chronic active cortical lesions, 25/mm3 in chronic inactive cortical lesions, 8/mm3 in myelinated MS cortex, and 1/mm3 in control cortex. In active and chronic active cortical lesions, activated microglia closely apposed and ensheathed apical dendrites, neurites, and neuronal perikarya. In addition, apoptotic neurons were increased significantly in demyelinated cortex compared to myelinated cortex. These data support the hypothesis that demyelination, axonal transection, dendritic transection, and apoptotic loss of neurons in the cerebral cortex contribute to neurological dysfunction in MS patients.

Effects of extracellular pH on tumour necrosis factor-alpha production by resident alveolar macrophages.

Cellular acid-base status has been found to exert selective actions on the effector functions of activated macrophages (mphi). We examined the effects of extracellular pH (pH(o)) on the production of tumour necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in resident alveolar mphi. Cells were obtained by bronchoalveolar lavage of rabbits, activated in vitro with LPS, and cultured at pH(o) 5.5, 6.5 or 7.4 for up to 18 h. The relative abundance of TNF-alpha mRNA peaked at approximately 2 h. The peak transcript abundance was increased at lower pH(o) values. This finding probably reflected pre-transcription/transcription effects of pH, in as much as the stability of TNF-alpha mRNA induced with phorbol ester was unaffected by the experimental pH(o) values. TNF-alpha secretion by LPS-treated mphi decreased at lower pH(o) values. The TNF-alpha content of mphi-conditioned media decreased progressively with decrements in pH(o). The reduced TNF-alpha secretion at pH(o) 5.5 was accompanied by an increase in the cytosolic TNF-alpha content (compared with that at pH(o) 7.4), indicating that pH(o) altered TNF-alpha secretion due, in part, to the intracellular retention of synthesized cytokine (i.e. a post-translation effect). The data show that pH(o) has multiple effects (pre-transcription/transcription and post-translation) on TNF-alpha production induced by LPS in resident alveolar mphi. These results suggest that the role of alveolar mphi in inflammatory responses is modulated by pH(o), which may be important in tumours/abscesses and sites of infection where the external milieu is acidic.

Cholera toxin-induced PGE(2) activity is reduced by chemical reaction with L-histidine.

Mediators of cholera toxin (CT)-induced fluid secretion include 3',5'-adenosine monophosphate (cAMP), prostaglandin E(2) (PGE(2)), and 5-hydroxytryptamine (5-HT). Administration of L-histidine significantly reduced the net secretory response of the small intestine of mice challenged with CT and reduced the capacity of PGE(2) to stimulate Na+ transport in Ussing chambers. We demonstrated that L-histidine chemically modified the structure of PGE(2) but had no direct effect on cAMP or 5-HT. L-Histidine and imidazole reacted with PGE(2) in vitro in cell-free mixtures incubated at 37 degrees C and pH 7.0 under an atmosphere of N(2) with the formation of PGE(2)-imidazole and PGE(2)-histidine covalent adducts. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analysis of the purified adduct showed that imidazole catalyzed the dehydration of PGE(2). A Michael adduct then was formed between C11 of 11-deoxy-Delta(10) PGE(2) (PGA(2)) and the tau nitrogen in the imidazole ring of L-histidine. Importantly, the isolated PGE(2)-imidazole and PGE(2)-histidine adducts inhibited CT-induced fluid loss and cAMP accumulation in mouse intestinal loops. The protection provided by PGE(2)-imidazole, PGE(2)-histidine, and L-histidine against intestinal fluid loss could provide a basis for future therapy against cholera.

Post-transcriptional effects of extracellular pH on tumour necrosis factor-alpha production in RAW 246.7 and J774 A.1 cells.

The present studies determined the effects of extracellular pH (pH(o)) on the production of tumour necrosis factor-alpha (TNF-alpha) in the macrophage-like cell lines RAW 246.7 and J774 A.1. The cells were activated with lipopolysaccharide (LPS) at pH(o) 5.5, 6.5 or 7.4. TNF-alpha gene transcription was monitored by Northern blot analysis. Synthesis of the cytokine was monitored by ELISA measurements of the TNF-alpha content of cell-conditioned media (extracellularly released TNF-alpha) and cell lysates (cytosolic TNF-alpha). The magnitude of the TNF-alpha response differed markedly between the two cell lines. RAW cells were more responsive to LPS than were J774 cells. However, the effects of pH(o) on TNF-alpha production were similar in the two cell lines. TNF-alpha gene transcription was insensitive to experimental pH(o). The pH(o) had no effect on the abundance of TNF-alpha mRNA at 2, 4 or 18 h. Nonetheless, synthesis of TNF-alpha was affected significantly by pH(o). The TNF-alpha contents of cell-conditioned medium and cell lysate at 18 h were reduced progressively at lower pH(o) values. The data indicate that pH(o) alters TNF-alpha production in RAW and J774 cells at a post-transcriptional level. These findings suggest that pH(o) influences the phenotypic responses of macrophages to activating stimuli and modifies the role that macrophages play in inflammatory and immune actions.

Prostaglandin levels in stimulated macrophages are controlled by phospholipase A2-activating protein and by activation of phospholipase C and D.

Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A2 (PLA2)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha increased PLAA levels at early time points (2-30 min), which correlated with an up-regulation in cytosolic PLA2 and PGE2 levels. Both COX-2 and secretory PLA2 were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4-24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503-538, PLAA exhibited homology with melittin, and increased PGE(2) production was noted in macrophages stimulated with melittin. In addition to PLA2, we demonstrated that activation of phospholipase C and D significantly controlled PGE2 production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.

The cytotoxic enterotoxin of Aeromonas hydrophila induces proinflammatory cytokine production and activates arachidonic acid metabolism in macrophages.

An aerolysin-related cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses multiple biological activities, which include its ability to lyse red blood cells, destroy tissue culture cell lines, evoke a fluid secretory response in ligated intestinal loop models, and induce lethality in mice. The role of Act in the virulence of the organism has been demonstrated. In this study, we evaluated the potential of Act to induce production of proinflammatory cytokines associated with Act-induced tissue injury and Act's capacity to activate in macrophages arachidonic acid (AA) metabolism that leads to production of eicosanoids (e.g., prostaglandin E(2) [PGE(2)]). Our data indicated that Act stimulated the production of tumor necrosis factor alpha and upregulated the expression of genes encoding interleukin-1beta (IL-1beta) and IL-6 in the murine macrophage cell line RAW264.7. Act also activated transcription of the gene encoding inducible nitric oxide synthase. Act evoked the production of PGE(2) coupled to the cyclooxygenase-2 (COX-2) pathway. AA is a substrate for PGE(2), and Act produced AA from phospholipids by inducing group V secretory phospholipase A(2). We also demonstrated that Act increased cyclic AMP (cAMP) production in macrophages. cAMP, along with PGE(2), could potentiate fluid secretion in animal models because of infiltration and activation of macrophages resulting from Act-induced tissue injury. After Act treatment of RAW cells, we detected an increased translocation of NF-kappaB and cAMP-responsive element binding protein (CREB) to the nucleus using gel shift assays. Act also upregulated production of antiapoptotic protein Bcl-2 in macrophages, suggesting a protective role for Bcl-2 against cell death induced by proinflammatory cytokines. The increased expression of genes encoding the proinflammatory cytokines, COX-2, and Bcl-2 appeared correlated with the activation of NF-kappaB and CREB. This is the first report of the detailed mechanisms of action of Act from A. hydrophila.

Melittin-mediated release of [3H]-oleic acid from E. coli cells is dependent upon heat- and trypsin-sensitive factor(s) in human serum.

Synthetic melittin mediated the release of [3H]-oleic acid ([3H]-OA) or its acylated lipids from [3H]-OA-labeled E. coli cells exposed to human serum. This phenomenon was not observed in the absence of serum and was calcium independent. The addition of serum was not required for melittin-mediated lysis of erythrocytes, although lysis was greater in the presence of serum than in its absence (P<0.001). Trypsin treatment of human serum reduced the melittin-mediated release of [3H]-OA/acylated lipids, and this effect was more pronounced upon boiling the serum (P<0.01). A kinetic study showed that maximum release of [3H]-OA/acylated lipids occurred within 3-6 min. Thin layer chromatography (TLC) analysis showed the lipids to be phosphatidyl ethanolamine (PE), phosphatidylethanol (PEt) and phosphatidic acid (PA). There was no detectable level of oleic acid (OA), diacylglycerol (DAG), phosphatidyl choline (PC) or phosphatidyl serine (PS). These findings suggested that a trypsin and heat-sensitive enzyme/factor present in the serum had a role in melittin-mediated action. These findings further showed that melittin activated phospholipase D (PLD), without affecting phospholipase A(2) (PLA(2)) or phospholipase C (PLC) activity.

Neutrophil activation by bacterial lipoprotein versus lipopolysaccharide: differential requirements for serum and CD14.

Neutrophil activation plays an important role in the inflammatory response to Gram-negative bacterial infections. LPS has been shown to be a major mediator of neutrophil activation which is accompanied by an early down-regulation of L-selectin and up-regulation of CD1lb/CD18. In this study, we investigated whether lipoprotein (LP), the most abundant protein in the outer membrane of bacteria from the family Enterobacteriaceae, can activate neutrophils and whether this activation is mediated by mechanisms that differ from those used by LPS or Escherichia coli diphosphoryl lipid A (EcDPLA). Neutrophil activation was assessed by measuring down-regulation of L-selectin and up-regulation of CD11b/CD18. When comparing molar concentrations of LP vs EcDPLA, LP was more potent (four times) at activating neutrophils. In contrast to LPS/EcDPLA, LP activation of neutrophils was serum independent. However, LP activation of neutrophils was enhanced by the addition of soluble CD14 and/or LPS-binding protein. In the presence of serum, LP activation of neutrophils was inhibited by different mAbs to CD14. This inhibition was significantly reduced or absent when performed in the absence of serum. Diphosphoryl lipid A from Rhodobacter spheroides (RaDPLA) completely inhibited LPS/EcDPLA activation of neutrophils but only slightly inhibited LP activation of neutrophils. These results suggest that LP activation of human neutrophils can be mediated by a mechanism that is different from LPS activation and that LP is a potentially important component in the development of diseases caused by Gram-negative bacteria of the family Enterobacteriaceae.

Thin and thick filament regulation of contractility in experimental cerebral vasospasm.

OBJECTIVE: Cerebral vasospasm is a potentially fatal consequence of aneurysmal subarachnoid hemorrhage and influences the prognosis of the patient. The purpose of this study was to evaluate the status of thin (actin) and thick (myosin) filament regulation of smooth muscle contraction in the double-subarachnoid hemorrhage canine model of cerebral vasospasm and to determine the effects of a kinase inhibitor reported to be effective in vasospasm, HA1077, on thin and thick filament regulation. METHODS: Cerebral vasospasm was assessed by vertebral angiography. Myosin regulatory light chain phosphorylation was measured using glycerol-urea gels, whereas protein levels of the thin filament-associated protein calponin were measured by Western blot. RESULTS: The basilar arteries of dogs in which subarachnoid hemorrhage was induced narrowed to 36% +/- 2.0% of their size on the first day (n = 12). The phosphorylation of the regulatory light chain tended to increase, but the change did not reach statistical significance (35% +/- 5.9% [n = 12] versus 25% +/- 4.8% [n = 10] in control arteries). In contrast to this increase, significant degradation of calponin was observed in the samples from vasospastic dogs (85.4% +/- 5.45% [n = 5] versus 15.2% +/- 6.21% [n = 5]; P < 0.01). Prophylactic treatment with intravenous injections of HA1077 at 0.67 mg/kg b.i.d. significantly inhibited vasospasm (diameters, 65% +/- 10.2% of Day 1 diameters [n = 5]; P < 0.05), and calponin degradation (57.8% +/- 13.9% [n = 4]) was substantially reduced. CONCLUSION: These data suggest that degradation of the thin filament-associated protein calponin plays a role in cerebral vasospasm and that the antivasospastic action of HA1077 is, at least in part, due to prevention of calponin degradation.

A laboratory simulation of toluene cleanup by air sparging of water-saturated sands.

Laboratory air sparging experiments were performed in narrow acrylic tanks to evaluate the cleanup of toluene in water-saturated sands. Air flow channels in the sediment were identified by way of a colorimetric visualization technique, which allowed pore water samples to be collected at a known horizontal distance from an air channel. Pore water was sampled at periodic intervals during sparging experiments and analyzed by gas chromatography to yield toluene concentration vs. time data. Results indicate that channelized air flow is effective in reducing toluene concentrations in the range of 36-3 ppm, within 2 to 5 days, at least up to 185 mm from an active air channel. While relatively rapid, these toluene reduction times are longer than previously published data, from similar type experiments. The discrepancy is likely a function of air delivery flow rate and proximity of sampling sites to active air channels. Data from the current investigation were used to attempt an estimate of effective diffusion coefficients (D*) for toluene in clean, well-characterized sands in which the concentration gradient was imposed by sparge air. Calculated D* values range from 2. 98x10(-8) m(2)/s to 5.74x10(-9) m(2)/s, and are significantly faster than previously published values of toluene diffusion in clay soils. However, the values are also slightly greater than diffusion coefficients for toluene in aqueous solutions, indicating that the calculations more likely estimate coefficients of hydrodynamic dispersion (D(L)).

Adjuvant effects of cholera toxin b subunit on immune response to recombinant thyrotropin receptor in mice.

We had previously shown that BALB/c mice immunized with the extracellular domain of human thyrotropin receptor (ETSHR) developed moderate hyperthyroxinemia. The antibody responses in these mice were predominantly of the IgG1 subclass. Since cholera toxin B subunit (CT-B) has direct effects on the thyroid, and is known to activate B lymphocytes and cause enhanced IgG1 production, we tested the ability of CT-B to modulate the antibody response to ETSHR. CT-B is unique in that it not only elicits a strong immune response to itself, but more importantly, when given with other antigens acts as a potent adjuvant. In the present study, BALB/c mice given ETSHR with CFA or CT-B via ip route showed higher titers of antibodies to ETSHR when compared to mice similarly immunized with ETSHR alone, or with IFA. Antibodies in ETSHR+CT-B immunized mice were mostly of the IgG1 subclass and reacted predominantly with ETSHR peptides 1 (aa 22-41), 21 (aa 322-341), and 23 (352-371). In contrast, animals immunized with ETSHR+CFA showed IgG1, IgG2a and IgG2b responses and reacted with peptides 1 and 21. Furthermore, mice immunized with ETSHR along with CT-B showed significantly higher levels of thyrotropin (TSH) binding inhibitory immunoglobulins (TBII) compared to those that did not receive CT-B. None of the mice immunized with a control antigen showed antibody response to ETSHR. These results suggested that CT-B could enhance and modulate immune response to ETSHR.

Surveillance of suicidal behavior in Kitsap County, Washington: a retrospective study.

Suicide is a major source of preventable morbidity and mortality in Kitsap County, Washington State. This article describes a study of suicidal behavior to identify risk groups in order to design intervention strategies. A retrospective study was conducted by reviewing the charts of individuals exhibiting suicidal behavior who had presented to the county's only civilian hospital emergency department over a 7 month period. Frequencies were calculated to identify at-risk populations and determine risk factors. One hundred forty-five charts were reviewed. Subjects were mostly female (69%), and ages ranged from 10 to 80 years with 73% between 15 and 44 years. Two-thirds of the subjects were not working. More than half had previously exhibited suicidal behavior and more than 75% had previous mental health encounter(s). Most admissions (67.6%) occurred between 4:00 p.m. and 4:00 a.m. The core public health functions of assessment, policy development, and assurance provided the framework for this community to explore the finding that suicide was a major source of preventable morbidity and mortality. Community-based intervention strategies have been developed in an effort to reach the Healthy People 2000 objective of reducing suicide deaths to no more than 10.5 per 100,000 residents.

Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells.

Human monocytic leukemia cells (U937) were challenged with synthetic melittin, and arachidonic acid (AA)/acylated lipids from both cells (pellet) and media (supernatant) were analyzed by thin layer chromatography (TLC). From these data, melittin-mediated activation/inhibition of major phospholipases in U937 cells was related to pore formation, permeabilization and cytolysis as determined by light microscopy. Also, the effect of melittin on acylhydrolase activity in the cell-free sonicated lysates of U937 cells was examined. Here we report that synthetic melittin (1 microM) caused cytolysis of U937 cells within 10-15 min. Cellular hypertrophy (5 min) and aggregation (1 min) preceded cytolysis. TLC analysis of these lipids showed that total levels (cellular + medium) of diacylglycerol (DAG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) decreased, while that of arachidonic acid (AA) increased continuously (5-30 min). However, levels of phosphatidylethanol (PEt) phosphatidic acid (PA) and phosphatidylserine (PS) were increased transiently at 5-10 min being maximal at 5 min. Taken together, the combined levels of PEt and PA (an end product of phopholipase D, PLD) were about 42-fold higher than the level of AA at 5-10 min. Enhancement of AA levels appeared to result from in vitro reactions of various acylhydrolases and their phospholipid substrates (free/membrane bound) liberated into the medium during pore formation/cell lysis. Incubation of sonicated cell lysates also enhanced release of AA, which decreased upon addition of melittin, indicating that melittin inhibited these acylhydrolases. A consistent decrease in the level of DAG showed that phospholipase C was unaffected. Hence, transient activation of PLD bymelittin at the point of initiation of cytolysis, suggested a role for PLD in melittin-mediated membrane disruption/cytolysis by an uncharacterized signal transduction mechanism.