Stabilization and formulation of a recombinant Human Cytomegalovirus vector for use as a candidate HIV-1 vaccine

Live attenuated viral vaccine/vector candidates are inherently unstable and infectivity titer losses can readily occur without defining appropriate formulations, storage conditions and clinical handling practices. During initial process development of a candidate vaccine against HIV-1 using a recombinant Human Cytomegalovirus vector (rHCMV-1), large vector titer losses were observed after storage at 4 °C and after undergoing freeze-thaw. Thus, the goal of this work was to develop candidate frozen liquid formulations of rHCMV-1 with improved freeze-thaw and short-term liquid stability for potential use in early clinical trials. To this end, a virus stability screening protocol was developed including use of a rapid, in vitro cell-based immunofluorescence focus assay to quantitate viral titers. A library of ∼50 pharmaceutical excipients (from various known classes of additives) were evaluated for their effect on vector stability after freeze-thaw cycling or incubation at 4 °C for several days. Certain additives including sugars and polymers (e.g., trehalose, sucrose, sorbitol, hydrolyzed gelatin, dextran 40) as well as removal of NaCl (lower ionic strength) protected rHCMV-1 against freeze-thaw mediated losses in viral titers. Optimized solution conditions (e.g., solution pH, buffers and sugar type) slowed the rate of rHCMV-1 titer losses in the liquid state at 4 °C. After evaluating various excipient combinations, three new candidate formulations were designed and rHCMV-1 stability was benchmarked against both the currently-used and a previously reported formulation. The new candidate formulations were significantly more stable in terms of reducing rHCMV-1 titer losses after 5 freeze-thaw cycles or incubation at 4 °C for 30 days. This case study highlights the utility of semi-empirical design of frozen liquid formulations of a live viral vaccine candidate, where protection against infectivity titer losses due to freeze-thaw and short-term liquid storage are sufficient to enable more rapid initiation of early clinical trials.     

Sodium chloride (NaCl) potentiates digoxin-induced anti-tumor activity in small cell lung cancer

Small cell lung cancer (SCLC) is a malignant neuroendocrine tumor with very high mortality. Effective new therapy for advanced SCLC patients is urgently needed. By screening a FDA-approved drug library, we identified a cardiac glycoside (CG), namely digoxin (an inhibitor of cellular Na⁺/K⁺ ATPase pump), which was highly effective in inhibiting SCLC cell growth. Intriguing findings showed that NaCl supplement markedly enhanced the anti-tumor activities of digoxin in both in vitro and in vivo models of SCLC. Subsequent analysis revealed that this novel combination of digoxin/NaCl caused an up-regulation of intracellular Na⁺ and Ca²⁺ levels with an induction of higher resting membrane potential of SCLC cells. We also found that this combination lead to morphological shrinking of SCLC cells, together with high levels of cytochrome C release. Lastly, our data revealed that NaCl supplement was able to induce the expression of ATP1A1 (a Na⁺/K⁺ ATPase subunit), in which contributes directly to the increased sensitivity of SCLC cells to digoxin. Thus, this is the first demonstration that NaCl is a potent supplement necessitating superior anti-cancer effects of digoxin for SCLC. Further, our study suggests that digoxin treatment could need to be combined with NaCl supplement in future clinical trial of SCLC, particularly where low Na⁺ is often present in SCLC patients.     

Recipient hypertonic saline infusion prevents cardiac allograft dysfunction

Objective

Hypertonic saline (HTS) has potent immune and vascular effects. We assessed recipient pretreatment with HTS on allograft function in a porcine model of heart transplantation and hypothesized that HTS infusion would limit endothelial and left ventricular (LV) dysfunction following transplantation.

GVHD after transfusion of stored RBC concentrates in a solution of mannitol, adenine, phosphate, citrate, glucose, and NaCl following trauma

BACKGROUND: It has not previously been reported that WBC-reduced RBC preparations can cause transfusion-associated GVHD, even in an immunocompetent individual. CASE REPORT: A 74-year-old man suffered a hemorrhage from the mesentery of the transverse colon after a traffic accident. During surgery, he received 10 units of RBCs from 10 donors in a solution containing mannitol, adenine, phosphate, citrate, glucose and NaCl (MAP). MAP RBCs had been stored for 7 to 8 days before use. On the 27th day after surgery, an erythematous, pruritic rash appeared over the face, neck, and trunk, which was associated with low-grade fever and pancytopenia. Transfusion-associated GVHD was strongly suspected and was confirmed by skin biopsy. To determine the origin of lymphocytes causing GVHD, several microsatellite loci were amplified from DNA of the patient's nails and blood and from blood samples of all 10 RBC donors by using PCR. Amplified alleles derived from the patient's blood were identical to those from one of the 10 samples. CONCLUSION: These findings indicate that transfusions of MAP-RBCs can cause transfusion-associated GVHD in an elderly but immunocompetent host.     

A high-concentration NaCl solution does not stimulate the human trigeminal nerve at the tip of the tongue

Conclusion. A 3 M NaCl solution does not stimulate the trigeminal nerve in the human tongue. Objectives. In rats, the trigeminal nerve has been reported to respond when the tongue is stimulated by a solution with an NaCl concentration of 0.4 M or greater. We have attempted to clarify whether or not relatively high concentrations of NaCl stimulate the trigeminal nerves of the human tongue. Materials and methods. We examined four patients whose bilateral chorda tympani nerves were resected during middle ear surgeries. We performed subjective tactile and taste tests. Next, we conducted objective examinations of the subjects’ tactile and gustatory functions by magnetoencephalography (MEG). Results. The subjective examination confirmed that all four subjects maintained normal tactile sensory functions in their tongues and that the gustatory sensation at their lingual apexes was totally abolished. Furthermore, the objective examination of the tactile function using MEG indicated that their brain responses to trigeminal nerve stimulations were normal. Further examination using MEG failed to produce brain responses to a 3 M NaCl solution in spite of their normally functioning trigeminal nerves. Therefore, we concluded that a 3 M NaCl solution does not stimulate the trigeminal nerve at the tip of the human tongue.     

Body Fluid Variations and Endogenous Digitalis-Like Compounds During Chronic NaCl Loading in Wistar Rats

Circulating digitalis-like compounds have been proposed to be raised in volume expanded hypertension and to participate in Na⁺ homeostasis. We have investigated the temporal relationships between the activity of these circulating digitalis-like compounds, blood pressure and body fluid volume variations during a chronic NaCl load in the Wistar rat.

Characteristics of salt-loaded rats were compared to those of weight-matched controls. At one week, when extracellular fluid volume (ECFV) was elevated, the capacity of plasma extracts to inhibit the Na⁺K⁺ATPase activity begun to rise. At two weeks, ECFV remained elevated, and plasma volume, blood pressure and the activity of plasma digitalis-like compounds increased. After 13 weeks, the continuous rise in plasma digitalis-like activity and in blood pressure was accompanied by the return of body fluid volumes towards control values. These changes in plasma digitalis-like activity, body fluid volumes, and systolic blood pressure during a high NaCl diet are compatible with the proposed role of circulating digitalis-like compounds as natriuretic and hypertensive factors.     

Short-term effects of electronic and tobacco cigarettes on exhaled nitric oxide

The objective of this study was to compare the short-term respiratory effects due to the inhalation of electronic and conventional tobacco cigarette-generated mainstream aerosols through the measurement of the exhaled nitric oxide (eNO). To this purpose, twenty-five smokers were asked to smoke a conventional cigarette and to vape an electronic cigarette (with and without nicotine), and an electronic cigarette without liquid (control session).     

3%氯化钠联合喜炎平治疗重症手足口病合并脑炎50例疗效观察

目的 研究3%氯化钠联合喜炎平治疗重症手足口病（HFMD）合并脑炎50例的临床疗效。方法 选取重症手足口病合并脑炎的患者100例,随机分为研究组和对照组,每组50例,对照组给予喜炎平,研究组给予3%氯化钠联合喜炎平,比较两组的临床疗效、退热时间、意识恢复时间以及住院时间。结果 研究组总有效率92.0%（46/50）显著高于对照组的总有效率80.0%（40/50）,两组比较差异具有统计学意义（P〈0.05）,研究组退热时间、意识恢复时间以及住院时间显著短于对照组,两组比较差异具有统计学意义（P〈0.05）。结论 3%氯化钠联合喜炎平治疗重症手足口病合并脑炎具有较好的临床疗效。     

Renal conservation of ketone bodies during starvation.

Renal handling of acetoacetate and beta-hydroxybutyrate was studied in 12 obese subjects undergoing total starvation. Simultaneously, the acetoacetate, beta-hydroxybutyrate, and inulin clearance rates were measured, and acetoacetate and beta-hydroxybutyrate reabsorption rates were calculated. Renal clearance of blood acetoacetate and beta-hydroxybutyrate remained constant. In contrast, acetoacetate reabsorption rate increased significantly from 47 plus or minus 10 mumoles/min on day 3 to 106 plus or minus 15, 89 plus or minus 10, and 96 plus or minus 10 mumoles/min on days 10, 17, and 24, respectively. Similarly, beta-hydroxybutyrate reabsorption rate increased significantly from 154 plus or minus 27 mumoles/min on day 3 to 419 plus or minus 53, 399 plus or minus 25, and 436 plus or minus 53 mumoles/min on days 10, 17, and 24, respectively. Both acetoacetate and beta-hydroxybutyrate reabsorption rates increased linearly when plotted against their filtered loads. Thus, no tubular maximal transport rate exists for acetoacetate or beta-hydroxybutyrate during physiologic ketonemia. Conservation 450-500 mmoles of ketone bodies/day prevents large urinary losses of cations during prolonged starvation. Since ammonium becomes the major cation excreted during prolonged fasting, the increased renal reabsorption of ketone bodies minimizes body protein loss and aids in maintaining high circulating acetoacetate and beta-hydroxybutyrate concentrations.     

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses

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