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Introducción. La alta tasa de contaminantes microbianos visibles en la fase de establecimiento, así como una baja tasa de multiplicación, enraizamiento y supervivencia ex vitro, constituyen los principales problemas durante la propagación in vitro en Bambusa vulgaris Schard. ex Wendl. El presente trabajo se realizó con el objetivo de proponer nuevas alternativas para la propagación de Bambusa vulgaris Schard. ex Wendl combinando técnicas tradicionales. Métodos. Se determinó la influencia de las condiciones climáticas en el establecimiento in vitro de los segmentos caulinares. Además, se estudió el efecto de las condiciones de cultivo en los brotes multiplicados en medio de cultivo líquido estático y Sistemas de inmersión temporal, así como su respuesta durante la fase de aclimatización. Resultados. Fueron identificados por primera vez los contaminantes microbianos de alta frecuencia de aparición asociados a las plantas en casa de cultivo e in vitro. Durante el establecimiento in vitro de las yemas axilares se logró un 99 % de brotación y 98 % de explantes libres de contaminantes microbianos visibles entre los meses de enero-abril y noviembre-diciembre. Se demostró que los brotes multiplicados entre el sexto y octavo subcultivo con 12 μM de 6-BAP pueden ser utilizados para la multiplicación en Sistemas de Inmersión Temporal. Se logró incrementar la calidad morfo-fisiológica y bioquímica de los brotes cultivados en los SIT con la menor concentración de 6-BAP (6,0 μM) y un tiempo de inmersión de 2 min, aspectos poco abordados en las especies de bambúes. Se describieron los primeros estudios en la especie sobre el efecto de la sacarosa en la respuesta morfo-fisiológica, anatómica y bioquímica de los brotes en los SIT durante la transición in vitro-ex vitro. Las plantas llevadas a la casa de cultivo pudieron emitir entre 4 y 7 nuevos brotes con altas tasas de supervivencia durante la extracción de retoños.

BACKGROUND: In acute lung injury positive end-expiratory pressure (PEEP) and recruitment maneuver are proposed to optimize arterial oxygenation. The aim of the study was to evaluate the impact of such a strategy on lung histological inflammation and hyperinflation in pigs with acid aspiration-induced lung injury. METHODS: Forty-seven pigs were randomly allocated in seven groups: (1) controls spontaneously breathing; (2) without lung injury, PEEP 5 cm H2O; (3) without lung injury, PEEP titration; (4) without lung injury, PEEP titration + recruitment maneuver; (5) with lung injury, PEEP 5 cm H2O; (6) with lung injury, PEEP titration; and (7) with lung injury, PEEP titration + recruitment maneuver. Acute lung injury was induced by intratracheal instillation of hydrochloric acid. PEEP titration was performed by incremental and decremental PEEP from 5 to 20 cm H2O for optimizing arterial oxygenation. Three recruitment maneuvers (pressure of 40 cm H2O maintained for 20 s) were applied to the assigned groups at each PEEP level. Proportion of lung inflammation, hemorrhage, edema, and alveolar wall disruption were recorded on each histological field. Mean alveolar area was measured in the aerated lung regions. RESULTS: Acid aspiration increased mean alveolar area and produced alveolar wall disruption, lung edema, alveolar hemorrhage, and lung inflammation. PEEP titration significantly improved arterial oxygenation but simultaneously increased lung inflammation in juxta-diaphragmatic lung regions. Recruitment maneuver during PEEP titration did not induce additional increase in lung inflammation and alveolar hyperinflation. CONCLUSION: In a porcine model of acid aspiration-induced lung injury, PEEP titration aimed at optimizing arterial oxygenation, substantially increased lung inflammation. Recruitment maneuvers further improved arterial oxygenation without additional effects on inflammation and hyperinflation. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the ability and accuracy of aortic flow velocity-time integral variation (ΔVTI) and peak aortic velocity variation (ΔVpeak) compared with pulse pressure variation (PPV) to predict fluid responsiveness in mechanically ventilated dogs., Study Design: Prospective clinical study., Animals: A group of 50 mechanically ventilated dogs with spontaneous hypotension during orthopedic or oncologic surgery., Methods: Investigations were performed in the surgery room. When mean arterial pressure (MAP) decreased to <65 mmHg, measurements were performed before and after a fluid challenge (lactated Ringer's solution 5 mL kg -1 over 15 minutes). Responders were defined as a change in stroke volume (SV; transesophageal Doppler) ≥15%. Data were analyzed using paired/unpaired t test or Mann-Whitney/Wilcoxon test when appropriate and receiver operating characteristics (ROC) curves; a p value <0.05 was considered statistically significant., Results: After the fluid challenge, 35 (70%) of 50 dogs were responders with significant increases in SV and decreases in PPV; 15 dogs were nonresponders. ΔVTI and ΔVpeak correlated with a 15% increase in SV. The optimum cut-off value for PPV was 15.6% (sensitivity, 88%; specificity, 100%), for ΔVTI was 10.65% (sensitivity, 65%; specificity, 100%) and for ΔVpeak was 10.15% (sensitivity, 80%; specificity, 100%). The area under the ROC curve for PPV was (0.93 ± 0.08) and for ΔVpeak was (0.89 ± 0.09), before fluid challenge. The gray zone area spread from 6.15% to 15.6% for PPV (18 dogs), 2.45% to 10.65% for ΔVTI (22 dogs) and 0.6% to 10.15% for ΔVpeak (25 dogs)., Conclusions: When using mechanical ventilation, ΔVTI and ΔVpeak predicted fluid responsiveness with the same ability as PPV, based on the area under the ROC curve analysis. However, PPV showed great accuracy demonstrated by a narrower gray zone that included fewer individuals., Clinical Relevance: ΔVTI and ΔVpeak can be used as indices of fluid responsiveness in anesthetized dogs., (Copyright © 2019. Published by Elsevier Ltd.)

Argentinian hemorrhagic Fever (AHF) is a febrile, acute disease caused by Junín virus (JUNV), a member of the Arenaviridae. Different approaches to obtain an effective antigen to prevent AHF using complete live or inactivated virus, as well as molecular constructs, have reached diverse development stages. This chapter refers to JUNV live attenuated vaccine strain Candid #1, currently used in Argentina to prevent AHF. A general standardized protocol used at Instituto Nacional de Enfermedades Virales Humanas (Pergamino, Pcia. Buenos Aires, Argentina) to manufacture the tissue culture derived Candid #1 vaccine is described. Intermediate stages like viral seeds and cell culture bank management, bulk vaccine manufacture, and finished product processing are also separately presented in terms of Production and Quality Control/Quality Assurance requirements, under the Adminitracion Nacional de Medicamentos, Alimentos y Tecnología Medica (ANMAT), the Argentine national regulatory authority.

Objective: To examine the intrapulmonary gas distribution of low and high tidal volumes (V T ) and to investigate whether this is altered by an alveolar recruitment maneuver (ARM) and 5 cmH 2 O positive end-expiratory pressure (PEEP) during anesthesia., Study Design: Prospective randomized clinical study., Animals: Fourteen client-owned bitches weighing 26 ± 7 kg undergoing elective ovariohysterectomy., Methods: Isoflurane-anesthetized dogs in dorsal recumbency were ventilated with 0 cmH 2 O PEEP and pressure-controlled ventilation by adjusting the peak inspiratory pressure (PIP) to achieve a low (7 mL kg -1 ; n = 7) or a high (12 mL kg -1 ; n = 7) V T . Ninety minutes after induction (T90), an ARM (PIP 20 cmH 2 O for 10 seconds, twice with a 10 second interval) was performed followed by the application of 5 cmH 2 O PEEP for 35 minutes (RM35). The vertical (ventral=0%; dorsal=100%) and horizontal (right=0%; left=100%) center of ventilation (CoV), four regions of interest (ROI) (ventral, central-ventral, central-dorsal, dorsal) identified in electrical impedance tomography images, and cardiopulmonary data were analyzed using two-way repeated measures anova., Results: The low V T was centered in more ventral (nondependent) areas compared with high V T at T90 (CoV: 38.8 ± 2.5% versus 44.6 ± 7.2%; p = 0.0325). The ARM and PEEP shifted the CoV towards dorsal (dependent) areas only during high V T (50.5 ± 7.9% versus 41.1 ± 2.8% during low V T , p = 0.0108), which was more distributed to the central-dorsal ROI compared with low V T (p = 0.0046). The horizontal CoV was centrally distributed and cardiovascular variables remained unchanged throughout regardless of the V T , ARM, and PEEP., Conclusions and Clinical Relevance: Both low and high V T were poorly distributed to dorsal dependent regions, where ventilation was improved following the current ARM and PEEP only during high V T . Studies on the role of high V T on pulmonary complications are required., (Copyright © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved.)

Candid#1 is the first live attenuated vaccine produced and registered in Argentina. Produced since 2003 at the INEVH to prevent Argentine hemorrhagic fever, it is obtained by harvesting supernatants of diploid cells infected with an attenuated strain of Junin virus and subsequent lyophilization. The stability of this vaccine is crucial to ensure its effectiveness. This study was aimed to evaluate the stability of Candid#1 by exposing it to different time and temperature conditions. Three vaccine batches produced in 2003 were analysed according to the following storage scheme: (a) reconstituted vaccine at 2 °C to 8 °C for 8 days; (b) lyophilized vaccine at 2 °C to 8 °C for 6 months; (c) lyophilized vaccine at -18 °C to -20 °C for 10 years. The potency was assessed in Vero cell monolayers under agar. The results were: (a) reconstituted vaccine was stable between 2 °C and 8 °C for 8 days, (b) lyophilized vaccine was stable between 2 °C and 8 °C for 2 months, and (c) lyophilized vaccine was stable 9 years between -18 °C and -20 °C, keeping all its properties. These results allowed us to establish the following storage conditions and expiration times for Candid#1: (a) reconstituted: 12 hours between 2 °C and 8 °C, (b) lyophilized: 30 days between 2 °C and 8 °C and (c) lyophilized: 9 years between -18 °C and -20 °C. Based on our results, favorable changes were made in the conditions of transport, storage and distribution of the vaccine. Domestic freezers in strategically located centers were installed, allowing the preservation of vaccine stocks for distribution to secondary vaccination centers.

The aim of this study was to evaluate the parents' perception of dental caries in children with intellectual disability. This cross-sectional study was conducted with 6 to 14 years old schoolchildren: Group 1 (50 children diagnosed with intellectual disabilities) and Group 2 (50 children without it). The dental caries was assessed by the World Health Organization (WHO) criteria for primary and permanent teeth. Parents' psychosocial perception was assessed by Early Childhood Oral Health Impact Scale (ECOHIS). Similar prevalence of caries free children was found between groups in both dentitions. In primary dentition the caries index was higher in Group 2, and the opposite occurred in permanent teeth. Group 1 presented higher impact (p < .05) in the dimension drinking, eating and pronunciation, whereas in Group 2 there was higher impact (p = .01) on pain, sleep, irritation, the smile and family finances. Findings showed significant impact of dental caries on parents' perception of the oral health related quality of life of children with intellectual disabilities., (© 2016 Special Care Dentistry Association and Wiley Periodicals, Inc.)

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

A clinical study in 946 human volunteers was done to compare Candid #1 vaccine manufactured in Argentina with the vaccine produced in USA that had been previously used. The efficacy was evaluated using immunogenicity measured by the detection of neutralizing antibodies as a subrogate marker. Safety was evaluated comparing the rate of adverse events. Both vaccines showed a comparable rate of seroconversion, slightly higher than the efficacy estimated from previous studies (95.5%). There were no severe adverse events related to the vaccines. The general events considered related to the vaccines were not clinically relevant and disappeared either spontaneously or with symptomatic treatment. Similar rates of adverse events (29.9% for the Argentine vaccine and 35.0% for the USA vaccine) were found for both vaccines. These included: headache, weakness, myalgias, mild low blood cell (< 4,000/mm(3)) and platelet (< 150,000/mm(3)) counts, nausea and/or vomiting, fever, retroocular pain, dizziness, microhematuria, low backache and exantema. These results indicate that the vaccine Candid#1 manufactured in Argentina is equivalent to the manufactured in USA. These results allowed the National Institute of Human Viral Diseases (INEVH) to register the vaccine produced locally under the National Regulatory Authority (ANMAT).

The V.A.C. Therapy System (KCI, San Antonio, TX) is an integrated wound management system that creates an environment that promotes wound healing. V.A.C. GranuFoam Silver dressing (S-ROCF) is a reticulated, open-celled polyurethane foam having a pore size range of 400-600 microm. The foam is coated with silver to impart antimicrobial properties throughout the dressing. The morphological and key mechanical properties including tensile strength, ultimate elongation, tear strength, and compression resistance of this dressing were compared to the original V.A.C. GranuFoam dressing (ROCF), which is also a reticulated, open-celled polyurethane foam, and were found to be comparable. Furthermore, the antimicrobial property of S-ROCF was evaluated in vitro for efficacy against pathogens commonly found in clinical settings, including Staphylococcus aureus and Pseudomonas aeruginosa. S-ROCF was found to be effective during antimicrobial testing with a 99.99% reduction in colony forming units, and remained effective after 72 h of simulated V.A.C. Therapy. The antimicrobial and mechanical characteristics of the foam were unaffected by aging as demonstrated through an accelerated aging process. The V.A.C. GranuFoam Silver dressing is a specialized antimicrobial dressing for use with the V.A.C. Therapy System that exhibits morphological and mechanical properties comparable to the V.A.C. GranuFoam dressing and provides the added benefit of antimicrobial efficacy.

The activity of LCM virus was first reported in Argentina at the beginning of the seventies and only five strains have been isolated from rodents Mus domesticus and two from humans. The objective of this paper was to find differential biological characteristics of Argentine strains of LCM virus comparing them in relation to the historical strains WE and Armstrong. Regarding the results obtained in tissue culture, when L 929 cells were used, plaque forming units (PFU) were obtained with human and mouse strains, whilst on Vero cells only human strains developed PFU. Differentials characteristics of historical and Argentine strain's plates were not found, neither differences related to the strain's origin. Neither historical nor Argentine strains were lethal to new-born mice giving a persistent infection, that was demonstrated when we inoculated new-born mouse by intracranial route with different strains of LCM virus and virus was isolated from brains harvested at different days post inoculation. The only exception was Cba An 13065 strain that exhibited virulence in new-born mice, only with 0.026 PFU was obtained 1 DL50. All the strains resulted lethal to adult mice. The mouse strains were more virulent than human strains, being Cba An 13065 the most virulent. These results demonstrate a different behavior in tissue culture between human and mouse strains and allow the identification of virulence markers by intracranial inoculation into new-born or adult mice.

Neutralizing antibody (NT Ab) titers to Candid #1 (C#1) vaccine against Argentine hemorrhagic fever were studied for 2 years post-vaccination in 330 volunteers, to assess whether the kinetics and/or magnitude of this immune response is modified by previous infection with the arena viruses Junin (JUN) and lymphocytic choriomeningitis (LCM). A total of 160 volunteers received C#1, distributed as follows: without detectable pre-infection with arenaviruses (n = 54); with pre-existing antibodies to JUN (n = 55); with pre-existing antibodies to LCM (n = 51). The remaining 170 individuals received placebo. Levels of anti-JUN NT Ab displayed a trend in which titers increased with the virulence of the infecting strain, from C#1 (X = 49), through subclinical JUN infection (X = 229), vaccination following subclinical infection (X = 367) to JUN clinical infection (X =773). It was also found that the mean titer of NT Ab to C#1 did not vary significantly during 2 years of study and was: a) significantly lower than that elicited by wild strains of JUN, both clinical and subclinical infections (p < 0.01); b) significantly increased the titers of pre-existing anti-JUN Ab (p < 0.01); and c) was not modified by pre-existing anti-LCM Ab (p > 0.05). These data indicate that the immune response to C#1 boosts pre-existing immunity to JUN virus and is not changed by previous experience with LCM virus.

We have recently shown by monitoring intracellular pHi with a stopped-flow fluorimeter, that when expressed in HEK293 kidney cells, two Rh glycoproteins, RhBG and RhCG, facilitated NH3 movement across the plasma membrane. Based on the results of 3D structure determination of AmtB, a bacterial member of the Amt/Mep/Rh superfamily, and of homology modeling of the human Rh proteins, we have attempted to determine if some selected residues predicted to be located in the pore or in the vestibule of the channel are essential for NH3 transport. Accordingly, wild type and mutant forms of RhCG were expressed in HEK293 cells and their ammonium function was analyzed with the stopped-flow fluorimeter. Some mutants that were not expressed at a significant level in HEK293 could not be tested for function, but mutations at positions F74, V137 and F235 (equivalent positions in AmtB: I28, L114, F215, respectively) resulted in a severe reduction of NH3 transport.

Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper, we report the nucleotide sequences of L RNA of Candid #1 and examine the relationship to its more virulent ancestors Junin virus XJ#44 and XJ 13 (prototype) and other closely and distantly related arenaviruses. Comparisons of the nucleotide and amino acid sequences of L and Z genes of Candid #1 and its progenitor strains revealed twelve point mutations in the L polypeptide that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype. In contrast, Z ORF was completely conserved among all strains.

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin-Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 microM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222-17227] constitute a family of NH3 channels in mammalian cells.

Lymphocytic choriomeningitis virus (LCMV) is the prototype of the family Arenaviridae and is associated with the natural reservoir, Mus domesticus (Md). It causes meningitis and a flu-like illness characterized by malaise, myalgia, retrorbital headache, and photophobia. This study presents the data obtained in a rodent and human serological study during 6 years (1998-2003) in the city of Rio Cuarto, Argentina. Antibodies anti-LCMV were sought by ELISA in rodents and humans. LCMV was found only in Md species in 9.4% of animals. The results also show some seasonal, no significant variations in the prevalence of the infection. Distribution of positive mice was not modified significantly by trapping sites, sex, or age of the animals. The prevalence of LCMV positive urban residents was found to be consistently low (1-3.6%) along the study period, with overage prevalence of 3.3% and values in males (4.6%) significantly higher than in females (2.6%) (P < 0.05). Seven of 432 pregnant women were found to be LCMV positive, but the absence of LCMV antibodies in the newborns demonstrated that the mothers were infected before pregnancy. This study is the first evidence on endemic LCMV in an Argentine city located outside the endemic area of Argentine hemorrhagic fever (AHF) and described the need to study other areas and increase awareness of this viral infection., (Copyright 2005 Wiley-Liss, Inc.)

Candid #1 vaccine against Argentine Hemorrhagic Fever produced in USA versus lots of the same vaccine made in Argentina were compared in guinea pigs regarding safety, immunogenicity and protective efficacy against a challenge with pathogenic Junin virus. Lots No Exp 3, 7A and 8A of Argentine origin as well as lot TSI 5-1-92 from USA were inoculated in guinea pigs of 250-400 g in two consecutive assays. Ten animals inoculated with saline performed as normal controls in each experiment. Parameters studied were: a) temperature; b) body weight; c) neutralizing antibodies to Junin virus; d) response to viral challenge. Animals gained weight and remained normothermic up to the challenge. Guinea pigs that received Candid #1 from any manufacturer elicited neutralizing antibodies to Junin virus (titles from 40 to 81920) and survived to challenge whilst 8/10 animals died in each control group. Data presented demonstrated that Candid #1 vaccines from USA or Argentine manufacturers were equally safe, immunogenic and protective in guinea pigs.

Tyrosine-functionalized polyphosphazenes were synthesized, and their hydrolytic stability, pH-sensitive behavior, and hydrogel-forming capabilities were investigated. The physical and chemical properties of the polymers varied with the type of linkage between the tyrosine unit and phosphazene backbone. Poly[(ethyl glycinat-N-yl)(ethyl tyrosinat-N-yl)phophazenes] (linkage via the amino group of tyrosine) were found to be hydrolytically erodible. The rate of hydrolysis was dependent on the ratio of the two side groups, the slowest rate being associated with the highest concentration of tyrosine. The hydrolysis products were identified as phosphates, tyrosine, glycine, ammonia, and ethanol derived from the ester group. The hydrolytically stable phenolic-linked tyrosine derivatives were prepared from N-t-BOC-L-tyrosine methyl ester and alkoxy-based cosubstituents. Polyphosphazenes with both propoxy and phenolic-linked tyrosine side groups showed a pH-sensitive solubility behavior, which was dependent on the ratio and nature of the two side groups. The polymer was soluble in aqueous media below pH 3 and above pH 4. From pH 3-4, the polymer was insoluble. Replacement of propoxy by trifluoroethoxy units yielded a polymer that was insoluble in aqueous media at all pH values. Replacement of propoxy by methoxyethoxyethoxy groups gave a polymer that was soluble at all pH values. Exposure of both the propoxy and methoxyethoxyethoxy polymers to calcium ions in aqueous media caused gel formation due to ionic cross-linking through the carboxylate groups.

Different proportions of IgG subclasses have previously been reported to distinguish the immune response elicited by primary and recurrent viral infections, as well as viral vaccines. The goal of this study was to study the IgG subclasses composition in the immune response of patients with Argentine hemorrhagic fever, and vaccinees with Candid #1 strain of Junin virus. Twenty-four individuals inoculated with Candid #1 vaccine and 67 patients with Argentine hemorrhagic fever were studied. Blood samples were drawn at 30, 60, and/or 180 days post-inoculation with Candid #1 and 30, 60, and 90 days after clinical onset of the disease. Specific anti-Junin virus IgG subclasses were titrated with specific human monoclonal antibody fluorescence isothiocyanate conjugate (FITC) by immunofluorescent assay (IFA). IgG(1) anti-Junin virus was found in every subject studied and IgG(3) was also detected in some patients with a severe form of Argentine hemorrhagic fever. IgG(2) and IgG(4) were not detected in any serum sample studied. The mean titer of specific IgG(1) in vaccinees was significantly lower than in patients with Argentine hemorrhagic fever (P < 0.05), but no difference was found between mild and severe cases of the disease (P > 0.05). The results of this study demonstrated a central role of IgG(1) in human recovery from infection with every strain of Junin virus, an observation stressed by the immune response to Candid #1 vaccine, which resulted in no difference in IgG subclasses composition from that found in mild cases of Argentine hemorrhagic fever., (Copyright 2003 Wiley-Liss, Inc.)

In most cases, the lack of Rh in Rh(null) red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter-based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

Junin virus is the etiological agent of Argentine hemorrhagic fever, a serious rodent-borne disease. An enzyme-linked immunosorbent assay (ELISA) to detect Junin virus IgG antibodies in rodents was evaluated using sera from 27 Calomys musculinus and five Calomys laucha, inoculated experimentally with a live attenuated strain of this arenavirus. The test performance was compared against an indirect immunofluorescence assay (IFA). The ELISA had a sensitivity and specificity of 100% and a reproducibility of 87.9% for samples with titers above the selected cut-off value. IFA had lower sensitivity (53%) with the same specificity. The ELISA results were similar, whether carried out on whole blood or serum samples, thus eliminating the need for serum separation. A high correlation (K=0.86) between ELISA and IFA results was obtained from 1011 wild sigmodontine and murine rodents collected within and outside of the Argentine hemorrhagic fever endemic area. These results indicate that Junin virus IgG ELISA is the most suitable assay for detection of Junin virus antibodies in rodent samples.

Biomaterials based on the polymers of lactic acid and glycolic acid and their copolymers are used or studied extensively as implantable devices for drug delivery, tissue engineering and other biomedical applications. Although these polymers have shown good biocompatibility, concerns have been raised regarding their acidic degradation products, which have important implications for long-term implantable systems. Therefore, we have designed a novel biodegradable polyphosphazene/poly(alpha-hydroxyester) blend whose degradation products are less acidic than those of the poly(alpha-hydroxyester) alone. In this study, the degradation characteristics of a blend of poly(lactide-co-glycolide) (50:50 PLAGA) and poly[(50% ethyl glycinato)(50% p-methylphenoxy) phosphazene] (PPHOS-EG50) were qualitatively and quantitatively determined with comparisons made to the parent polymers. Circular matrices (14mm diameter) of the PLAGA, PPHOS-EG50 and PLAGA-PPHOS-EG50 blend were degraded in non-buffered solutions (pH 7.4). The degraded polymers were characterized for percentage mass loss and molecular weight and the degradation medium was characterized for acid released in non-buffered solutions. The amounts of neutralizing base necessary to bring about neutral pH were measured for each polymer or polymer blend during degradation. The poly(phosphazene)/poly(lactide-co-glycolide) blend required significantly less neutralizing base in order to bring about neutral solution pH during the degradation period studied. The results indicated that the blend degraded at a rate intermediate to that of the parent polymers and that the degradation products of the polyphosphazene neutralized the acidic degradation products of PLAGA. Thus, results from these in vitro degradation studies suggest that the PLAGA-PPHOS-EG50 blend may provide a viable improvement to biomaterials based on acid-releasing organic polymers.

The active coexistence of two pathogenic arenaviruses, Junin (JUNV) and lymphocytic choriomeningitis (LCMV), in the same region of Argentina, has been known since the early 70's, and records of clinical and subclinical human infections by one and/or the other agent have been continuously produced for the last 25 years. Anti-LCMV antibody is currently searched only by indirect immunofluorescence, a test that shows cross reactions among a number of arenaviruses yielding, in the cases of LCMV and JUNV consecutive infections, a concomitant seroconversion for both viruses, as an inconclusive diagnostic result. In contrast, neutralization (NT) tests reveal arenavirus antibodies directed to unique epitopes on these virus envelopes, thus allowing to disclose the sequence in the cases of consecutive infections. In this paper, the characteristics of neutralization (NT) test for LCMV in cell cultures are described, as well as its performance in the field diagnosis of LCMV human infections. The native LCMV strain Cba An 13065 was inoculated on L-929 cell (ATCC CCL 1), and procedures were followed to perform a constant virus-variable serum NT test. Final points of sera titrations were expressed as the maximal serum dilution that yielded 75% of pfu inhibition. This NT test was assayed on paired serum samples of 36 patients with confirmed Argentine hemorrhagic fever (AHF) (a disease caused by JUNV), who had had a known previous contact with LCMV through IFI. The use of this one test led to confusing diagnosis of the disease due to concomitant seroconversion for JUNV and LCMV. By using NT test, it was shown that: some of them were possibly not infected by LCMV, and that 30/36 cases (83.3%) had a pre-existing level of LCMV antibody, with titers in the range of 5 to 640, remaining unchanged 60 days after the clinical AHF. This shows that NT antibodies to LCMV are not influenced by the outcome of the immune response to JUNV, thus confirming the efficiency of NT test as identificator among arenaviruses. To assess the performance of this NT test in individuals having only IFI antibodies to LCMV, 126 serum samples obtained through serological surveillance in a rural area of Argentina, were used. It was found that NT had improved coincidence with IFI as IFI titers increased. Interpretations were based on the pan-arenavirus antibody response obtained by using IFI as the only test. Results presented herein prove that the described NT test is a valuable tool for the detection of LCMV infections, particularly when a previous infection with LCMV has to be demonstrated during the acute phase of Argentine hemorrhagic fever.

Lutheran (Lu) blood group antigens and the basal cell adhesion molecule antigen reside on two glycoproteins that belong to the Ig superfamily (IgSF) and carry five Ig-like extracellular domains. These glycoproteins act as widely expressed adhesion molecules and represent the unique receptors for laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF domains responsible for binding to laminin. In plasmonic resonance surface experiments, only recombinant Lu proteins containing the N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to inhibit binding of laminin to Lu-expressing K562 cells. Mutant recombinant proteins containing only IgSF domain 1, domains 1 + 2, domains 1 + 3, domains 2 + 3, domain 3, domain 4, domain 5, and domains 4 + 5 failed to bind laminin as well as a construct containing all of the extracellular domains except domain 3. Altogether, these results indicate that IgSF domains 1-3 are involved in laminin binding and that a specific spatial arrangement of these three first domains is most probably necessary for interaction. Neither the RGD nor the N-glycosylation motifs present in IgSF domain 3 were involved in laminin binding.

Traditional materials for bone repair or replacements such as autografts and allografts have a limited supply and other complications. Thus, alternative materials need to be explored. Three-dimensional, porous composites prepared from bioresorbable polymers and hydroxyapatite or other calcium phosphate ceramics are promising materials for the repair or replacement of diseased or damaged bone. However, in many cases the ceramic component of these composites is crystalline in nature, while bone apatite is made of a poorly crystalline, carbonated phosphate system. In this study, we synthesized a noncrystalline, carbonated calcium phosphate ceramic by carrying out the reaction within bioresorbable PLAGA microspheres using a modified emulsion/solvent evaporation technique, making each individual microsphere a composite. Sintering the composite microspheres together yielded a bioresorbable, porous, 3-dimensional scaffold that may be ideal for tissue ingrowth, making this composite scaffold potentially suitable for bone repair applications., (Copyright 2001 John Wiley & Sons, Inc.)

The activity of lymphocytic choriomeningitis virus (LCMv) in Argentina has been previously reported on the basis of serological evidence in rodents and humans and the isolation of only one strain of LCMv from a Mus domesticus captured in the province of Córdoba. The aim of this paper was to register patients with serological diagnosis of LCM, to isolate and to identify human strains of LCMv in Argentina. During the last 19 years, 15 cases were diagnosed as LCM by immunoflourescent indirect assay (IFI) and enzyme-linked immunosorbent assay (ELISA) but when neutralizing assay (NT) was incorporated, eight cases were classified as confirmed, three as probable and four as negative. The geographic distribution of the cases included three provinces: Córdoba, Buenos Aires and Santa Fe. Viral isolation was attempted in five patients classified as confirmed and only two resulted positive (P5226 and P8573). They were identified as LCMv by IFI and NT. The coexistence of LCMv with other arenaviruses, such as Junin and Oliveros viruses, in the same area, raises the probability of interactions between them, which could modify the virulence and/or pathogenicity for humans associated to genomic changes. Future studies of antigenic, genomic and virulence variability of different Argentine strains of LCMv, as well as the systematic search for human infection, will contribute to define the importance of this viral agent in our country and to implement control measures.

Because of an aging population and increased occurrence of sports-related injuries, musculoskeletal disorders have become one of the major health concerns in the United States. Current treatments, although fairly successful, do not provide the optimum therapy. These treatments typically rely on donor tissues obtained either from the patient or from another source. The former raises the issue of supply, whereas the latter poses the risk of rejection and disease transfer. This has prompted orthopedic surgeons and scientists to look for viable alternatives. In recent years, tissue engineering has gained increasing support as a method to treat orthopedic disorders. Because it uses principles of engineering, biology, and chemistry, tissue engineering may provide a more effective approach to the treatment of musculoskeletal disorders than traditional methods. This chapter presents a review of current methods and new tissue-engineering techniques for the treatment of disorders affecting bone, ligament, and cartilage.

A colchicine release system utilizing biodegradable poly(phosphazenes) was investigated in vitro for intra-articular administration. Polymer degradation and drug release studies were performed on colchicine-loaded poly(phosphazenes) containing either imidazolyl (I-PPHOS) or ethyl glycinato (EG-PPHOS) side chain substituents over a 21-day period. To study the effects of an implantable colchicine-PPHOS delivery system on local musculoskeletal tissue in vitro, osteoblast-like cells were grown on the matrices. Colchicine release was 20% for I-PPHOS and 60% for EG-PPHOS over the 21-day period. Release appeared to proceed through a combination of diffusional and degradative mechanisms. Environmental scanning electron microscopy (ESEM) studies revealed large pores in the drug-depleted devices in contrast to the control matrices without drug, which may have contributed to the release seen, especially with ethyl glycinato-containing matrices. Cell growth on matrices containing colchicine was significantly (p < 0.05) inhibited in contrast to growth on tissue culture polystyrene (TCPS) and EG-PPHOS matrices without drug. The in vitro cell kinetic data suggest that designs for in vivo studies must take into account possible toxicity of colchicine and the polymer matrix on local tissue. Biodegradable PPHOS systems are promising candidates for use as intra-articular delivery vehicles for drugs with potential for systemic toxicity.

Argentine hemorrhagic fever (AHF), caused by the arenavirus Junin, is a major public health problem among agricultural workers in Argentina. A prospective, randomized, double-blind, placebo-controlled, efficacy trial of Candid 1, a live attenuated Junin virus vaccine, was conducted over two consecutive epidemic seasons among 6500 male agricultural workers in the AHF-endemic region. Twenty-three men developed laboratory-confirmed AHF during the study; 22 received placebo and 1 received vaccine (vaccine efficacy 95%; 95% confidence interval [CI], 82%-99%). Three additional subjects in each group developed laboratory-confirmed Junin virus infection associated with mild illnesses that did not fulfill the clinical case definition for AHF, yielding a protective efficacy for prevention of any illness associated with Junin virus infection of 84% (95% CI, 60%-94%). No serious adverse events were attributed to vaccination. Candid 1, the first vaccine for the prevention of illness caused by an arenavirus, is safe and highly efficacious.

To elaborate a set of serological tests for the diagnosis of Argentine haemorrhagic fever (AHF), an enzyme-linked immunosorbent assay (ELISA) for detection of specific anti-Junin virus (JV) IgG is described, and its performance is compared with that of the plaque reduction neutralization test (PRNT). The reproducibility, sensitivity, specificity, and confidence limits for positive and negative results for ELISA were statistically analysed. The value of 800 was demonstrated as the lowest positive titer. Titers > or = 800 varied within one (two-fold) dilution in 95.6% of the tests, while the sensitivity and specificity were 99.2% and 98.8%, respectively. The assay yielded 1% of false positives and 0.05% of false negatives. A comparison of ELISA to PRNT in detecting the seroconversion for JV was studied by the chi square test (comparison of proportions in paired samples) and the K parameter for agreement proportion. Comparison of ELISA to PRNT showed no significant difference in the proportions of positive and negative results of these assays (P < 0.01), demonstrating an equivalent performance (K = 0.98) in the diagnosis of AHF. In addition, the simplicity and safety of the procedures involved make this ELISA the most suitable test to detect natural human JV infections.

A novel biodegradable polymer blend was developed for potential biomedical applications. A 50:50 poly(lactide-co-glycolide) (PLAGA) was blended in a 50:50 ratio with the followiing polyphosphazenes (PPHOS): poly[(25% ethyl glycinato)(75% p-methylphenoxy)phosphazene[, poly[(50% ethyl glycinato)(50% p-methylphenoxy)phosphazene], and poly[(75% ethyl glycinato)(25% p-methylphenoxy)phosphazene] to obtain Blends A, B, and C, respectively, using a mutual solvent technique. The miscibility of these blends was determined by measuring their glass transition temperature (Tg) using differential scanning calorimetry. After fabrication using a casting technique, the degradation of the matrices was examined. Differential scanning calorimetry showed one glass transition temperature for each blend which was between the Tg's of their respective parent polymers indicating miscibility of the blends. Surface analysis by scanning electron microscopy showed the matrices to have smooth uniform surfaces. Degradation studies showed near-zero order degradation kinetics for the blends with Blends A and B losing 10% of their mass after two weeks and Blend C degrading more rapidly (30% mass loss during the same period). These findings suggest that these novel biodegradable PLAGA/PPHOS blends may be useful for biomedical purposes.

The Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper we report the nucleotide sequences of S RNA of Candid #1 and its more virulent ancestors XJ#44 and XJ (prototype). Their relationship to Junin virus wild-type MC2 strain and other closely and distantly related arenaviruses was also examined. Comparisons of the nucleotide and amino acid sequences of N and GPC genes from Candid #1 and its progenitor strains revealed some changes that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype.

For Argentine Hemorrhagic Fever, a disease caused by Junin virus (JV), there is an effective treatment, consisting of the transfusion of immune plasma (IP). This plasma is obtained from individuals who have had the disease. Since Hepatitis C virus (HCV) is transmitted parenterally, this study was aimed to estimate the prevalence of anti-HCV in a population of IP donors. In this study, 376 donors (47 females and 329 males) were studied: 95 individuals (24 females and 71 males) who had had FHA but had not received treatment and 88 laboratory workers (57 females and 31 males) who were included as controls. Serum samples were tested by EIA (Abbott, Germany) for HCV, and later confirmed by LIATEK (Organon, Ireland). Antibodies to HCV were detected in 29/376 donors (7.7%), in only 1/95 (1.0%) untreated convalescents of AHF and in 1/ 88 (1.1%) of laboratory workers. Retrospective analysis of the seroconversion for HCV in these individuals demonstrated that in 16/24 donors (66.6%) the infection by HCV was probably associated with the IP transfusion. The data presented herein show how the infection with HCV was disseminated among donors of IP, stressing the risk associated to transfusional practices, and emphasizing the need of vaccination to prevent AHF and also the risk inherent to its treatment.

A new class of pH-sensitive hydrogels has been designed and synthesized. These are novel polyphosphazenes that bear various ratios of sodium oxybenzoate and methoxyethoxyethoxy side groups. These water-soluble macromolecules were cross-linked by 60Co gamma irradiation and the products were allowed to absorb water to form hydrogels. The hydrogels had higher equilibrium degrees of swelling in basic than in acidic buffer solutions, and polymers with a higher loading of the ionic side group showed higher swellability than those with a lower loading of this side group. The effects of ionic strength, cation charge and radiation dose on the degree of swelling were also studied. A study of the diffusion of the dye Biebrich Scarlet from the hydrogels showed complete release of the dye in 4-12 h in pH 7.4 buffer solution but significantly lower release at pH 2 even after 48 h. The release rate also varied as the side-group ratios were changed. The prehydrogel polymers were synthesized via the macromolecular substitution reactions of poly(dichlorophosphazene) with sodium methoxyethoxyethoxide and the sodium salt of propyl 4-hydroxybenzoate, followed by ester hydrolysis to yield the sodium carboxylate. The hydrogels are of interest for possible use as pH-sensitive membranes and for a number of potential biomedical applications.

The activity of lymphocytic choriomeningitis virus (LCMV) in the endemic area of Argentine hemorrhagic fever has been previously reported and represents the first evidence of the coexistence of two arenaviruses pathogenic for humans, Junin and LCMV, in the same geographic area. Data are presented on the prevalence of LCMV human infection in a 10,000-km2 area located in Santa Fe Province, Argentina. Study subjects were males, 15-65 years old, living and/or working in the rural area of 41 localities. One serum sample was obtained from each 7,227 volunteers from a total population of 21,340 individuals with the described features. Antibodies to LCMV were assessed by means of an indirect immunofluorescence assay. These antibodies were found in 172 serum samples, with titers ranging from 1:8 to 1:128 (geometric mean titer = 15.03), and a mean percentage of infection of 2.38%. A significantly different distribution of positive individuals was found between the eastern (1.54%) and western (3.07%) borders of the region studied (P < 0.0003). The higher percentage of infection on the western side was due to the existence of two clusters of counties with a mean percentage of 6.06% that was significantly different from the 1.67% obtained in the rest of the study area (P < 0.0003). These results provide new information on the LCMV activity in Argentina, and update the evidence on the coexistence of two arenaviruses in the same region of Argentina. This circumstance increases the probability of generation of viral reassortants with changes that could determine the need for new therapeutic and/or preventive strategies for arenaviral diseases.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMNL) of patients with Argentine hemorrhagic fever (AHF) were tested as effectors (E) of antibody-dependent cell cytotoxicity (ADCC). 51Cr labeled chicken red blood cells (CRBC) coated with anti-CRBC or normal rabbit serum were used as targets (T). Three ADCC assays were performed with both effectors from patients: on admission (I), 4 days after the transfusion of immune plasma (II), and 30 days after the clinical onset (III). The ADCC values obtained displayed high variation between individuals. From the linear portions in the curves representing specific 51Cr release vs. E:T ratio plots, extrapolations were made to determine lytic units (LU), defined here as effector concentrations required to lyse 50% of the targets. The results were expressed as LU in 10(6) effector cells. The killing activity ranges of patients' PMNL (I = 1.04 +/- 0.34; II = 2.22 +/- 0.66; and III = 2.08 +/- 1.18) were not significantly different from that of 21 normal controls (1.19 +/- 0.36), except for range II (P less than 0.01). ADCC activity ranges of patients' PBMC (I = 3.40 +/- 1.06; II = 3.16 +/- 1.60; and III = 1.93 +/- 0.42) were not significantly different from that determined in 12 healthy subjects (1.86 +/- 0.40). These results demonstrate that patients' PBMC and PMNL can perform ADCC with efficiency comparable to normal effector cells, during the acute period of AHF, and in early convalescence. Consequently, ADCC can be a relevant mechanism in the clearance of Junin virus-infected cells.

Cold lymphocytotoxins have been described extensively in many situations. In the present work, this kind of antibody has been found less frequently than previously in some diseases like infectious mononucleosis, rheumatoïd arthritis, or kidney transplanted patients. These discrepancies may be due to technical considerations. In contrast, cold lymphocytotoxins, in the present work, have been frequently detected in auto-immune hemolytic anemias with cold agglutinins, and in patients having immuno-deficiencies.

Peripheral blood lymphocyte subpopulations were studied in 15 patients with Argentine Hemorrhagic Fever (AHF), during the acute period of the disease and in early convalescence. Anti-human Ig antibodies were used to identify B cells and monoclonal antibodies to assess T4 and T8 subsets. During the acute period of the disease, significant alterations were found in B, T4, and T8 lymphocytes (P less than .001), as well as in T4/T8 ratios (P less than .001). These abnormalities disappeared in early convalescence, around 30 days after the clinical onset. Diminished numbers of T4 lymphocytes are interpreted as relevant to the immunodepression that characterizes the acute phase of AHF.

The possibility that a plasmid carried by Klebsiella pneumoniae plays a role in the pathogenesis of ankylosing spondylitis was explored. K. pneumoniae K21 contains a congruent to 25-kb plasmid, but this plasmid is not present in lymphocyte DNAs of ankylosing spondylitis HLA-B27 patients, as demonstrated by molecular hybridization experiments.

An enzyme-linked immunosorbent assay (ELISA) was evaluated for the quantitation of anti-Junin virus (JV) antibodies, in 83 selected cases of Argentine haemorrhagic fever (AHF). Serum samples were studied in two groups to facilitate comparative analysis; the first group was ELISA with indirect immunofluorescence (IF) test, in the second ELISA with plaque reduction neutralization test (PRINT). From the results obtained by using ELISA and IF on the same serum samples, a clear tendency of ELISA to demonstrate seroconversion for JV earlier and at higher frequency than IF test was noted. Simultaneous titration of specific antibodies by ELISA and PRNT tests rendered significantly correlated titers (r = 0.81), both methods being equivalently specific (100%). The demonstration of specific antibodies by ELISA in two cases that were undetected by the PRNT test resulted in a higher sensitivity index for ELISA than for PRNT (100% vs 97%). It is concluded that ELISA could efficiently replace IF and PRNT tests for the diagnosis of AHF.

Detection of viremia was attempted by three different methods in 30 cases of Argentine hemorrhagic fever (AHF). Cocultivation of peripheral blood mononuclear cells (PBMC) with Vero cell monolayers was the most sensitive, detecting Junin virus (JV) in 96% of the cases. Inoculation of whole blood into suckling mice and on Vero cells rendered 53 and 46% of positive isolations, respectively. The results presented suggest that PBMC are infected with JV during the acute period of AHF. JV was isolated with decreasing frequency up to 3 days after treatment with immune plasma, but no virus was recovered from PBMC during early convalescence.