UK B.1.1.7 variant exhibits increased respiratory replication and shedding in nonhuman primates.

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, African green monkeys were infected intranasally with either a contemporary D614G or the UK B.1.1.7 variant. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tract tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases. ONE-SENTENCE SUMMARY: UK B.1.1.7 infection of African green monkeys exhibits increased respiratory replication and shedding but no disease enhancement.

Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Umbilical cord blood graft engineering: challenges and opportunities.

We are entering a very exciting era in umbilical cord blood transplantation (UCBT), where many of the associated formidable challenges may become treatable by ex vivo graft manipulation and/or adoptive immunotherapy utilizing specific cellular products. We envisage the use of double UCBT rather than single UCBT for most patients; this allows for greater ability to treat larger patients as well as to manipulate the graft. Ex vivo expansion and/or fucosylation of one cord will achieve more rapid engraftment, minimize the period of neutropenia and also give certainty that the other cord will provide long-term engraftment/immune reconstitution. The non-expanded (and future dominant) cord could be chosen for characteristics such as better HLA matching to minimize GvHD, or larger cell counts to enable part of the unit to be utilized for the development of specific cellular therapies such as the production of virus-specific T-cells or chimeric-antigen receptor T-cells which are reviewed in this study.

Robust T cell responses to aspergillosis in chronic granulomatous disease: implications for immunotherapy.

Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus-specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus-specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4(+) T cell responses to aspergillus were unimpaired: aspergillus-specific T cell frequencies were even elevated in CGD mice (P < 0·01) and humans (P = 0·02), compared to their healthy counterparts. CD4-depleted murine models suggested that the role of T cells might be redundant because resistance to aspergillus infection was conserved in CD4(+) T cell-depleted mice, similar to wild-type animals. In contrast, mice depleted of neutrophils alone or neutrophils and CD4(+) T cells developed clinical and pathological evidence of pulmonary aspergillosis and increased mortality (P < 0·05 compared to non-depleted animals). Our findings that T cells in CGD have a robust aspergillus CD4(+) T cell response suggest that CD4(+) T cell-based immunotherapy for this disease is unlikely to be beneficial.

Spontaneous high-grade glial intramedullary tumor of the spine in a rhesus macaque (Macaca mulatta).

BACKGROUND: A 4-year-old rhesus macaque presented with acute, progressive paresis of the extremities. METHODS: A complete blood count, serum biochemical analysis, neurologic exam and necropsy were performed. RESULTS: The clinical, histopathological, and immunohistochemical findings confirmed a high-grade intramedullary glial tumor of the spinal cord that was most consistent with an ependymoma. CONCLUSIONS: We describe a case of a naturally occurring spontaneous spinal cord neoplasia in a non-human primate.

In vitro and in vivo activities of E-101 solution against Acinetobacter baumannii isolates from U.S. military personnel.

We evaluated the in vitro and in vivo activity of a novel topical myeloperoxidase-mediated antimicrobial, E-101 solution, against 5 multidrug-resistant Acinetobacter baumannii isolates recovered from wounded American soldiers. Time-kill studies demonstrated rapid bactericidal activity against all A. baumannii strains tested in the presence of 3% blood. The in vitro bactericidal activity of E-101 solution against A. baumannii strains was confirmed in a full-thickness excision rat model. Additional in vivo studies appear warranted.

Differential effects of volatile and intravenous anesthetics on the activity of human TASK-1.

Volatile anesthetics have been shown to activate various two-pore (2P) domain K(+) (K(2P)) channels such as TASK-1 and TREK-1 (TWIK-related acid-sensitive K(+) channel), and mice deficient in these channels are resistant to halothane-induced anesthesia. Here, we investigated whether K(2P) channels were also potentially important targets of intravenous anesthetics. Whole cell patch-clamp techniques were used to determine the effects of the commonly used intravenous anesthetics etomidate and propofol on the acid-sensitive K(+) current in rat ventricular myocytes (which strongly express TASK-1) and selected human K(2P) channels expressed in Xenopus laevis oocytes. In myocytes, etomidate decreased both inward rectifier K(+) (K(ir)) current (I(K1)) and acid-sensitive outward K(+) current at positive potentials, suggesting that this drug may inhibit TASK channels. Indeed, in addition to inhibiting guinea pig Kir2.1 expressed in oocytes, etomidate inhibited human TASK-1 (and TASK-3) in a concentration-dependent fashion. Propofol had no effect on human TASK-1 (or TASK-3) expressed in oocytes. Moreover, we showed that, similar to the known effect of halothane, sevoflurane and the purified R-(-)- and S-(+)-enantiomers of isoflurane, without stereoselectivity, activated human TASK-1. We conclude that intravenous and volatile anesthetics have dissimilar effects on K(2P) channels. Human TASK-1 (and TASK-3) are insensitive to propofol but are inhibited by supraclinical concentrations of etomidate. In contrast, stimulatory effects of sevoflurane and enantiomeric isoflurane on human TASK-1 can be observed at clinically relevant concentrations.

Multidetector CT coronary angiography: have we found the holy grail of non-invasive coronary imaging?

Is technology about to deliver on the long awaited goal of effective non-invasive methods for visualising and assessing coronary arteries?

The stipulations of one institutional review board: a five year review.

OBJECTIVES: This study was designed to explore the prevalence and types of stipulations (such as clarifications or changes) required of investigators by the institutional review board (IRB) of one institution over a five year period. DESIGN: Stipulations to research proposals (n = 124) were documented from the minutes of the IRB meetings. SETTING: Community hospital. PARTICIPANTS: IRB submissions. MAIN MEASUREMENTS: Number and type of IRB stipulations. RESULTS: Nineteen research submissions (15.3%) were approved without any stipulations. For the remainder, the majority of stipulations related to consent forms (74.2%). CONCLUSIONS: Consent forms appear to be at highest risk for IRB stipulations. Being aware of high risk areas before submission of research proposals may reduce the frequency of stipulations required of investigators.

Extracellular Ca2+ is obligatory for ouabain-induced potentiation of cardiac basal energy expenditure.

1. The method of action of cardiac glycosides is commonly explained by the 'pump-inhibition hypothesis': inhibition of the Na+/K+-ATPase allows [Na+]i to rise, eventually reversing Na+/Ca2+ exchange. The resulting influx of Ca2+o increases [Ca2+]i, thereby activating intracellular Ca2+-dependent ATPases and, hence, energy demand. This sequence has been presumed to occur during diastole as well as systole. However, it has been reported that dihydro-ouabain-induced potentiation of heat production by quiescent ventricular trabeculae persists in the absence of Ca2+o. This implies that the pump-inhibition hypothesis is inapplicable during diastole. 2. We tested this implication by: (i). measuring the rate of oxygen consumption (Vo2) of arrested guinea-pig whole-hearts; (ii). measuring[Ca2+]i in quiescent ventricular trabeculae; and (iii). mathematical modelling using software (Oxsoft Heart, Oxford Software, Oxford, UK) based on DiFrancesco-Noble formalism. 3. Upon induction of arrest, whole heart Vo2 fell to one-quarter of its 'beating' value. Subsequent perfusion with ouabain (20 micromol/L), in the presence of Ca2+o, increased Vo2 fourfold. This increase was prevented by withholding Ca2+o. Comparable results were obtained in quiescent trabeculae: ouabain increased [Ca2+]i only if Ca2+o was present. Mathematical modelling readily simulated these experimental results. 4. We conclude that influx of Ca2+o is mandatory for potentiation of cardiac basal metabolism by cardiac glycosides.

Long-chain acyl-coenzyme A esters and fatty acids directly link metabolism to K(ATP) channels in the heart.

ATP-sensitive K (K(ATP)) channels are inhibited by cytosolic ATP, a defining property that implicitly links these channels to cellular metabolism. Here we report a direct link between fatty acid metabolism and K(ATP) channels in cardiac muscle cells. Long-chain (LC) acyl-coenzyme A (CoA) esters are synthesized from fatty acids and serve as the principal metabolic substrates of the heart. We have studied the effects of LC acyl-CoA esters and LC fatty acids on K(ATP) channels of isolated guinea pig ventricular myocytes and compared them with the effects of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Application of oleoyl-CoA (0.2 or 1 micromol/L), a naturally occurring acyl-CoA ester, to the cytosolic side of excised patches completely prevented rundown of K(ATP) channels, but not of Kir2 channels. The open probability of K(ATP) channels measured in the presence of oleoyl-CoA or PIP(2) was voltage dependent, increasing with depolarization. Oleoyl-CoA greatly reduced the ATP sensitivity of K(ATP) channels. At a concentration of 2 micromol/L, oleoyl-CoA increased the half-maximal inhibitory concentration of ATP >200-fold. The time course of the decrease in ATP sensitivity was much faster during application of oleoyl-CoA than during application of PIP(2). The effects of PIP(2), but not of oleoyl-CoA, were inhibited by increasing Ca(2+) to 1 mmol/L. Oleate (C18:1; 10 micromol/L), the precursor of oleoyl-CoA, inhibited K(ATP) channels activated by oleoyl-COA: Palmitoleoyl-CoA and palmitoleate (C16:1) exerted similar reciprocal effects. These findings indicate that LC fatty acids and their CoA-linked derivatives may be key physiological modulators of K(ATP) channel activity in the heart.

Metabolic consequences of a species difference in Gibbs free energy of Na+/Ca2+ exchange: rat versus guinea pig.

The Gibbs free energy of the sarcolemmal Na+/Ca2+ exchanger (DeltaG(Na/Ca)) determines its net Ca2+ flux. We tested the hypothesis that a difference of diastolic DeltaG(Na/Ca) exists between rat and guinea pig myocardium. We measured the suprabasal rate of oxygen consumption (VO2) of arrested Langendorff-perfused hearts of both species, manipulating DeltaG(Na/Ca) by reduction of extracellular Na+ concentration, [Na+](o). Hill equations fitted to the resulting VO2-[Na+](o) relationships yielded Michaelis constant (K(m)) values of 67 and 25 mM for rat and guinea pig, respectively. We developed and tested a simple thermodynamic model that attributes this difference of K(m) values to a 7.84 kJ/mol difference of DeltaG(Na/Ca). The model predicts that reversal of Na+/Ca2+ exchange, leading to diastolic Ca2+ influx, should occur at a value of [Na+](o) about three times higher in rat myocardium. We verified this quantitative prediction using fura 2 fluorescence to index intracellular Ca2+ concentration in isolated ventricular trabeculae at 37 degrees C. The postulated difference in free energy of Na+/Ca2+ exchange explains a number of reported disparities of Ca2+ handling at rest between rat and guinea pig myocardia.

A study of the efficacy and safety of irbesartan in combination with conventional therapy, including ACE inhibitors, in heart failure. Irbesartan Heart Failure Group.

Because heart failure therapy with angiotensin-converting enzyme (ACE) inhibitors may not be optimal, owing to persistent levels of angiotensin II occurring through incomplete blockade and alternate pathways, the benefit of adding irbesartan, an angiotensin receptor antagonist, to conventional therapy, including ACE inhibitors, was examined. In this multicentre, randomised, double-blind, placebo-controlled study, 109 patients with heart failure (New York Heart Association functional class II and III) and left ventricular ejection fraction (LVEF) < or = 40% received stable doses of ACE inhibitors and diuretics before and throughout the study. Irbesartan was titrated as tolerated to 150 mg once daily in all patients. Exercise tolerance time (ETT), LVEF and clinical status were assessed at baseline and after 12 weeks. Compared with placebo, irbesartan in combination with conventional therapy, including ACE inhibitors, produced favourable trends in ETT and LVEF and was well tolerated in patients with mild to moderate heart failure.

Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area.

A randomized, double-blind, placebo-controlled efficacy trial of one dose of CVD 103-HgR live oral cholera vaccine was performed in Indonesia from 1993 to 1997. 67,508 persons aged 2-41 years ingested vaccine or placebo and were followed for four years, detecting cholera cases using hospital-based surveillance. A nested reactogenicity study (538 vaccinees, 535 controls) revealed no vaccine-attributable side effects. A nested immunogenicity study (N=657) showed vibriocidal seroresponses in 64-70% of vaccinees vs 1-2% of controls. Cholera incidence was lower than expected. 103 cases of Vibrio cholerae O1 El Tor diarrhea were detected, 93 evaluable for vaccine efficacy (43 vaccine, 50 placebo; efficacy=14%). A suggestion of protection was observed among persons with blood group O [P=0.12]. Only seven cases occurred within six months of vaccination, precluding assessment of short-term efficacy. In Jakarta, single-dose CVD 103-HgR did not confer long-term protection. Short-term protection from a single-dose and long-term protection from two doses have yet to be studied.

3-Dimensional configuration of perimysial collagen fibres in rat cardiac muscle at resting and extended sarcomere lengths.

1. We have used fluorescence confocal laser scanning microscopy to attain the three-dimensional (3-D) microstructure of perimysial collagen fibres over the range of sarcomere lengths (1.9-2.3 micrometers) in which passive force of cardiac muscle increases steeply. 2. A uniaxial muscle preparation (right ventricular trabecula of rat) was used so that the 3-D collagen configuration could be readily related to sarcomere length. Transmission electron microscopy showed that these preparations were structurally homologous to ventricular wall muscle. 3. Trabeculae were mounted on the stage of an inverted microscope and fixed at various sarcomere lengths. After a trabecula was stained with the fluorophore Sirius Red F3BA and embedded in resin, sequential optical sectioning enabled 3-D reconstruction of its perimysial collagen fibres. The area fraction of these fibres, determined from the cross-sections of seven trabeculae, was 10.5 +/- 3.9 % (means +/- s.d.). 4. The reconstructed 3-D images show that perimysial collagen fibres are wavy (as distinct from coiled) cords which straighten considerably as the sarcomere length is increased from 1.85 +/- 0.06 micrometer (near-resting length) to 2.3 +/- 0.04 micrometer (means +/- s.d., n = 4). These observations are consistent with the notion that the straightening of these fibres is responsible for limiting extension of the cardiac sarcomere to a length of approximately 2.3 micrometers.

Early experience using duplex ultrasonography in the diagnosis of deep venous thrombosis; a prospective evaluation.

Duplex ultrasound is used in many radiology departments as the first line of investigation for symptomatic deep venous thrombosis. Before changing the practice of our department from venography to duplex ultrasonography, we wanted to assess our ability to identify deep venous thrombosis on ultrasound. Thirty-eight patients were investigated for suspected deep venous thrombosis by venography and duplex ultrasound. The results were compared using venography as the 'gold standard'. Duplex ultrasound correctly identified 13 out of 16 limbs with deep venous thrombosis. Four of the 38 duplex ultrasound examinations (11%) were described as inadequate at the time of examination, and when these are excluded from the analysis a sensitivity of 93 %, and specificity of 80 % are achieved. We conclude that there is a significant learning curve when performing duplex ultrasound of the lower limb, and that change-over from venography to ultrasound should include a period during which both examinations are routinely performed.

Seizures associated with buspirone overdose: case report and literature review.

Buspirone is a novel anxiolytic agent that is commonly used in the treatment of generalized anxiety disorder. Many studies have found it to be equal in efficacy to the benzodiazepines in the treatment of this illness. Its use has gained favor over the past few years because of its relatively mild and tolerable side-effect profile. The authors report a case of buspirone overdose that resulted in a generalized tonic clonic seizure approximately 36 hours after ingestion. Although there have been no previous cases of seizure induction by buspirone in humans, there are animal models of this phenomenon. The authors describe the case, review the literature regarding buspirone overdose and toxicity, and discuss treatment recommendations.

Mechanisms of force inhibition by halothane and isoflurane in intact rat cardiac muscle.

1. We investigated the mechanisms underlying the negative inotropic effect of the volatile anaesthetics halothane and isoflurane using twenty-two intact, right ventricular trabeculae of rat. [Ca2+]1 was measured qualitatively using either fluo-3 or fura-2, loaded into the cytosol via the acetoxymethyl (AM) ester form. Diastolic sarcomere length was adjusted to 2.1-2.2 micrograms and experiments were performed at 21-23 degrees C. 2. Halothane (0.25-3%) and isoflurane (0.48-4%) produced dose-dependent decreases in the amplitudes of the intracellular Ca2+ transients and twitch force. When the fluorescent Ca2+ indicator signals were corrected for changes in autofluorescence, neither volatile anaesthetic significantly changed diastolic [Ca2+]. 3. The ability of halothane and isoflurane to induce Ca2+ release from the sarcoplasmic reticulum of quiescent trabeculae was examined. When the superfusate was Ca2+ ad Na+ free (thereby preventing Na(+)-Ca2+ exchange and Ca2+ influx), 2% halothane, but not 4% isoflurane, evoked a transient increase in [Ca2+]i. 4. Halothane and isoflurane produced reversible, dose-dependent changes in cellular autofluorescence, the pattern of which was consistent with an increase in concentration of the reduced forms of nicotinamide adenine nucleotides and flavoproteins. This observation supports the putative inhibitory action of volatile anaesthetics at the site of Complex I of the mitochondrial electron transport chain. 5. Addition of the fatty acid hexanoate, a substrate that can be metabolized in the face of Complex I inhibition, did not appreciably attenuate the anaesthetic-induced negative inotropy; however, it greatly diminished autofluorescence changes. 6. To determine whether direct actions of the volatile anaesthetics on the contractile system contributed to the negative inotropy, external [Ca2+] was varied to modulate the amplitude of the Ca2+ transient. In the presence of 2% halothane or 4% isoflurane, restoration of the peak Ca2+ transient to control levels did not restore peak force. Moreover, halothane (1%) and isoflurane (16%) each reduced maximal Ca2(+)-activated force (attained using ryanodine tetani and a high external [Ca2+]) by around 15%. 7. We conclude that the negative inotropic actions of halothane and isoflurane on intact cardiac muscle reflect both reduced availability of Ca2+ and decreased responsiveness of the contractile system to Ca2+. The inhibitory action of the volatile anaesthetics on mitochondrial function does not contribute significantly to the negative inotropy but may lead to changes in cellular autofluorescence and misinterpretation of fluorescent Ca2+ indicator signals.