Robert W. Wannemacher

Gluconeogenesis, Ureagenesis and Ketogenesis During Sepsis.

Abstract : Acute sepsis is characterized by an accelerated catabolism of somatic proteins, an increased rate of oxidation and transamination of branched-chain amino acids within muscle, an enhanced formation of lactic acid, alanine and glutamine in muscle, an enhanced flux of these substrates from muscle to liver, and an acceleration of the synthesis and release of glucose from the liver. Endocrine responses which influence these metabolic changes include an enhanced secretion of both insulin and glucagon from pancreatic islet cells and a resultant decrease in the molar ratio of insulin to glucagon in plasma. The secretion of the adrenal glucocorticoids and growth hormone are increased as may be the secretion of catecholamines. The accelerated release of glucose from the liver is generally accompanied by an increase in glucose pool size and an increase in the rate of glucose utilization as a cellular fuel. The hormonal, enzymatic, and substrate interrelationships within the liner combine in their effects to inhibit Ketogenesis and stimulate hepatic lipogenesis during acute sepsis.

In vivo effects of T-2 mycotoxin on synthesis of proteins and DNA in rat tissues.

Rats were given an ip injection of T-2 mycotoxin (T-2), the T-2 metabolite, T-2 tetraol (tetraol), or cycloheximide. Serum, liver, heart, kidney, spleen, muscle, and intestine were collected at 3, 6, and 9 hr postinjection after a 2-hr pulse at each time with [14C]leucine and [3H]thymidine. Protein and DNA synthesis levels in rats were determined by dual-label counting of the acid-precipitable fraction of tissue homogenates. Rats given a lethal dose of T-2, tetraol, or cycloheximide died between 14 and 20 hr. Maximum inhibition of protein synthesis at the earliest time period was observed in additional rats given the same lethal dose of the three treatments and continued for the duration of the study (9 hr). With sublethal doses of T-2 or tetraol, the same early decrease in protein synthesis was observed but, in most of the tissues, recovery was seen with time. In the T-2-treated rats. DNA synthesis in the six tissues studied was also suppressed, although to a lesser degree. With sublethal doses, complete recovery of DNA synthesis took place in four of the six tissues by 9 hr after toxin exposure. The appearance of newly translated serum proteins did not occur in the animals treated with T-2 mycotoxin or cycloheximide, as evidenced by total and PCA-soluble serum levels of labeled leucine. An increase in tissue-pool levels of free leucine and thymidine in response to T-2 mycotoxin was also noted. T-2 mycotoxin, its metabolite, T-2 tetraol, and cycloheximide cause a rapid inhibition of protein and DNA synthesis in all tissue types studied. These results are compared with the responses seen in in vitro studies.

Fate and distribution of 3H-labeled T-2 mycotoxin in guinea pigs

T-2 toxin is a potent cytotoxic metabolite produced by the Fusarium species. The fate and distribution of 3H-labeled T-2 toxin were examined in male guinea pigs. Radioactivity was detected in all body tissues within 30 min after an im injection of an LD50 dose (1.04 mg/kg) of T-2 toxin. The plasma concentration of trichothecene molar equivalents versus time was multiphasic, with an initial absorption half-life equal to or less than 30 min. Bile contained a large amount of radioactivity which was identified as HT-2, 4-deacetylneosolaniol, 3-hydroxy HT-2, 3-hydroxy T-2 triol, and several more-polar unknowns. These T-2 metabolites are excreted from liver via bile into the intestine. Within 5 days, 75% of the total radioactivity was excreted in urine and feces at a ratio of 4 to 1. The appearance of radioactivity in the excreta was biphasic. Metabolic derivatives of T-2 excreted in urine were T-2 tetraol, 4-deacetylneosolaniol, 3-hydroxy HT-2, and several unknowns. These studies showed a rapid appearance in and subsequent loss of radioactivity from tissues and body fluids. Only 0.01% of the total administered radioactivity was still detectable in tissues at 28 days. The distribution patterns and excretion rates suggest that liver and kidney are the principal organs of detoxication and excretion of T-2 toxin and its metabolites.

Relationship between serum chromium concentrations and glucose utilization in normal and infected subjects.

Studies in healthy individuals demonstrate that serum chromium concentrations fall precipitously following the intravenous administration of a 30-gm. glucose load. Significant decreases from baseline control fasting serum Cr concentrations were also observed when intravenous glucose was given during sandfly fever. Glucose disappearance rates also decreased significantly to approximately one half of pre-illness control values while serum Cr values declined still further. In addition, serum Cr disappearance rates could be calculated. When individual preexposure and postexposure serum glucose and Cr disappearance rates were compared, a significant linear correlation was found (P <0.05). Acute infection appears to reduce the availability of circulating Cr, which may contribute to the altered glucose metabolism characteristic of acute infections even in the presence of elevated insulin levels and other hormonal changes.

[3H]-Saxitoxinol metabolism and elimination in the rat

Tritiated saxitoxinol was used to obtain preliminary information on saxitoxin metabolism in the rat. Sublethal doses of tritiated saxitoxinol (18.9-microCi/kg; 3.8 micrograms/kg) were injected i.v. into each of six rats. Urine and fecal samples were collected up to 144 hr post-injection. Within 4 hr, 60% of injected radioactivity was excreted in urine. No radioactivity was found in feces. High performance liquid chromatography analyses of urine showed that saxitoxinol was not metabolized by the rats.

Process development and cGMP manufacturing of a recombinant ricin vaccine: An effective and stable recombinant ricin a‐chain vaccine—RVEc™

Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A‐chain (RTA), which is an N‐glycosidase enzyme, and the B‐chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1‐33/44‐198 protein, herein denoted RVEa™) expressed in Escherichia coli using a codon‐optimized gene was shown to be non‐toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale‐up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)‐compliant production of RVEc™ at the 40 L scale. The average yield of the final purified bulk RVEc™ is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc™ was >99% pure by three HPLC methods and SDS‐PAGE. The intact mass and peptide mapping analysis of RVEc™ confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc™ was immunoprotective against lethal ricin challenge and elicited neutralizing anti‐ricin antibodies in 95–100% of the vaccinated mice. Published 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.

The hemostatic derangement produced by T-2 toxin in cynomolgus monkeys.

T-2 toxin, a mycotoxin produced by several strains of the genus Fusarium, has been implicated as a cause of serious illness in both animals and man. Hemorrhage is a feature of the syndromes which have been described. An LD20 dose of T-2 was administered im to adult cynomolgus monkeys. This resulted in prolongation of prothrombin and activated partial thromboplastin times and a decrease in multiple coagulation factors. These changes were detected within hours of toxin administration, were maximal at 24 hr, and returned to normal over the next 3 days. Fibrin-fibrinogen degradation products were not detected at any time point. Repeated phlebotomy produced a significantly greater increase in platelet count in control monkeys, which could be taken as evidence for an effect of toxin on platelet kinetics. In treated animals, the hematocrit level declined by about 10%, but a similar decrease occurred in control animals. The white blood cell count increased 4 to 5 times over pretreatment values. Despite the changes in multiple laboratory parameters, treated monkeys did not exhibit clinical evidence of hemorrhage. In three animals which died as a result of toxicosis, necropsy revealed mild petechial hemorrhage involving the colon and heart, as well as necrosis of lymphoid tissues.