Increased length-dependent activation of human engineered heart tissue after chronic α1A-adrenergic agonist treatment: testing a novel heart failure therapy.

Chronic stimulation of cardiac α1A-adrenergic receptors (α1A-ARs) improves symptoms in multiple preclinical models of heart failure. However, the translational significance remains unclear. Human engineered heart tissues (EHTs) provide a means of quantifying the effects of chronic α1A-AR stimulation on human cardiomyocyte physiology. EHTs were created from thin slices of decellularized pig myocardium seeded with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and fibroblasts. With a paired experimental design, EHTs were cultured for 3 wk, mechanically tested, cultured again for 2 wk with α1A-AR agonist A61603 (10 nM) or vehicle control, and retested after drug washout for 24 h. Separate control experiments determined the effects of EHT age (3-5 wk) or repeat mechanical testing. We found that chronic A61603 treatment caused a 25% increase of length-dependent activation (LDA) of contraction compared with vehicle treatment (n = 7/group, P = 0.035). EHT force was not increased after chronic A61603 treatment. However, after vehicle treatment, EHT force was increased by 35% relative to baseline testing (n = 7/group, P = 0.022), suggesting EHT maturation. Control experiments suggested that increased EHT force resulted from repeat mechanical testing, not from EHT aging. RNA-seq analysis confirmed that the α1A-AR is expressed in human EHTs and found chronic A61603 treatment affected gene expression in biological pathways known to be activated by α1A-ARs, including the MAP kinase signaling pathway. In conclusion, increased LDA in human EHT after chronic A61603 treatment raises the possibility that chronic stimulation of the α1A-AR might be beneficial for increasing LDA in human myocardium and might be beneficial for treating human heart failure by restoring LDA.NEW & NOTEWORTHY Chronic stimulation of α1A-adrenergic receptors (α1A-ARs) is known to mediate therapeutic effects in animal heart failure models. To investigate the effects of chronic α1A-AR stimulation in human cardiomyocytes, we tested engineered heart tissue (EHT) created with iPSC-derived cardiomyocytes. RNA-seq analysis confirmed human EHT expressed α1A-ARs. Chronic (2 wk) α1A-AR stimulation with A61603 (10 nM) increased length-dependent activation (LDA) of contraction. Chronic α1A-AR stimulation might be beneficial for treating human heart failure by restoring LDA.

Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia.

Schizophrenia is a neurodevelopmental disorder that affects up to 1% of the general population. Various genes show associations with schizophrenia and a very weak nominal association with the tight junction protein, claudin-5, has previously been identified. Claudin-5 is expressed in endothelial cells forming part of the blood-brain barrier (BBB). Furthermore, schizophrenia occurs in 30% of individuals with 22q11 deletion syndrome (22q11DS), a population who are haploinsufficient for the claudin-5 gene. Here, we show that a variant in the claudin-5 gene is weakly associated with schizophrenia in 22q11DS, leading to 75% less claudin-5 being expressed in endothelial cells. We also show that targeted adeno-associated virus-mediated suppression of claudin-5 in the mouse brain results in localized BBB disruption and behavioural changes. Using an inducible 'knockdown' mouse model, we further link claudin-5 suppression with psychosis through a distinct behavioural phenotype showing impairments in learning and memory, anxiety-like behaviour and sensorimotor gating. In addition, these animals develop seizures and die after 3-4 weeks of claudin-5 suppression, reinforcing the crucial role of claudin-5 in normal neurological function. Finally, we show that anti-psychotic medications dose-dependently increase claudin-5 expression in vitro and in vivo while aberrant, discontinuous expression of claudin-5 in the brains of schizophrenic patients post mortem was observed compared to age-matched controls. Together, these data suggest that BBB disruption may be a modifying factor in the development of schizophrenia and that drugs directly targeting the BBB may offer new therapeutic opportunities for treating this disorder.

Heparin versus placebo for acute coronary syndromes.

BACKGROUND: Acute coronary syndromes (ACS) represent a spectrum of disease including unstable angina (UA) and non-ST segment myocardial infarction (NSTEMI). Despite treatment with aspirin, beta-blockers and nitroglycerin, UA/NSTEMI is still associated with significant morbidity and mortality. Although emerging evidence suggests that low molecular weight heparin (LMWH) is more efficacious compared to unfractionated heparin (UFH), there is limited data to support the role of heparins as a drug class in the treatment of ACS. OBJECTIVES: To determine the effect of heparins (UFH and LMWH) compared with placebo for the treatment of patients with ACS. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials on The Cochrane Library (issue 4, 2002), MEDLINE (1966 to May 2002), EMBASE (1980 to May 2002) and CINAHL (1982 to May 2002). Authors of included studies and pharmaceutical industry representatives were contacted to determine if unpublished studies which met the inclusion criteria were available. SELECTION CRITERIA: Randomized controlled trials of parenteral UFH or LMWH versus placebo in people with ACS (UA or NSTEMI). DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed quality of studies. Data were extracted independently by two reviewers. Study authors were contacted to verify and clarify missing data. MAIN RESULTS: Eight studies (3118 participants) were included in this review. We found no evidence for difference in overall mortality between the groups treated with heparin and placebo (RR = 0.84, 95% CI 0.36 to 1.98). Heparins reduced the occurrence of MI (RR = 0.40, 95% CI 0.25 to 0.63, NNT = 33). An increase in the incidence of minor bleeds (RR = 6.80, 95% CI 1.23 to 37.49, NNH = 17). AUTHORS' CONCLUSIONS: Compared to placebo, patients treated with heparins had similar risk of mortality, revascularization, recurrent angina, major bleeding and thrombocytopenia. However, those treated with heparins had decreased risk of MI and a higher incidence of minor bleeding.

Emergency department patients' opinions of screening for intimate partner violence among women.

BACKGROUND: Universal screening for intimate partner violence (IPV) in the emergency department (ED) has been advocated by many medical institutions. Policies implemented for IPV screening have met with numerous obstacles. One such obstacle is the perception by emergency personnel that patients might be offended by such screening if they presented to the ED for problems unrelated to trauma. OBJECTIVES: To assess opinions of adult ED patients regarding a policy of universal IPV screening for women presenting to the ED. METHODS: This study was conducted in EDs in Halifax, Nova Scotia, and St John's, Newfoundland. Patients were questioned as to whether it was appropriate for all women to be asked if they had experienced violent or threatening behaviour from someone close to them. Patients in significant pain or in extremis were not approached. RESULTS: The data consist of a convenience sample of 514 adult ED patients, aged 16-95 years. Two (0.4%) were excluded from the analysis. Of 512 analysed, 442 (86.0%) answered "yes" to the question, 53 (10.3%) answered "no", 17 (3.3%) had no opinion. There were no significant differences between the proportion of "yes" and "no" answers in the male and female groups. CONCLUSION: Universal screening for IPV of adult female patients presenting to the ED was supported by most patients. Patient objections should not be seen as a reason to withhold questioning on this issue.

Patients with community acquired pneumonia discharged from the emergency department according to a clinical practice guideline.

OBJECTIVES: To assess the safety of discharging patients with community acquired pneumonia (CAP) according to a clinical practice guideline. METHODS: A systematic retrospective review of medical records of 867 adult patients discharged from an emergency department (ED) with CAP between 3 January 1999 and 3 January 2001. Readmission or death rates within 30 days of discharge were evaluated, using data from all local hospitals and from the provincial coroner. RESULTS: Of 685 patients with pneumonia severity index (PSI) scores of <91, 13 (1.9%) were readmitted and five (0.76%) died within 30 days of the ED visit. Thirty day readmission and death rates for patients with PSI >90 were 7.14% (13 of 182) and 9.34% (17 of 182), respectively. CONCLUSION: Adult patients with CAP discharged from the ED according to the recommendations of a clinical practice guideline based on the PSI have low readmission and death rates, and are generally safely managed as outpatients.

Loss of translational control in yeast compromised for the major mRNA decay pathway.

The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways. In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress. In the past, the paradigm of the eIF2alpha kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2alpha phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.

Utility of blood cultures in the management of adults with community acquired pneumonia discharged from the emergency department.

STUDY OBJECTIVE: To assess the clinical value of blood cultures (BCs) in the management of adult patients discharged from the emergency department (ED) with a diagnosis of community acquired pneumonia (CAP). METHODS: The courses of antibiotic regimens and outcomes of patients with positive BC results were examined to assess their influence on BCs. RESULTS: BCs were obtained from 289 outpatients. Six clinically significant organisms were identified (a yield of 2.1%). Outpatients with CAP who had blood cultures performed had a 0.69% (2 of 289) chance of having a change of treatment directed by the results of the culture. CONCLUSION: BCs have little utility in the ambulatory management of CAP.

Rib fractures following minor trauma in older patients: a not-so-benign injury.

Two older adults presented to the emergency department with rib fractures following minor trauma. Both were discharged on oral analgesics and died within 2 days. Rib fractures more often lead to adverse outcomes in older adults. Emergency physicians should consider admitting any such patient who presents with two or more rib fractures.

The Internet and electronic transmission of medical records.

OBJECTIVE: To review, from a legal perspective the potential for using the Internet for inter-institutional transfer of patient medical records. METHODS: Basic issues and recent legislation that relate to protection of both medical data, and those transferring that data over public network systems is reviewed. RESULTS: Many laws already in existence can be applied to Internet transmission, but questions of jurisdiction remain. Providing signatures on requests for information, which are in essence contracts, is a problem. Signatures must both prove the identity of the participants and provide for non-repudiation of the agreement. Cryptographic digital signatures appear secure and effective, but their use is difficult to implement. Simpler methods are fraught with risks, yet are more easily accomplished. The patient's rights of privacy must be balanced against the need for access by government, physician, or healthcare institutions to confidential information. In general, information holders must put forth reasonable efforts to keep information confidential. The development of acknowledged standards will provide guidance. Multiple laws provide some deterrence and hence some reassurance to healthcare institutions, for example, by criminalizing acts of electronic interception of patient records in transit. CONCLUSION: Some believe the expense of secure transfer of medical records by electronic means is a major obstacle; this is false: such transfers are now technologically quite easy. The greatest obstacle to electronic transfer of medical records at this point is the development of workable standards for signing agreements and protecting transmissions, but the perceived advantages will likely drive the necessary developments.

Characterization of a New York ovine lentivirus isolate.

A lentivirus has been isolated from a Finnish ewe with ovine progressive pneumonia in a closed upstate New York flock. We demonstrated that the virus, designated ovine lentivirus strain CU1 (OLV-CU1), is biologically, biochemically and molecularly related to, but distinct from, previously described sheep and goat lentiviruses. Nine of 32 ewes (from the affected flock) with precipitating antibodies for ovine lentivirus also produced antibodies that were able to neutralize the infectivity of OLV-CU1. The virus replicated in cultured sheep fibroblasts and caused the formation of large multi-nucleated cells. OLV-CU1-specific RNA transcripts found in infected cells and virion antigenic proteins were similar to those of other small ruminant lentiviruses. However, the virus was distinguished from other isolates at the DNA level by nucleic acid hybridization, restriction endonuclease mapping and partial sequencing of the virus genome.

The influence of opsonins on the bactericidal effect of bovine alveolar macrophages against Pasteurella multocida.

Monolayers of bovine alveolar macrophages were infected with smooth and rough strains of Pasteurella multocida to determine their bactericidal function. Heat inactivated normal bovine serum, fresh bovine serum, specific immune serum and a combination of specific immune serum and fresh bovine serum were used as opsonins. A substantial difference in the bactericidal effect of macrophages against the logarithmic and stationary phase p. multocida was observed. Opsonization of the rough strain of P. multocida with the combination of specific immune and fresh serum increased their association with alveolar macrophages compared to those opsonized with either normal serum, fresh serum or specific immune serum. Opsonization of the smooth strain of P. multocida with normal bovine serum resulted in substantially fewer bacteria associated with macrophages at time zero, compared with other opsonins. Regardless of the kind of opsonins used in these experiments, the number of the rough strain of P. multocida killed by macrophages was always higher than those of the smooth strain.

Immunization against experimental murine salmonellosis with liposome-associated O-antigen.

Partially delipidated Salmonella typhimurium (O-1,4,5,12) lipopolysaccharide was incorporated into small multilamellar liposomes composed of either naturally occurring or synthetic phospholipids. Vaccination of mice with the liposome-lipopolysaccharide complexes induced a cellular response specific for O-1,4,5,12 determinants, as determined by the development of a delayed-type hypersensitivity reaction. The liposome-lipopolysaccharide vaccines were significantly more effective, compared with other nonviable vaccines tested, in protecting mice against a lethal intravenous challenge infection with virulent S. typhimurium. Protection afforded by the liposome-lipopolysaccharide vaccines was comparable to that conferred by a live S. typhimurium vaccine. Results suggest that liposome-induced modulation of the host immune response in favor of cell-mediated immunity may be more efficacious in preventing diseases in which cell-mediated immunity is of prime importance.

Liposome-encapsulated cephalothin in the treatment of experimental murine salmonellosis.

The effectiveness of liposome-encapsulated cephalothin was compared with free cephalothin for the treatment of mice experimentally infected with Salmonella typhimurium. Compared with free cephalothin, following intravenous administration, liposome-encapsulated cephalothin was cleared from the circulation more rapidly and concentrated in the liver and spleen. Treatment of infected mice with the liposome antibiotic complex was more efficacious in terms of reducing the number of S. typhimurium in these organs as compared with the free antibiotic. The results suggest that liposome-encapsulated antimicrobial agents may possess a therapeutic advantage in the treatment of diseases caused by facultative intracellular bacteria since this manipulation favors delivery of the entrapped antibiotic to intracellular sites occupied by S. typhimurium.

Intraphagocytic killing of Salmonella typhimurium by liposome-encapsulated cephalothin.

Multilamellar liposomes (lipid bilayer vesicles) composed of phosphatidylcholine, cholesterol, and phosphatidylserine (molar ratio, 6:3:1) were produced and then made to entrap an aqueous solution of cephalothin. Resident murine peritoneal macrophages were shown to be capable of interiorizing the liposome-antibiotic complex; this event resulted in a relatively high intracellular concentration of cephalothin. In macrophages infected in vitro with Salmonella typhimurium, intracellular killing of the bacteria was maximal at 60 min of incubation; at this time, 60% of the interiorized organisms had been killed. Treatment of infected macrophages with liposome-encapsulated cephalothin enhanced the intraphagocytic killing of S typhimurium over that by macrophages treated with free cephalothin. These results demonstrate the superiority of liposome-encapsulated antibiotics to free antibiotics in effecting the elimination of a facultative intracellular bacterium from its intracellular site. This type of complex may find application in the treatment of diseases caused by this group of microorganisms.

Bactericidal activity of alveolar and peritoneal macrophages exposed in vitro to three strains of Pasteurella multocida.

Normal ICR mice were infected intravenously, intraperitoneally, or aerogenically with Pasteurella multocida strains isolated from a turkey (S68), calf (V90), or rabbit (J20) lung. Both the turkey and calf isolates were highly virulent for mice and multiplied logarithmically in the lungs, liver, and spleen, resulting in death of the animals in 18 to 36 h. The rabbit strain was avirulent for mice, but repeated passage in mice did result in some increased virulence. All three strains of P. multocida were inactivated rapidly by normal mouse peritoneal macrophages, provided that the organisms were opsonized with specific hyperimmune serum before being exposed to the macrophage monolayers. P. multocida was slowly inactivated by normal mouse alveolar macrophages when the organisms were preopsonized. However, the surviving organisms later multiplied extensively in vitro. Macrophages harvested from hyperimmunized mice were no better at inactivating opsonized P. multocida cells than were normal mouse cells. The relative importance of the different phagocytic cell populations in the uptake and killing of opsonized P. multocida cells is discussed in relation to immunity to this important animal pathogen.

Fate of Pasteurella hemolytica in conventionally raised and germfree mice.

When Pasteurella hemolytica was introduced into conventionally raised ICR mice by a variety of routes (intraperitoneal, aerogenic, and oral), the inoculum was rapidly eliminated, and none of the mice died. Even when the inoculum was injected intraperitoneally into sublethally irradiated (600 rads) mice, the organisms were eliminated rapidly unless suspended in 10% hog gastric mucin. When germfree ICR mice were orally infected with P. hemolytica, the infection established itself in the intestinal tract and spread to the mesenteric lymph nodes but did not progress beyond this point. Despite the inability of P. hemolytica to establish itself systemically, the organism multiplied freely in mouse blood and a homogenate of normal mouse lung in vitro. Normal mouse peritoneal macrophages could phagocytose P. hemolytica in vitro, although not as efficiently as the control Listeria monocytogenes suspensions. The addition of hyperimmune bovine serum (opsonin) to the P. hemolytica suspension increased phagocytosis but did not greatly affect the subsequent bactericidal activity of the macrophages in vitro. The reason for the lack of pathogenicity shown by P. hemolytica in normal mice remains enigmatic.