Safety of snake antivenom immunoglobulins: efficacy of viral inactivation in a complete downstream process.

Viral safety remains a challenge when processing a plasma-derived product. A variety of pathogens might be present in the starting material, which requires a downstream process capable of broad viral reduction. In this article, we used a wide panel of viruses to assess viral removal/inactivation of our downstream process for Snake Antivenom Immunoglobulin (SAI). First, we screened and excluded equine plasma that cross-reacted with any model virus, a procedure not published before for antivenoms. In addition, we evaluated for the first time the virucidal capacity of phenol applied to SAI products. Among the steps analyzed in the process, phenol addition was the most effective one, followed by heat, caprylic acid, and pepsin. All viruses were fully inactivated only by phenol treatment; heat, the second most effective step, did not inactivate the rotavirus and the adenovirus used. We therefore present a SAI downstream method that is cost-effective and eliminates viruses to the extent required by WHO for a safe product.

Trends in adjuvant development for vaccines: DAMPs and PAMPs as potential new adjuvants

Aluminum salts have been widely used in vaccine formulations and, after their introduction more than 80 years ago, only few vaccine formulations using new adjuvants were developed in the last two decades. Recent advances in the understanding of how innate mechanisms influence the adaptive immunity opened up the possibility for the development of new adjuvants in a more rational design. The purpose of this review is to discuss the recent advances in this field regarding the attempts to determine the molecular basis and the general mechanisms underlying the development of new adjuvants, with particular emphasis on the activation of receptors of innate immune recognition. One can anticipate that the use of these novel adjuvants will also provide a window of opportunities for the development of new vaccines.

Cloning of an unusual natriuretic peptide from the South American coral snake Micrurus corallinus.

In the course of cloning abundant cDNAs from the South American coral snake Micrurus corallinus venom gland, we characterized a cDNA coding for a putative natriuretic peptide. All the natural natriuretic peptides described so far, possess a ring structure composed of 17 amino acids formed through an S-S bridge which is extended at the N-terminus by few to several amino acids and may be extended at the C-terminus, usually 4-7 amino acids. In contrast, the M. corallinus natriuretic peptide presents several distinct features: (a) the proform of the deduced natriuretic peptide displays an unusual C-terminus extension. This implies that the mature peptide has a long C-terminal tail or it is further extensively processed to result in the mature natriuretic peptide with the expected 4-7 amino-acid extension. (b) the deduced natriuretic peptide presents an unusual internal Cys within the ring structure. This raises the possibility of natriuretic peptides with a smaller ring structure. (c) the putative natriuretic peptide is flanked by two homologous peptides of unknown function. In addition, an analogous peptide was synthesized and assayed on perfused rat kidney, showing a dose-dependent response in urinary volume and sodium excretion. Moreover, northern-blot studies showed that M. corallinus natriuretic peptide transcripts were highly expressed in venom glands, but they were not detectable in other tissues like heart and brain, suggesting a main role for this M. corallinus natriuretic peptide in the venom gland or in the envenomation by this coral snake's bite.

The use of frozen inoculum and 0,2 por cento formaldehyde for inactivation of Pertussis vaccine production

The use of frozen seeds and the effect of the concentration of formaldehyde, and the removal of 2/3 of the supernatant were investigated in order to facilitate the production of pertussis cellular vaccine. Results indicated that is possible to replace fresh seeds by frozen ones, and the formaldehyde concentration can be increased to 0,2 por cento after the remotion of 2/3 of supernatant, resulting in a good vaccine preparation in a shorter time

Effect of saponin from Quillaja saponaria (molina) on antibody, tumour necrosis factor and interferon-gamma production.

Saponin has been described to contain adjuvant activity in vaccination protocols, in protection against disease, and on humoral immune response. In this paper we describe the effect of a pure saponin from Quillaja saponaria (molina) on the immune response elicited in mice by two antigens, BSA and Crotalus durissus terrificus (South American rattlesnake) venom. Antibody production as measured by ELISA shows that saponin was able to increase antibody synthesis to both antigens. Moreover, mice immunized with verom plus saponin were completely protected against the lethal effects of the venom. The effect of saponin was also evaluated for cytokine production. Tumour necrosis factor activity about 2.9 times higher than in control mice was detectable in sera from animals immunized with saponin. Interferon-gamma was produced only when BSA and saponin were injected together into the mice.

Characterization of an endooligopeptidase A-like protein in PC12 cells: activity modulation by cAMP but not by basic fibroblast growth factor.

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.

Kinetic studies on the membrane form of intestinal alkaline phosphatase

We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane formof alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzylphosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 µM, and Vmax 240 µmol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 µM of pNPP at 37§C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43 percent and 84 percent, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23 percent and 17 percent. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30 percent compared to p-nitrophenylphosphate

A Bordetella pertussis acellular vaccine candidate: antigenic characterization and antibody induction

A single-step chromatography on Matrex-Gel Blue A has been employed to obtain soluble extracts containing some of the most important antigens of Bordetella pertussis, pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (69-kDa outer membrane protein), fimbriae (FIM2 and FIM3) and adenylate cyclase (AC). Two supernatants, P19 (48.8 mg PT, 6.8 mg FHA, 17.3 mg AC, 13 mg FIM2 and 4.9 mg FIM33 per liter) and P21 (0.1 mg PT, 0.07 mg FHA, 0.46 mg FIM2 and 0.94 mg FIM3 per liter), resulting from bacteria grown in Stainer-Scholte medium, were submitted to chromatography. Fractions with the antigens were obtained after stepwise elution with 60 mM sodium phosphate buffer, pH 6.0; 50 mM Tris-HC1, pH 7.4; 50 mM Tris-HC1, pH 7.4/0.75 M MgCl2; 50mM Tris-HCl, pH 7.4/4 M MgCl2 and 4 M urea. Preparations from P19 (containing 4.05 µg PT, 8.14 µg FHA, 6.3 µg AC, 3.37 µg 69-kDA, 9.54 µg FIM2 and 2.23 µg FIM3) and P21 (with 0.175 µg PT, 0.28 µg PT, 0.28 µg FHA, 0.002 µg69-kDa, 0.005 µg FIM2 and 0.122 µg FIM3) were detoxified with glutaraldehyde and tested as an acellular pertussis vaccine. These products were non-toxic for mice and induced high levels of antibodies against purified pertussis antigens, as judged by ELISA

Preparation of human rabies vaccine in VERO cell culture using a microcarrier system

1. The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor. Virus titers of about 10(6) LD50/ml were obtained regularly. 2. Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose). The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA. 3. The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2). 4. This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available

Cyclic AMP potentiates bFGF-induced neurite outgrowth in PC12 cells.

We report here that basic fibroblast growth factor (bFGF)-elicited neurite outgrowth in PC12 cells is potentiated by dibutyryl cyclic adenosine monophosphate (dbcAMP) or forskolin. This property was also described for nerve growth factor (NGF), suggesting that both NGF and bFGF may share common intracellular events leading to neurite outgrowth and synergism with dbcAMP and forskolin. The synergistic effect of dbcAMP and forskolin is specific, since treatment of PC12 cells with bFGF and dibutyryl cyclic guanosine monophosphate (dbcGMP) or phorbol ester did not change the neurite outgrowth response of cells treated with bFGF alone. Furthermore, neurite outgrowth depends on cellular adhesion. Increasing adhesion by plate treatment with poly-d-lysine increases the neurite outgrowth elicited by bFGF alone or bFGF plus dbcAMP. On the other hand, decreasing cellular adhesiveness by plating PC12 cells in semi-solid agarose renders the cells unable to develop neuritic processes. In addition, 3H-methylthymidine incorporation studies showed that bFGF-treated PC12 cells cease growth only when they become fully differentiated after 3-5 days of treatment. In contrast, dbcAMP, which is a poor differentiation factor, is able to block cellular growth after 24 hour treatment. These results suggest that when PC12 cells become differentiated, they stop growing. However, growth inhibition does not necessarily lead to differentiation.

Bioactivity of skeletal muscle proteolysis-inducing factors in the plasma proteins from cancer patients with weight loss.

We determined the circulating level of bioactivity for skeletal muscle proteolysis-inducing factors (PIF) in the blood samples from cancer patients whose body weight loss was greater than 10%. The level of bioactivity was estimated by measurement of tyrosine release from isolated 1at diaphragm muscles incubated with an ultrafiltered fraction of plasma or serum proteins containing molecules from 0 to 25 kDa in molecular weight. Significant levels of bioactivity were detected in 25 of the 50 cancer samples. No activity was found in 18 of the samples from healthy human blood donors. The ability of 13 of the cancer samples to induce muscle proteolysis was significantly inhibited by incubation of muscles in presence of indomethacin (10 microM). The neutralisation of 12 of the cancer samples with the antibodies to recombinant human interleukin-1 (IL-1), alpha and beta forms, partially abrogated the activity in five samples. These results suggest that the accelerated breakdown of proteins induced by the cancer plasma factors is at least in part mediated by IL-1 in cooperation with other active factors not yet defined. Additionally, we have shown that the increased breakdown of proteins induced by PIF in the crude supernatant derived from activated mouse peritoneal macrophages is prevented by the treatment of muscles with either indomethacin or quin-2 (1 microM). These observations provide indirect evidence for a possible causal relationship between the production of PIF and the body-weight loss of cancer patients.

Effect of extra-hepatic Walker sarcoma 256 on the synthesis and degradation of liver cytochromes P-450 and b5.

Extra-hepatic Walker sarcoma 256 produced a marked decrease (approximately 60%) in the levels of cytochrome P-450 and NADP-cytochrome P-450 reductase in rat liver endoplasmic reticulum, and a lesser decrease (approximately 20%) of cytochrome b5 and NADH-cytochrome b5 reductase. Polychlorinated biphenyls induced the synthesis of these cytochromes and reductases to approximately the same extent both in normal and tumor-bearing rats. The double-label technique was used to demonstrate that the synthesis of cytochromes P-450 and b5 was reduced in the liver of tumor-bearing rats. The turnover of P-450 was not affected by the tumor, whereas cytochrome b5 turnover was decreased. It is proposed that Walker sarcoma 256 mainly affects the transcription of cytochromes P-450 and b5 through a toxohormone, and that a regulatory mechanism coordinates the level of each cytochrome and its respective reductase.

The possible role of ATP-dependent proteolysis on the solubilization of methemoglobin reductase during reticulocyte maturation.

The ATP-dependent proteolytic system present in reticulocytes can release the active hydrophilic domain of cytochrome b5 and NADH-cytochrome b5 reductase from the endoplasmic reticulum, that in mature erythrocytes act as methemoglobin reductase.

The integration of nutrition education in the basic biomedical sciences.

At the Center for Biomedical Education at the City University of New York, nutrition is integrated into the chemistry-biochemistry sequence of a six-year B.S.-M.D. program. Students perform an actual analysis of a sample of their own food, learning a number of basic techniques and concepts. At the same time they carry on experiments with rats on diets similar to those used by some people. Those activities are complemented by a dietary survey on themselves and other college students. The fundamentals of nutrition are taught as part of the biochemistry course, while the interpretation of the nutrition survey will be conducted in the course titled Health, Medicine and Society, in which students become acquainted with health problems in the community they will serve as general practitioners.

Some properties of cytochrome b5 from liver microsomes of man, monkey, pig and chicken.

1. Cytochrome b(5) was released from liver microsomes of man, monkey, pig and chicken by incubation with a crude lipase preparation. 2. By using DEAE-cellulose chromatography, ammonium sulphate fractionation, Sephadex-gel filtration and a final gradient elution on DEAE-Sephadex A-50, cytochromes b(5) were obtained from the four species studied, all possessing similar spectral properties. 3. Stokes radii of the cytochromes were measured by gel filtration. 4. N-Terminal amino acids for the different cytochromes were serine for man and monkey, alanine for pig and glycine for chicken. 5. Amino acid analyses of the cytochromes are presented. 6. Peptide ;fingerprint' patterns of tryptic digests of the different cytochromes are discussed and clearly show increasing similarity for more closely related species.