Autoimmunity in Human CE: Correlative with The Fertility Status of The CE Cyst.

Cystic echinococcosis is speculated to exert several immune-evasion strategies involving autoimmune-phenomena. We evaluated the hypothesizes that the prevalence of autoantibodies increases in the sera of CE patients that may evidence the association between the parasite and autoimmune diseases. Sera from 63 subjects at distinct types of CE cyst fertility were investigated for antinuclear antibodies (ANA), and anti-CCP antibodies. Plasma levels and cellular production of IL-17A cytokine were specifically defined as being assumed to prime for autoimmunity. Healthy-controls were age and gender-matched to test sera. ANA expressions inside the surgically removed metacestode and adventitial layer were also assayed. Out of 63 patients, 35 % had fertile highly viable cysts (group-1), 41 % had fertile low viable cysts (group-2) and 24 % had non-fertile cysts (group-3). A four-fold increase in ANA sera-levels was detected in group-1 compared with their controls (p-value 0.001) while anti-CCP levels were of insignificant differences. In group-2 and group-3, no significant differences were detected between ANA and anti-CCP sera-levels in CE patients and their controls. IL-17A sera-levels in group-1 and group- 2 were significantly higher than their healthy-controls while being of insignificant differences in group-3, p-value= 0.300. No association was detected between sera-levels of IL-17A and ANA as well as anti-CCP antibodies. Interestingly, relative IL-17A cellular expression associated positive ANA deposition in the parasite cells and adventitial layer. Collectively, based on the parasite fertility, IL-17A and ANA seemed to be involved in the host immune defenses against CE. There is no association between CE and anti-CCP antibodies.

Preparation of a novel antivenom against Atractaspis and Walterinnesia venoms.

The two deadly snakes, Walterinnesia aegyptia (black desert cobra) and Atractaspis microlepidota (mole viper) share a common habitat in the central, eastern and western provinces of Saudi Arabia. Bites by either snake were characterized by rapid death, sometimes before reaching any medical facility. Confusing reports of "a black snake bite" are frequently found. The NAVPC had succeeded in preparing a highly effective antivenom against W. aegyptia venom which is now available in the market, but no antivenom against Atractaspis venom is found worldwide. This is probably because of the low molecular weight of sarafotoxins in the venom and hence their poor antigenic properties. At the NAVPC, sarafotoxins were separated by sequential gel filtration of A. microlepidota venom, while toxin T(III) of W. aegyptia venom obtained by cation exchange chromatography and gel filtration. Conjugation of the two toxins was carried out using glutaraldehyde in a two-step procedure followed by exhaustive dialysis. The conjugate was utilized to hyperimmunize 3-years old horses for 10 months, applying a low-dosage protocol and immunostimulants; the crude venoms of both snakes being added during the last 2 months. The F(ab')2 fraction of the antivenom was obtained by pH-guided salt precipitation, enzyme digestion and tangential desalting and filtration. The bivalent antivenom obtained protected mice and rats against the lethal effects of both venoms and rescued the rats challenged with lethal doses of the venoms in recovery experiments. It also neutralized the haemorrhagic, necrotizing and the cardiotoxic effects of A. microlepidota venom and the neuromuscular blocking effect of W. aegyptia venom. The antivenom offers a good rescue potential to those who are bitten by "a black snake" in Saudi Arabia.

Are the toxicological effects of scorpion envenomation related to tissue venom concentration?

The pharmacokinetics of 125I-labelled Androctonus amoreuxi venom and its lethal fraction was studied in rabbits. Comparative pharmacokinetic studies of labelled A. amoreuxi, Leisurus quinquestriatus and Buthotus judaicus venoms were carried out in guinea-pigs. The pharmacokinetics of A. amoreuxi venom was also studied in rats. Groups of rats were injected with labelled A. amoreuxi venom and killed at frequent time intervals for the determination of the relative tissue venom concentration as a function of time. Several groups of rabbits were injected with A. amoreuxi venom and serial blood samples withdrawn at time intervals comparable with those used in the pharmacokinetic studies for the determination of serum glucose, insulin, cortisol, total proteins, albumin, globulins, cholesterol, total bilirubin, urea, uric acid, bicarbonate, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, sodium, potassium, calcium and phosphorus. The packed cell volume, and total and differential leucocyte counts were also determined. In another series of experiments continuous monitoring of the electrocardiograms of rabbits following venom injection was made to correlate any abnormalities with tissue venom concentration. All three venoms and the lethal fraction showed an open two-compartment behaviour with rapid distribution half-lives ranging between 4 and 7 min and overall elimination half-lives of 4.2 to 13.4 hr. The behaviour of A. amoreuxi venom was not markedly different in the three species of animals used. In a given species (guinea-pigs) the behaviour of the three venoms was not markedly different. Correlation of the ECG changes with cardiac venom concentration showed that arrhythmias and infarction occurred at times when cardiac concentration was very low, indicating that the cardiac abnormalities might result from indirect factors. Comparison of the course of the biochemical changes with venom concentration in the central compartment indicated that the site of action of the venom is not located in the central compartment. Correlation of the intensity of the biochemical effects with venom concentration in the peripheral compartment revealed an apparent delay in the onset and peak of action. This was explained by assuming that the tissue compartment could be divided into a rapidly accessible and a slowly accessible compartment with the venom acting through the slowly accessible compartment. There was also the possibility of the venom acting indirectly through the release of other substances or transformation to an intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics of 125I-labelled IgG, F(ab')2 and Fab fractions of scorpion and snake antivenins: merits and potential for therapeutic use.

The immunoglobulin fractions IgG, F(ab')2 and Fab of scorpion and snake antivenoms possess pharmacokinetic characteristics that are significantly different from their respective venoms. The venoms (and their toxins) are several fold faster in their distribution into the tissues than any of the immunoglobulin fraction. In rabbits, F(ab')2 possessed the fastest disposition rate constants and the longest distribution half lives. In the physiologically based pharmacokinetic experiments carried out in mice F(ab')2 possessed the highest Cp(max), smallest AUC and the shortest t1/2beta in the different tissues while Fab had values in between IgG and F(ab')2. Rescue experiments in anaesthetized rats challenged with lethal doses of venoms or toxins and infused with border-line neutralizing doses of antivenoms, showed that rats infused with F(ab')2 completely recovered, those infused with IgG partially rescued and none of the rats infused with Fab survived. It is concluded that F(ab')2 of scorpion and snake antivenoms possess pharmacokinetic characteristics that render it the most suitable for use in serotherapy of scorpion and snake envenoming.

Androctonus crassicauda (Olivier), a dangerous and unduly neglected scorpion--I. Pharmacological and clinical studies.

Androctonus crassicauda venom has an i.v. LD50 in mice of 0.32 +/- 0.02 mg/kg, which makes the scorpion among the most toxic species in the world. Fifty-one non-fatal and one fatal cases of scorpion sting were presented. Pain and tenderness were very common following the sting. Generalized erythema occurred in 20-25% of all infants and children below the age of 5 years. Severe CNS manifestations including seizures, unconsciousness and marked irritability occurred mainly in infants and young children, while hypertension occurred in the majority of victims below the age of 11 years. Two pregnant victims were treated with antivenom with no bad consequences on mothers or foetuses. The fatal case described was inadequately treated with antivenom and presented a rare situation of intracranial coagulation in the basal cisterns or low in the cranial subarachnoid space. The victim developed moderate hydrocephalus of the communicating type with clear ventricular CSF and strongly xanthocromic fluid from lumbar puncture. The effects of A. crassicauda venom on isolated hearts, atria and anaesthetized rat blood pressure appeared to be mediated largely through stimulation of the autonomic nervous system with predominance of sympathetic stimulation and release of tissue catecholamines. Electrocardiograms recorded simultaneously with blood pressure changes showed evidence of ectopic foci during the hypertensive phase and ischaemia, inferior wall infarction and different degrees of heart block during the late hypotensive phase. Androctonus crassicauda venom was unique in following a three-compartment open model comprising a central compartment 'blood', a rapidly equilibrating 'shallow' tissue compartment and a slowly equilibrating 'deep' tissue compartment. The overall elimination half-life, t1/2 beta, was 24 hr, indicating that the venom has the slowest elimination among all known scorpion venoms. The long stay of the venom in the body might explain the increased risk of toxicity and the good potential for treatment with serotherapy even hours after the sting.

Pharmacokinetics of 125I-labelled Walterinnesia aegyptia venom and its distribution of the venom and its toxin versus slow absorption and distribution of IGG, F(AB')2 and F(AB) of the antivenin.

A three-compartment open pharmacokinetic model best fitted the data obtained following the i.v. injection of the venom, toxin and the immunoglobulin fractions into either rabbits or mice. The venom and toxin, however, possessed pharmacokinetic characteristics that were significantly different from the immunoglobulin fractions. The venom and toxin had very highly significantly greater disposition rate constants to the shallow and deep tissue compartments and overall elimination rate constant from the central compartment than any of the immunoglobulin fractions. This was reflected in other pharmacokinetic parameters, including highly significantly smaller areas under the curve (AUC) and highly significantly greater volumes of the central compartment (Vc), shallow tissue compartment (Vt shallow), deep tissue compartment (Vt deep) and total body clearance (TBC). In rabbits, F(ab')2 possessed the fastest disposition rate constants and the shortest distribution half-lives, while Fab showed the slowest disposition rate constants and the longest distribution half-lives. The same picture occurred in mice except that the values for Fab were between those of F(ab')2 and IgG. The time needed by the venom and toxin to reach maximum tissue concentration (tmax) ranged between 7 and 15 min and 60 and 180 min for the shallow and deep tissue compartments, respectively. The immunoglobulin fractions required 8-26-fold these times to attain tmax; F(ab')2 was the fastest to achieve its maximal concentration. Following i.m. injection, very fast absorption of venom and toxin took place, with the toxin reaching tmax within 5-20 min and 90% of the injected dose absorbed within 60 min. The bioavailability factor (F) was 0.82 and 0.88 for the venom and toxin, respectively. Fab had an F-value of 0.36 and required 4.3 and 47.4-fold the time taken by the venom and toxin to achieve tmax. The calculated values of F for F(ab')2 and IgG were 0.25 and 0.26, respectively. In the physiologically based pharmacokinetics (PBPK), the venom and toxin reached tmax in the different organs studied very rapidly while the immunoglobulin fractions required several-fold this time to attain tmax. F(ab')2 possessed the highest CPmax, the smallest AUC and the shortest t1/2 beta in the different tissues; Fab had values between F(ab)2 and IgG. It is concluded that F(ab')2 possesses pharmacokinetic characteristics that render it most suitable for use in serotherapy of snake and scorpion envenoming. It should be injected i.v. in doses higher than calculated neutralizing doses to compensate for the slow rate of distribution. Because of slow and incomplete absorption, the i.m. injection of the immunoglobulin fractions would be of little value in serotherapy.

Do changes in body temperature following envenomation by the scorpion Leiurus quinquestriatus influence the course of toxicity?

Four fatal cases following scorpion sting in children are presented. Two victims had rectal temperature above 41 degrees C, the third exhibited a temperature of 40.9 degrees C from the combined effects of scorpion sting and heat stroke, while the fourth was hypothermic. All victims developed hypothermia 48 hr following the sting. The hyperthermia was effectively treated by acetaminophen suppositories, ice packs and water sponges. All victims showed late hypotension that was refractory to dopamine infusion. This was explained by bradykinin released by the venom blocking the dopaminergic receptors. Deterioration of the cortical activity of the victims maintained on mechanical ventilation before the incidence of asystole suggests a central component in the cardiovascular manifestations of envenomation. A. amoreuxi venom was selected as a model for the pharmacokinetic and quantitative toxicological studies since it has no effect on body temperature. In hyperthermic rabbits injected with labelled lethal fraction of A. amoreuxi venom, there was a significant decrease in the elimination half-life, t1/2 beta, the apparent volume of the tissue compartment, Vt, the apparent volume of distribution, Vdss, and the intercompartmental rate constant, kCT. Hypothermic rabbits showed a significant decrease in the apparent first-order elimination rate constant, kd, and a significant increase in the elimination half-life. In both states a higher concentration of the lethal fraction in the blood was calculated. This would explain the rapidity of onset of the electrocardiographic effects and the decreased survival time in both the hyperthermic and hypothermic rabbits injected with venom when compared to normothermic animals. The s.c. LD50 in mice and the i.v. MLD in rats were significantly reduced in the hypothermic mice and hypothermic and hyperthermic rats.

A three-compartment open pharmacokinetic model can explain variable toxicities of cobra venoms and their alpha toxins.

The pharmacokinetic profiles of labelled Naja melanoleuca, Naja nivea, Naja nigricollis and Naja haje venoms and their alpha neurotoxins were determined following rapid i.v. injection into rabbits. The data obtained fitted a triexponential equation characteristic of a three-compartment open pharmacokinetic model comprising a central compartment 'blood', a rapidly equilibrating 'shallow' tissue compartment and a slowly equilibrating 'deep' tissue compartment. The distribution half-lives for the shallow compartment ranged from 3.2 to 5 min, reflecting the rapid uptake of venoms and toxins compared with 22-47 min for the deep tissue compartment denoting much slower uptake. The overall elimination half-lives, t1/2 beta, ranged from 15 to 29 hr, indicating a slow body elimination. Peak tissue concentration was reached within 15-20 min in the shallow tissue compartment. The corresponding values for the deep tissue compartment were 120 min for N. melanoleuca and N. nigricollis venoms and their toxins and 240 min for N. nivea and N. haje venoms and their toxins. Steady-state distribution between the shallow tissue compartment and the blood gave values of 0.50 and 0.92 (N. melanoleuca), 1.64 and 1.05 (N. nivea), 0.78 and 0.92 (N. nigricollis) and 1.70 and 1.03 (N. haje) for the venoms and their toxins, respectively. The corresponding values for the deep tissue compartment gave ratios of 3.31 and 3.44 (N. melanoleuca), 2.99 and 1.68 (N. nivea), 3.74 and 3.79 (N. nigricollis) and 1.39 and 2.46 (N. haje) for the venoms and their toxins, respectively. Ratios lower than unity indicate lower venom and toxin concentrations in the tissues than in the blood, while larger ratios denote higher tissue concentrations. The values thus reflect a higher affinity of the venoms and their toxins for the central than the shallow tissue compartment and for the deep tissue than the central compartment. The sites of action of the venoms seem to be located in the deep tissue compartment since most of the pharmacological, biochemical and electrocardiographic effects of the venoms started 30-60 min after i.v. injection. The mean residence time in the body, MRTb, ranged from 20.8 to 51.8 hr, which correlated well with the long duration of the pharmacological and biochemical effects induced by the venoms. The tissue distribution of the venoms and toxins was similar, with the highest uptake being in the kidneys, followed by the stomach, lungs, liver, spleen, intestine, heart and diaphragm. Very high radioactivity was found in the stomach contents, which reached values higher than the kidneys. Some of the biochemical markers were significantly changed by one or more venoms but the grouped parameters did not reflect significant changes in cardiac, renal, hepatic or electrolyte profiles as a function of time. It is concluded that antivenom, even if injected several hours after a cobra bite, is still capable of neutralizing the slowly eliminating venom. To speed up neutralization of the venom effects, doses of antivenom higher than the calculated in vitro neutralizing dose ought to be injected to compensate for the slow rate of transfer of antivenom to the tissues.

Bosentan, a selective and more potent antagonist for Atractaspis envenomation than the specific antivenom.

Preparation of an antivenom against Atractaspis had been done for the first time in the year 2007 in NAVPC in Riyadh, however, it was a lengthy and expensive process. In this study, an alternative treatment was tested using: 1- Nitroglycerin to antagonize the coronary vasospasm induced by the venom, 2- Bosentan to block the endothelin receptors since there is a similarity in structure and effect between the toxic fraction of venom (Sarafotoxins) and endothelins and 3- The specific Antivenom in comparison to nitroglycerin and bosentan. Pretreatment of rabbits with nitroglycerin, antivenom or bosentan completely protected all rabbits from the minimal lethal doses of venom or its toxic fraction. On the other hand, injecting any of the three drugs a few minutes (min) after injecting one minimal lethal dose (MLD) of the venom or the toxic fraction and at the first signs of ischemia, just before death, showed that bosentan completely saved all rabbits. In case of nitroglycerin all rabbits died and in case of antivenom, only 5 out of 10 rabbits were rescued. It is clear that bosentan is superior to the specific antivenom in protecting rabbits; this may be due to its higher affinity to endothelin receptors than sarafotoxins. This preclinical study shows a good potential in using bosentan as a selective antidote for atractaspis envenomation, especially in the African continent.

What is the optimum concentration of m-cresol in antivenoms?

Antivenoms against snake and scorpion envenomations are usually equine in nature and composed mostly of F(ab')2; additionally, phenol and m-cresol are mainly employed for their preservation. Although there is no study on this subject, m-cresol is utilized by most manufacturers in a concentration that ranges from 0.15 to 0.35 g percent. Decreasing the concentration of m-cresol to its minimal effective level may protect victims from its toxic effects and keep the antivenom stable during its shelf life without forming any aggregates. In the present work, different concentrations of m-cresol, ranging from 0.1 to 0.35 g percent, were used with some selected batches of snake and scorpion antivenoms. A low concentration of 0.15 g percent showed an acceptable preserving result that complies perfectly with antimicrobial specifications stated by the British Pharmacopoeia. Tested antivenoms (in 12 batches), when kept in a cold room for 39 months (more than their shelf life), retained their physical, chemical and microbiological activities according to the specifications of pharmacopeias. The present data demonstrated that reduction of m-cresol concentration to 0.15 g percent in case of equine F(ab')2 antivenoms will improve safety of such preparations and preserve their stability during their shelf life.(AU)