Dante M. Beltramo

Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose‐ and time‐dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan‐induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3‐(3‐acetamide)‐1‐benzyl‐2‐ethylindolyl‐5‐oxy‐propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage‐activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.

Superstimulation of ovarian follicular development in beef cattle with a single intramuscular injection of Folltropin-V.

The need to inject FSH twice daily for superstimulation of ovarian follicular development in cattle necessitates frequent attention by farm-personnel and increases the possibility of failures due to mishandling and errors in administration of treatments. A series of three experiments were designed to evaluate the feasibility of superstimulation in beef cattle with a single intramuscular (IM) injection of Folltropin-V diluted in a hyaluronan-based slow-release formulation (SRF). In Experiment 1, cows were assigned to one of three treatment groups to compare two methods of injection as compared to the twice daily IM injection protocol. Superovulatory response of cows (n=6) treated with twice daily IM injections over 4 days (Control) was greater than of cows treated with a single subcutaneous (SC) injection in SRF (n=6), while superovulatory response of cows treated with a single IM injection in SRF (n=6) was intermediate. Experiment 2 was designed to compare two concentrations of SRF (20mg/mL hyaluronan, 100% compared to 10mg/mL hyaluronan, 50%) in a single IM injection protocol. The mean number of corpora lutea (CL) were not significantly different (P≥0.05), but the numbers of total ova/embryos (P<0.05), fertilized ova (P<0.01) and transferable embryos (P<0.001) were greater in cows treated with FSH in 100% SRF (n=20) than cows treated with FSH in 50% SRF (n=20). Experiment 3 was designed to compare superovulatory response in Red Angus donor cows treated with a single IM injection of Folltropin-V diluted in 100% solution of SRF with those treated with the traditional twice-daily IM injection protocol over 4 days. Mean (±SEM) numbers of CL (13.7±1.2 compared to 13.8±1.2), total ova/embryos (12.3±1.5 compared to 13.7±2.1), fertilized ova (7.2±1.1 compared to 8.4±1.4) and transferable embryos (4.9±0.8 compared to 6.4±1.3) were not significantly different between Control (n=29) and Single injection (n=29) groups, respectively. In summary, superstimulation of beef donor cows with a single IM injection of Folltropin-V diluted in 100% solution of SRF resulted in a comparable superovulatory response to the traditional twice-daily IM administration of Folltropin-V diluted in saline over 4 days.

Growth of milk fermentative bacteria in the presence of chitosan for potential use in cheese making

The effects of different molecular weight chitosans alone and their complexes with casein on the growth of three representative milk fermentative bacteria (Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Propionibacterium freudenreichii) were compared. In nutrient broth, all chitosans showed a dose dependent inhibition of S. thermophilus and L. delbrueckii ssp. bulgaricus growth. Chitosan of high and low molecular weight, but not chitosan oligosaccharides, showed a dose dependent inhibition of P. freudenreichii that was dependent on the initial bacterial load. The inhibiting effects of chitosan were greatly reversed when the biopolymers were incubated with milk before interaction with bacteria. Under these conditions a cheese like product was made with high molecular weight chitosan as the coagulating agent. The product had lower firmness than the respective cheese control, probably because of a higher water retention. The effect of chitosan on milk fermentative processes appeared to be dependent not only on its molecular weight and concentration, but also on the presence of casein micelles or milk fat, that, acting in a competitive manner, could prevent the inhibitory activity of these biopolymers on bacterial growth.

Rapid HCP5 single-nucleotide polymorphism genotyping: A simple allele-specific PCR method for prediction of hypersensitivity reaction to Abacavir

BACKGROUND The most important factor limiting the success of an antiretroviral therapy is toxicity. The HLA-B*5701 allele is predictive of hypersensitivity reaction to Abacavir, and this gene is in a perfect linkage disequilibrium with the rs2395029 SNP present in the HCP5 gene. METHODS Genomic DNA was extracted from blood obtained from 201 unrelated healthy Argentinean volunteers. The DNA was subjected to an allele-specific PCR method. Sequencing was performed to validate the test results. RESULTS We were successful to amplify specific fragment of interest from the DNA samples. The method is easy, specific and reproducible. CONCLUSIONS The application of this methodology is a rapid and simple method to detect the HCP5 polymorphism (rs2395029) previous to administration of Abacavir in patients with HIV infection.

Vantris®, A biocompatible, synthetic, non-biodegradable, easy-to-inject bulking substance: Evaluation of local tissular reaction, localized migration and long-distance migration

Biodegradable injectable bulking agents of animal origin present a fast rate of bio-reabsorption and may cause an allergic reaction. Biodegradable elements of synthetic origin have a high rate of reabsorption after a year. Non-biodegradable agents of synthetic origin lead to the formation of a fibrotic capsule, giving stability and long-term permanence. VANTRIS is categorized into this last group; it belongs to the family of Acrylics, particles of polyacrylate polyalcohol copolymer immersed in a glycerol and physiological solution carrier. Molecular mass is very high. When injected in soft tissues, this material causes a bulkiness that remains stable through time. The carrier is a 40% glycerol solution with a pH of 6. Once injected, the carrier is eliminated by the reticular system through the kidneys, without metabolizing. Particles of this polyacrylate polyalcohol with glycerol are highly deformable by compression, and may be injected using a 23-gauge needle. The average of particles size is 320 mm. Once implanted, particles are covered by a fibrotic capsule of up to 70 microns. Particles of this new material are anionic with high superficial electronegativity, thus promoting a low cellular interaction and low fibrotic growth. The new polyacrylate polyalcohol copolymer with glycerol was tested for biocompatibility according to ISO 10993-1:2003 in vitro, showing that they are not mutagenic for the Salmonella T. strains analyzed. The extract turned out to be non-cytotoxic for cell lines in culture and non-genotoxic for mice. In in vivo studies, acrylate did not cause sensitization in mice. The macroscopic reaction of tissue irritation was not significant in subcutaneous implants and in urethras of rabbits. Seven female dogs were injected transurethrally with VANTRIS to evaluate short and long-term migration (13 weeks and 12 months respectively). No particles or signs of inflammation or necrosis are observed in any of the organs examined 13 weeks and 12 months after implantation. To conclude, this new material meets the conditions of ideal tissue bulking material.

Self-assembled micelles of monosialogangliosides as nanodelivery vehicles for taxanes.

We demonstrate herein that taxanes (paclitaxel (Ptx) and docetaxel (Dtx)) can be spontaneously loaded into ganglioside nanomicelles. The efficiency of gangliosides to solubilize taxanes was highly dependent on their self-aggregating structure. Thus, GM3 that forms unilamellar vesicles was less efficient to solubilize taxanes than gangliosides that form micelles (i.e. GM1 and GM2). Sialic acid cyclization of GM1 by acid treatment led to an important reduction in its capacity to solubilize taxanes, as also did the replacement of the fatty acid of ceramide by a dicholoracetyl group. Water solubility of paclitaxel (Ptx) is less than 1 μg mL⁻¹ and increased up to 6.3mg.mL⁻¹ upon its association with GM1 micelles. The incorporation of Ptx in GM1 reached an optimum at GM1/Ptx 20/1 molar ratio when performed at room temperature. An increase in the solubilization capacity of GM1 micelles was observed upon dehydration of their polar head group by pre-treatment at 55 °C. Loading of Ptx into the micelle induced a structural reorganization that led to an important protection of Ptx reducing its hydrolysis at alkaline pH. Diffusion of either GM1 or Ptx was restricted upon mixed-micelle formation indicating that they are kinetically more stable than pure ganglioside micelles. X-ray powder diffraction of lyophilized GM1 micelles with Ptx showed a change in their internal structure from a crystalline state to completely amorphous. Taxane-ganglioside mixed micelles were stable in solution for at least 4months and also upon freeze-thawing or lyophilization-solubilization cycles. Upon mixing with human blood constituents, GM1/Ptx micelles did not induce hemolysis or platelet aggregation and were spontaneously covered with human serum albumin (HSA), which could aid in the delivery of micellar content to tumors. In vitro antimitotic activity of GM1/Ptx mixed micelles was qualitatively equivalent to that of free drug in DMSO solution.

A simple allele-specific polymerase chain reaction method to detect the Gly143Glu polymorphism in the human carboxylesterase 1 gene: importance of genotyping for pharmacogenetic treatment.

Human carboxylesterases 1 and 2 (CES1 and CES2) catalyze the hydrolysis of many exogenous compounds. Alterations in CES sequences could lead to variability in both the inactivation of drugs and the activation of prodrugs. The human CES1 gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. Some of theses drugs are the antiviral oseltamivir used to treat some types of influenza infections and the methylphenidate employed in the treatment of patients with attention deficit. The Gly143Glu polymorphism in CES1 gene has been shown to reduce enzyme activity. The aim of the present study was to develop an easy and cheap method to detect this polymorphism. For this, we studied a group of people from Córdoba, a Mediterranean area from Argentina. Our results show that our methodology could detect the presence of this polymorphism with a frequency around 1.8%, only in the heterozygote form. These results could be relevant to patients before the treatment with some drugs where the CES1 enzyme is involved.

Eudragit E100 surface activity and lipid interactions.

Eudragit E100 (E100) is a cationic methacrylate polymer that interacts with viral and cell membranes. We studied the effect of pH, ionic strength and the presence of lipid monolayers on the surface activity of the polymer. E100 forms stable monolayers at the air-water interface, either by spreading or when added into the subphase. This behavior is highly influenced by the pH and saline concentration of the subphase. At pH 5 or higher, the adsorption of the polymer to the air-water interface begins immediately after its injection into the subphase, while at pH below 5 E100 remains in the subphase with a particularly slow adsorption to the interface. In addition, low ionic strength (10 mM) in the subphase results in a fast adsorption of the polymer to the interface, even at pH under 5. On the other hand, in the presence of non-ionic (cholesterol) or anionic (monosialoganglioside) lipid monolayers, E100 shows a fast adsorption to the interface, [comma] reaching surface pressures of 25 and 36 mN m(-1), respectively. However, E100 barely interacts with monolayers of a zwitterionic lipid (hydrogenated soy lecithin) with a cut-off pressure of 11 mN m(-1). The interaction of E100 with GM1 micelles in the subphase reduces its surface activity. Altogether these results show that E100 can effectively penetrate into model membranes and that its amphipathic character is largely dependent on the chemical composition of the aqueous environment and the lipid composition of the membrane.

Renal tubular acidosis and vasculitis associated with IgE deposits in the kidney and small vessels

We report a woman with a history of allergies, polyuria, polydipsia, proteinuria, renal loss of electrolytes, renal tubular acidosis, nephrocalcinosis, and palpable purpura. A proximal defect was excluded by a normal bicarbonate reabsorption curve, and a distal tubular defect was shown because urine pH did not decrease to less than 6.4 despite ammonium chloride-induced systemic acidosis. Moreover, furosemide failed to improve urinary acidification. Urine-to-blood PCO(2) gradient was less than 14 mm Hg, although the urine bicarbonate level reached values as high as 89 mEq/L. Combining bicarbonate and neutral phosphate infusions increased the urine-to-blood PCO(2) gradient to only 20 mm Hg. These subnormal PCO(2) gradient values point to proton-pump dysfunction in the collecting tubule. Histological evidence of tubulointerstitial disease accompanied the tubular defects. The striking histological feature was the presence of immunoglobulin E (IgE) deposits in glomeruli, tubuli, and vessels. Concurrent with these findings, she had high serum IgE titers and CD23 levels. IgE antibodies from her serum were reactive against human renal tubuli, with binding to two regions that matched two different proteins present in cortex and medulla. One of these proteins corresponded to carbonic anhydrase II (31 kd); the second, to an unidentified protein that seems attached to cell membranes. We suggest that these IgE antibodies could have had a pathogenic role in this patients glomerular, tubular, and small-vessel disease.

Distribution of Polymorphisms in Cytochrome P450 2B6, Histocompatibility Complex P5, Chemokine Coreceptor 5, and Interleukin 28B Genes in Inhabitants from the Central Area of Argentina

AIMS The selection of the most appropriate treatment for several diseases relies on a number of factors such as environment, age, gender, and nutrition. Additionally, the contribution of different genetic polymorphisms to treatment efficacy has been largely recognized. The lack of information on the pharmacogenetic profile of our population prompted us to analyze the frequency of polymorphisms known to be relevant to achieve treatment efficacy with different therapeutic agents in viral infectious diseases, such as Hepatitis C and AIDS. RESULTS The allelic frequencies for the wild-type variant of the genes analyzed were cytochrome P450 2B6 (CYP2B6; rs3745274; 516G) 0.618 (95% confidence interval [CI]: 0.523, 0.711), chemokine coreceptor 5 (CCR5; rs333) 0.961 (95% CI: 0.942, 0.98), histocompatibility complex P5 (HCP5; rs2395029; 335T) 0.971 (95% CI: 0.937, 1), and interleukin 28B (IL28B; rs12979860; 12007005C) 0.656 (95% CI: 0.564, 0.747), respectively. CONCLUSIONS Our data indicate that the genetic profile of the population studied is similar to that reported for other Caucasian populations, with only slight differences for CYP2B6. Noteworthy, the considerable number of patients carrying CYP2B6 (516T) and IL28B (12007005T) alleles underlies the importance of considering pharmacogenetic testing before starting drug therapy protocols to prevent toxicity and/or lack of effectiveness in AIDS or hepatitis C virus infections.