Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus.

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (P<0.05). The neutralization potency of the product against the venom of Echis carinatus (E. carinatus) was about 7 LD50/ml (54.6 µg/ml) when tested on mice. Based on the results, the Fab fragment antivenom had the ability to neutralize the in vivo biological activity of the venom of Iranian E. carinatus. However, further studies are recommended to reach a suitable concentration of antivenom fragment.

Evaluation of the efficacy of a new oil-based adjuvant ISA 61 VG FMD vaccine as a potential vaccine for cattle.

Foot-and-mouth disease is an important viral disease of cloven-hoofed animals. Inactivated whole particle virus vaccines are still widely used in prophylactic vaccination campaigns. The choice of adjuvant is a very important factor in enhancing immune responses and the efficacy of inactivated vaccines. Montanide ISA 61 VG is a new ready-to-use mineral oil-based adjuvant developed by SEPPIC Inc. (SEPPIC, France) with high-potential immune responses needed for clinical protection against FMD infection. In this study, we compared the efficacy of two FMD vaccines either formulated with the new oil-based adjuvant ISA 61 VG and saponin, or with aluminum hydroxide gel and saponin. Both vaccines contained the same antigen payloads of O2010/IR. Two groups of 15 naive cattle received a single vaccination with different doses (full dose, 1/3 dose and 1/9 dose) to calculate their PD50 (50% protective dose) after being challenged with the homologous virulent virus. The mean neutralizing antibody titer was determined at 0, 7, 14 and 21 days after vaccination, measured by a micro neutralization test. The new vaccine improved humoral immune responses by 19%, while inducing a higher geometric mean. The titer for neutralizing antibodies was 2.91 log10 compared to the alum-gel based adjuvant vaccine which was 2.44 log10 (P-value=0.1782). The new vaccine showed a PD50 value of 10.05 as compared to a PD50 value of 4.171, respectively. According to the results, the FMD vaccine formulated with the new oil adjuvant, ISA 61 VG, shows potential as an alternative vaccine for routine and emergency vaccinations in the FMD enzootic region.

Differentially androgen-modulated genes in ovarian epithelial cells from BRCA mutation carriers and control patients predict ovarian cancer survival and disease progression.

Epidemiological studies have implicated androgens in the etiology and progression of epithelial ovarian cancer. We previously reported that some androgen responses were dysregulated in malignant ovarian epithelial cells relative to control, non-malignant ovarian surface epithelial (OSE) cells. Moreover, dysregulated androgen responses were observed in OSE cells derived from patients with germline BRCA-1 or -2 mutations (OSEb), which account for the majority of familial ovarian cancer predisposition, and such altered responses may be involved in ovarian carcinogenesis or progression. In the present study, gene expression profiling using cDNA microarrays identified 17 genes differentially expressed in response to continuous androgen exposure in OSEb cells and ovarian cancer cells as compared to OSE cells derived from control patients. A subset of these differentially affected genes was selected and verified by quantitative real-time reverse transcription-polymerase chain reaction. Six of the gene products mapped to the OPHID protein-protein interaction database, and five were networked within two interacting partners. Basic leucine zipper transcription factor 2 (BACH2) and acetylcholinesterase (ACHE), which were upregulated by androgen in OSEb cells relative to OSE cells, were further investigated using an ovarian cancer tissue microarray from a separate set of 149 clinical samples. Both cytoplasmic ACHE and BACH2 immunostaining were significantly increased in ovarian cancer relative to benign cases. High levels of cytoplasmic ACHE staining correlated with decreased survival, whereas nuclear BACH2 staining correlated with decreased time to disease recurrence. The finding that products of genes differentially responsive to androgen in OSEb cells may predict survival and disease progression supports a role for altered androgen effects in ovarian cancer. In addition to BACH2 and ACHE, this study highlights a set of potentially functionally related genes for further investigation in ovarian cancer.

Canister tip orientation and residual volume have significant impact on the dose of benzocaine delivered by Hurricaine spray.

Delivered quantities of 20% benzocaine spray (Hurricaine; Beutlich L.P. Pharmaceuticals, Waukegan, IL) are estimated by counting the number of sprays or the spraying time. Because Hurricaine spray supplies a continuous (albeit nonmetered) stream of benzocaine, neither method addresses delivered dose. We hypothesized that dose per time is a function of canister content and orientation. Thirty full canisters of Hurricaine were placed into three equal orientations (upright, inverted, or horizontal). Extrapolating from a full canister, four different estimates of benzocaine residual volume were determined before spraying out the contents (80%, 60%, 40%, and 20% full). Each canister was then sprayed for 10-s intervals, and the quantity delivered was calculated and compared statistically. Upright canisters 100% full emitted more benzocaine than canisters with residual volume 20% full (190 +/- 10 vs 172 +/- 10 mg/s). Inverted canisters emitted significantly less benzocaine from 100% full to residual volume 20% full (188 +/- 14 vs 70 +/- 10 mg/s). Oriented horizontally, two full canisters emitted <76 mg/s benzocaine, contrasted with the remaining eight in that group (186 +/- 20 mg/s). We conclude that the benzocaine (Hurricaine) sprayed in milligrams per second depends on canister content and orientation. When residual volumes diminish, there is a reduction in spraying volume per time. This diminution occurs progressively from larger to smaller residual volumes with canisters oriented horizontally, inverted, or upright. Arbitrary documentation of spraying time bears no relationship to dose delivered. Perhaps affixing an atomization device to a graduated syringe filled with benzocaine will help increase accuracy and precision in dosing.

Cholesterol inhibitory effects on human sperm-induced acrosome reaction.

Progesterone (P4) is known to induce an acrosome reaction in mammalian sperm in vitro, whereas cholesterol is a major inhibitor of acrosome reaction. This study had three objectives: to study the in vitro effects of exogenous cholesterol on acrosome reactions in human sperm, to study the mechanism by which cholesterol affects P4-induced acrosome reaction and those induced by dibutyryl cyclic adenosine monophosphate (db-cAMP), and to study the status of the P4 surface receptor during capacitation and acrosome reaction and its relationship with cholesterol and different acrosome reaction inducers. Acrosome reaction was induced with exposure to 10 microg/mL of P4 for 30 minutes and 1 mM of db-cAMP for 30 minutes in motile sperm either in the presence or absence of 0.1-1 microg/mL of cholesterol for 30 minutes. The effects of a 30-minute exposure to 1 microg/mL of beta-sitosterol, a cholesterol plant analogue, as well as the effects of cholesterol on P4-induced acrosome reactions were compared. Fluorescein isothiocyanate-labeled albumin-progesterone conjugate (P4-FITC-BSA) was used as the probe in order to quantify the percentage of sperm in which the P, surface receptor was exposed. The results of this study indicate that cholesterol inhibited P4-induced acrosome reactions when added to the sperm during capacitation (long incubation) and when it was added with P4 during the induction of acrosome reactions (short incubation). Similarly, acrosome reaction that was induced by db-cAMP was also inhibited by cholesterol. Fifty percent of P4-induced acrosome reaction was inhibited by a cholesterol concentration of 0.2 microg/mL. Cholesterol's inhibition of induced acrosome reaction was independent of P4 concentration. Beta-sitosterol inhibited P4-induced acrosome reaction in a dose-dependent manner that was identical to that of cholesterol. We observed that increases in the P4 surface receptor exposure were time-dependent and receptors migrated toward the equatorial segment during the first 2 hours of capacitation. We also found that db-cAMP induced the appearance of the P4 surface receptor in the sperm plasma membrane and that cholesterol inhibited it. The results of this study suggest that cholesterol inhibits acrosome reaction in a noncompetitive manner by modifying the structure of the sperm plasma membrane, which prevents exposure of the P4 surface receptor for P4 binding.

Current concepts in cardiopulmonary resuscitation in adults.

Cardiopulmonary resuscitation (CPR) provides artificial circulation and ventilation during cardiopulmonary arrest. CPR is further categorised as basic life support (BLS), advanced cardiac life support (ACLS) and postresuscitation support. BLS consists of provision of a patent upper airway, ventilation and circulation of blood by closed chest cardiac compressions. ACLS includes use of specialised equipment to maintain the airway, early defibrillation and pharmacologic therapy. Successful outcome from an arrest depends on the total duration of an arrest and early defibrillation, as ventricular fibrillation is the most common cardiac rhythm found in adult cardiac arrest. Initial drug therapy during CPR aims at correction of arterial hypoxaemia and restoring coronary and cerebral perfusion. Oxygen and epinephrine constitute the mainstay of drug therapy during CPR. In patients with ventricular tachycardia, lidocaine is the drug of choice, followed by bretylium. Magnesium has proved to be useful in both refractory pulseless ventricular tachycardia and fibrillation. Atropine has not been demonstrated to improve outcome from arrest but can be administered in bradyasystolic cardiac arrest. The routine administration of bicarbonate and calcium is no longer recommended but situations exist where they can be used appropriately. Administration of drugs during CPR should preferably be via a central route, but epinephrine, lidocaine and atropine can be administered via the endotracheal tube if intravenous access has not been established. Postresuscitation care includes mechanical ventilation if necessary to optimise oxygenation and ventilation and steps to maintain vital organ and optimal brain protection, which includes avoidance of hypertension, hypotension and hyperglycaemia.

Assessment of progesterone-induced acrosome reaction by biotinylated monoclonal antibody probes.

PROBLEM: To develop an immunohistochemical assay for determination of acrosome-reacted human sperm and to study the effects of progesterone and cholesterol treatment on human sperm acrosome reaction. METHOD OF STUDY: Three distinct anti-sperm monoclonal antibodies were biotinylated and used as probes for assessment of acrosome reaction in a 30-min immunohistochemical assay. Progesterone and/or cholesterol were added to sperm preparation to influence the acrosome reaction in different experimental conditions. RESULTS: Percentages of acrosome-intact sperm decreased significantly during the 18-hr incubation. Acrosome reaction could be induced by progesterone as early as 2 hr after sperm incubation in human tubal fluid. The degree of progesterone-induced acrosome reaction was time dependent and the optimal effect was reached by adding 10 micrograms/ml progesterone for 30 min incubation. Progesterone-induced acrosome reaction was shown to be hormone-concentration dependent with 50% stimulation at 1 microgram/ml. Cholesterol (1 microgram/ml) was found to inhibit progesterone-induced acrosome reaction either by co-incubation with human sperm during capacitation, or by simultaneous incubation with progesterone during acrosome reaction induction. CONCLUSIONS: Assessment of human sperm acrosomal status by avidin-biotin immunohistochemical assay can be a routine in clinical laboratories for male infertility services. Cholesterol can inhibit progesterone-induced acrosome reaction, possibly by its modifications of sperm plasma membrane and/or interference of progesterone binding to its surface receptors.

Bifurcated anastomosis.

Bifurcated anastomosis is a technique wherein geometrically tailored flaps of a vein graft are anastomosed to an arteriotomy in a vessel and its two branches. Although this technique may be useful for the surgical management of any diseased arterial bifurcation, it is particularly valuable in coronary endarterectomy or when complete revascularization of a coronary artery with proximal and bifurcation disease would otherwise require three separate bypass grafts.