Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.

Esophageal histoplasmosis in a renal allograft recipient.

Histoplasmosis is a progressive granulomatous disease caused by the intracellular dimorphic fungus Histoplasma capsulatum. We report a rare case of esophageal histoplasmosis in a renal allograft recipient. A 55-year-old male who received a live, unrelated renal allograft 20 years ago presented with complaints of recurrent fever for ten to 12 months, weight loss over six months, progressive dysphagia more for solids for five to six months and joint pain and swelling involving the bilateral metacarpo-phalangeal and proximal interphalangeal joints. Biopsy from the esophageal ulcers revealed dense inflammation infiltrated with lymphocytes and macrophages with clusters of strongly positive intracellular fungal spores with a clear area or "halo-like" zone suggestive of Histoplasma capsulatum invasion. The patient was treated with intravenous liposomal amphotericin B for ten days and later switched over to oral itraconazole. Repeated endoscopy revealed significant improvement of the lesions.

RBx 7,796: A novel inhibitor of 5-lipoxygenase.

OBJECTIVE: To evaluate the pharmacological profile of RBx 7,796, a novel 5-lipoxygenase inhibitor. MATERIALS AND METHODS: RBx 7,796 was evaluated for 5- lipoxygenase inhibitory potential using human recombinant enzyme and profiled for selectivity against 12 and 15 lipoxygenase. RBx 7,796 was evaluated in cell based assay for inhibition of A23,187 induced LTB(4) release from isolated neutrophils. Ex vivo activity was evaluated for inhibition of A23,187 induced LTB(4) release in blood from treated rats. In vivo efficacy of RBx 7,796 was profiled in LPS induced neutrophilia model in rats and also in ovalbumin induced bronchoconstriction and airway inflammation models in guinea pigs. RESULTS: RBx 7,796, a novel chemotype, showed competitive inhibition of 5-lipoxygenase enzyme with an IC(50) of 3.5 +/- 1.1 microM. RBx 7,796 offered >100 fold selectivity against other related enzymes - 12 and 15 lipoxygenase. RBx 7,796 inhibited release of LTB(4) from human and rat neutrophils in vitro. Upon administration to rats, RBx 7,796 inhibited A23,187 induced LTB(4) release from rat neutrophils. Upon repeated administration, dosed once daily, RBx 7,796 inhibited LPS induced neutrophil influx in rat airway. RBx 7,796 also inhibited allergen induced bronchoconstriction and eosinophil influx in guinea pig airway in a dose dependent manner. CONCLUSION: The results suggest that RBx 7,796, a novel chemotype, is an orally efficacious inhibitor of 5-lipoxygenase enzyme that is effective against both neutrophilic and eosinophilic airway inflammation and shows potent inhibition with once daily administration.

Triple A syndrome.

Triple A syndrome (Allgrove syndrome) is an autosomal recessive disorder consisting of achalasia, alacrima and Addison insufficiency. We report an 11-year-old girl with predominant symptom of achalasia who was diagnosed as Triple A syndrome almost 3 years after initial presentation.

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Prevalence of coronary heart disease and risk factors in an urban Indian population: Jaipur Heart Watch-2.

BACKGROUND: The prevalence of risk factors for coronary heart disease has been inadequately studied in India. A repeat cross-sectional survey was carried out to evaluate the changes in the major coronary risk factors in the urban population of Jaipur previously studied in the early 1990s. METHODS AND RESULTS: Randomly selected adults > or =20 years of age were studied using stratified sampling. The target study sample was 1800 with a population proportionate gender distribution (males 960, females 840). Coronary risk factors, anthropometric variables, blood pressure, ECG, fasting blood glucose and lipids were evaluated. A total of 1123 subjects (62.4%) (males 550, females 573) were examined. Fasting blood samples were available in 523 males and 559 females. Overall coronary heart diesase prevalence, diagnosed by history or ECG changes, was found in 34 males (6.18%) and 58 females (10.12%). Risk factor prevalence showed that smoking/tobacco use was present in 201 males (36.5%) and 67 females (11.7%). Physical inactivity, either work-related or leisure time, was seen in 157 males (28.5%) and 130 females (22.7%). Hypertension (> or =140 and/or 90 mmHg) was present in 200 males (36.4%) and 215 females (37.5%). Diabetes diagnosed by history or fasting glucose > or =126 mg/dl was found in 72 males (13.1%) and 65 females (11.3%). Obesity, body mass index > or =27 kg/m2 was present in 135 males (24.5%) and 173 females (30.2%), while truncal obesity (waist:hip >0.9 males, >0.8 females) was found in 316 males (57.4%) and 392 females (68.4%). The most common dyslipidemia in both males and females was low HDL-cholesterol (<40 mg/dl: males 54.9%, females 54.2%). High total cholesterol levels of > or =200 mg/dl (males 37.4%, females 4.1%), high LDL-cholesterol levels of > or =130 mg/dl (males 37.0%, females 45.8%) and high levels of triglycerides > or = 150 mg/dl (males 32.3%, females 28.6%) were also seen in a significant number. Hypertension, obesity, truncal obesity, diabetes and dyslipidemias increased significantly with age in both males and females (Mantel-Haenzel chi2 for trend, p<0.05). CONCLUSIONS: There is a high prevalence of standard coronary risk factors--smoking, physical inactivity, hypertension, hypercholesterolemia, diabetes and obesity--as well as factors peculiar to south Asians--truncal obesity, low HDL-cholesterol and high triglycerides--in this urban Indian population. As compared to a previous study in the early 1900s in a similar population, there is a significant increase in the number of people with obesity, diabetes and dyslipidemias.

Oxidative stress as a mechanism of cardiac failure in chronic volume overload in canine model.

We investigated the effects of chronic volume overload in the absence or presence of vitamin E supplements on the cardiac function and contractility, cardiac malondialdehyde (MDA)--a lipid peroxidation product--cardiac antioxidant enzyme activity and antioxidant reserve in canine model. The dogs were divided into three groups of seven dogs each: group I, control; group II, mitral regurgitation (MR) of 4 months duration; and group III, MR of 4 months duration receiving vitamin E (40 U/kg/daily) orally. MR was created by detaching two or more chordae tendinae to raise left atrial pressure to 2.5 to three times normal. MR produced a decrease in the index of myocardial contractility with little change in myocardial function. Decrease in myocardial (left and right ventricles) contractility was associated with an increase in cardiac MDA, and a decrease in cardiac antioxidant reserve and antioxidant enzyme activity. Prevention of volume overload-induced decrease in myocardial contractility by vitamin E was associated with a decrease in cardiac MDA and an increase in cardiac antioxidant reserve and glutathione peroxidase activity towards control levels. Superoxide dismutase and catalase activity remained depressed in vitamin E-treated group. The results indicate that chronic volume overload decreases the contractility of both right and left ventricles and is associated with oxidative stress in both ventricles. These results support the hypothesis that oxygen free radicals are involved in the chronic volume overload-induced cardiac depression.

Platelet-activating-factor-induced changes in cardiovascular function and oxyradical status of myocardium in presence of the PAF antagonist CV-6209.

The effects of platelet-activating factor (PAF) on cardiac function and contractility and its mechanism of action are not fully understood. The authors investigated the effects of PAF in the absence or presence of a potent PAF antagonist CV-6209 on the cardiac function and contractility; lipid peroxidation product malondialdehyde (MDA), an indirect measure of oxygen free radicals; serum creatine kinase (CK); blood lactate; and pH in anesthetized dogs. CV-6209 (1 mg/kg, IV) did not produce significant changes in the various parameters studied. PAF (1 microgram/kg, IV) produced decreases in the cardiac function (cardiac index, left ventricular work index) and indices of cardiac contractility [(+) and (-) dp/dt, dp/dt at CPIP, (dp/dt)/IP, dp/dt at CPIP/IP, Vmax] and increases in the systemic and pulmonary vascular resistance. It also produced increases in cardiac MDA, serum CK, blood lactate, and H+ and decreases in blood pH and HCO3-. CV-6209 completely prevented the PAF-induced changes in the hemodynamic and biochemical parameters. These results suggest that PAF-induced cardiac depression may be due to PAF-induced release of oxyradicals from neutrophils and that PAF antagonist may be useful in counteracting the deleterious effects of PAF on the cardiovascular system.

Influence of hydroxyl radical on rabbit airway smooth muscle chronically exposed to H2O2 in vivo.

The effects of .OH on the isolated tracheal smooth muscles (TSM), from control, polyethylene glycol (PEG)-glucose oxidase (GO)-, and GO+PEG catalase-treated rabbits were investigated. GO or GO+catalase were given intravenously each week for 4 mo. .OH produced relaxation of basal and ACh-precontracted tension in TSM of control rabbits. The relaxant effect was attenuated by removal of epithelium, whereas it was converted to contraction in the indomethacin-pretreated muscle. .OH produced contraction of TSM and ACh-precontracted muscle in GO-treated rabbits. The contractile response was abolished in preparations denuded of epithelium or pretreated with indomethacin. .OH produced relaxation in basal tension, but a small contraction in ACh-precontracted muscle of GO+catalase-treated rabbits. The contractile response to .OH was unaffected by indomethacin pretreatment; however, it was converted to relaxation in the preparations denuded of epithelium. Contractile response of TSM to ACh was augmented in deepithelialized, indomethacin-, or GO-treated preparations. H2O2 damaged the tracheal epithelium. These results suggest that 1) .OH-induced relaxation/contraction of TSM is partly epithelium dependent and is mediated by bronchodilator/bronchoconstrictor arachidonic acid metabolites, 2) the airway smooth muscle with healthy epithelium responds to .OH differently from those with dysfunctional or damaged epithelium, and 3) hyperresponsiveness of the airways to ACh may be related to the epithelial dysfunction.

Mechanism of H2O2-induced modulation of airway smooth muscle.

We investigated the effects of H2O2 generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit tracheal smooth muscle suspended in Krebs-Ringer solution. H2O2 generated by G+GO was measured with luminol-dependent chemiluminescence. G+GO in the concentrations of 1x (1.80 microM G, 0.075 U/ml GO) and 2, 4, and 8x generated 1.35, 3.2, 6.10, and 6.00 microM of H2O2, respectively. H2O2 produced relaxation of rabbit tracheal smooth muscle, relaxed acetylcholine (ACh)-precontracted muscle, and reduced muscle responsiveness to ACh. These effects were concentration dependent. H2O2, however, produced contraction of guinea pig tracheal smooth muscle. Catalase completely inhibited the H2O2-induced relaxation of ACh-precontracted tracheal smooth muscle. H2O2-induced relaxation was greater in preparations with intact epithelium (65%) than in those denuded of epithelium (40%). The relaxant effects of H2O2 in the presence of an inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine), an inhibitor of guanylate cyclase (methylene blue), an inhibitor of cyclooxygenase (indomethacin), and an ATP-sensitive K+ channel blocker (glipizide) were 44, 44, 39, and 48%, respectively. H2O2-induced relaxation in the presence of indomethacin in preparations with denuded epithelium was 29%. These results suggest that H2O2-induced relaxation of tracheal smooth muscle is partly epithelium dependent and is mediated by inhibitory arachidonic acid metabolites, epithelium-derived relaxing factor (nitric oxide), ATP-sensitive K+ channels, and the synthesis and release of prostaglandins from epithelium and the underlying smooth muscle.

Detection of ischemia-reperfusion cardiac injury by cardiac muscle chemiluminescence.

Various methods have been used in the past to assess the implication of oxygen free radicals (OFR) in ischemia-reperfusion-induced cardiac injury. Luminol-enhanced tert-butyl-initiated chemiluminescence in cardiac tissue reflects oxidative stress and is a very sensitive method. It was used to elucidate the role of OFR in cardiac injury due to ischemia and reperfusion. Studies were conducted on perfused isolated rabbit hearts in three groups (n = 8 in each): I, control; II, submitted to global ischemia for 30 min; III, submitted to ischemia for 30 min followed by reperfusion for 60 min. The heart tissue was then assayed for chemiluminescence (CL); content of malondialdehyde (MDA), an indicator of OFR-induced cardiac injury; and activity of tissue levels of antioxidants [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)]. The control values for left and right ventricular CL and malondialdehyde were 81.1 +/- 15.4 (S.E.) and 182.4 +/- 50.3 (S.E.), mv.min.mg protein-1; and 0.024 +/- 0.006 (S.E.) and 0.324 +/- 0.005 (S.E.) nmoles.mg protein-1 respectively. Ischemia produced an increase in the cardiac CL (3.3 to 4.4 fold) and MDA content (2 to 2.6 fold). Reperfusion following ischemia also produced similar changes in CL and MDA content. The control values for activity of left ventricular SOD, catalase, and GSH-Px were 45.77 +/- 1.73 (S.E.) U.mg protein-1, 5.35 +/- 0.51 (S.E.) K.10(-3).sec-1.mg protein-1, and 77.50 +/- 7.70 (S.E.) nmoles NADPH.min-1.mg protein-1 respectively. Activities of SOD and catalase decreased during ischemia but were similar to control values in ischemic-reperfused hearts. The GSH-Px activity of left ventricle was unaffected by ischemia, and ischemia-reperfusion. GSH-Px activity of the right ventricle increased with ischemia, and ischemic-reperfusion. These results indicate that cardiac tissue chemiluminescence would be a useful and sensitive tool for the detection of oxygen free radical-induced cardiac injury.

Oxygen free radicals in volume overload heart failure.

It has been suggested that oxygen free radicals (OFR) depress the excitation-contraction coupling in cardiac muscle. It is possible that a decrease in the cardiac contractility in the failing heart may be due to an increased OFR producing activity of polymorphonuclear (PMN) leukocytes. We studied the OFR producing activity (chemiluminescence) of PMN leukocytes from blood in dogs with heart failure due to chronic volume overload. The animals were divided into two groups: I) normal, (n = 10): II) dogs with mitral insufficiency (MI) of 6 to 9 months duration, (n = 10). Hemodynamic studies were done to establish the presence of heart failure. Blood samples were collected to measure PMN leukocyte chemiluminescence. There was a decrease in the cardiac index and index of myocardial contractility (dp/dt/IIP) and an increase in the left ventricular end-diastolic pressure in dogs with MI indicating left ventricular failure. The peak chemiluminescent activity of the PMN leukocytes in blood of dogs with failure was about four folds greater than that in the blood from normal dogs. These results suggest that there may be an increased OFR generation in dogs with volume overload heart failure. The decrease in the myocardial contractility in the failing heart might be due to an increase in the OFR produced by the PMN leukocytes.

Observations on human bone-marrow for Leishmania donovani.

During the recent epidemic in North Bihar, a total of 65 cases which were clinically designated as Kala-azar had been subjected to bone-marrow aspiration from the iliac crest, out of which the L.D. body was demonstrated in 55 cases (84.61 per cent). Culture analysis of bone-marrow on modified Tobie's medium, revealed that ten cases (15.39 per cent) which were negative for L.D. body did not show any leptomonads, whereas out of 55 L.D. body positive cases, the promastigote forms developed in 22 cases (40.00 per cent). The probable reasons for the negative findings and also the age and sex distribution in Kala-azar are discussed in brief.

Effects of dilazep in evolving myocardial infarction in dogs: a biochemical study.

Mongrel dogs of either sex with ligation of their left anterior descending coronary artery were employed for the present study to investigate the biochemical basis of the cardioprotective action of dilazep. Dilazep (0.2 mg/kg intravenous) was administered 20 minutes after coronary arterial occlusion. Ligation of the artery for 40 minutes increased the tissue lactate concentration and decreased the adenosine triphosphate molecules within the occluded bed. Dilazep decreased the lactate concentration and improved the adenosine triphosphate content of this bed significantly. Dilazep also lowered the tissue cyclic adenosine monophosphate concentration and free fatty acid extraction of the ischemic myocardium as studied 40 minutes after coronary arterial ligation. It can be inferred that less free fatty acid extraction and calcium antagonistic action of dilazep helps in restoring mitochondrial function. Furthermore, decreased tissue lactate concentration resulted after better perfusion of the occluded bed and helped in greater generation of adenosine triphosphate molecules. These favorable biochemical and metabolic changes contribute to the cardioprotective action of dilazep.