Generation of a human control iPS cell line (ESi080-A) from a donor with no rheumatic diseases.

Here, we report the establishment of the human iPS cell line N1-FiPS4F#7 generated from skin cells of a patient with no rheumatic diseases, thus obtaining an appropriate control iPS cell line for researchers working in the field of rheumatic diseases. The reprogramming factors Oct4, Sox2, Klf4 and c-Myc were introduced using a non-integrating reprogramming strategy involving Sendai Virus.

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research.

The unavailability of sufficient numbers of human primary cells is a major roadblock for in vitro repair of bone and/or cartilage, and for performing disease modelling experiments. Immortalized mesenchymal stromal cells (iMSCs) may be employed as a research tool for avoiding these problems. The purpose of this review was to revise the available literature on the characteristics of the iMSC lines, paying special attention to the maintenance of the phenotype of the primary cells from which they were derived, and whether they are effectively useful for in vitro disease modeling and cell therapy purposes. This review was performed by searching on Web of Science, Scopus, and PubMed databases from 1 January 2015 to 30 September 2019. The keywords used were ALL = (mesenchymal AND ("cell line" OR immortal*) AND (cartilage OR chondrogenesis OR bone OR osteogenesis) AND human). Only original research studies in which a human iMSC line was employed for osteogenesis or chondrogenesis experiments were included. After describing the success of the immortalization protocol, we focused on the iMSCs maintenance of the parental phenotype and multipotency. According to the literature revised, it seems that the maintenance of these characteristics is not guaranteed by immortalization, and that careful selection and validation of clones with particular characteristics is necessary for taking advantage of the full potential of iMSC to be employed in bone and cartilage-related research.

Induced pluripotent stem cells for cartilage repair: current status and future perspectives.

The establishment of cartilage regenerative medicine is an important clinical issue, but the search for cell sources able to restore cartilage integrity proves to be challenging. Human mesenchymal stromal cells (MSCs) are prone to form epiphyseal or hypertrophic cartilage and have an age-related limited proliferation. On the other hand, it is difficult to obtain functional chondrocytes from human embryonic stem cells (ESCs). Moreover, the ethical issues associated with human ESCs are an additional disadvantage of using such cells. Since their discovery in 2006, induced pluripotent stems cells (iPSCs) have opened many gateways to regenerative medicine research, especially in cartilage tissue engineering therapies. iPSCs have the capacity to overcome limitations associated with current cell sources since large numbers of autologous cells can be derived from small starting populations. Moreover, problems associated with epiphyseal or hypertrophic-cartilage formation can be overcome using iPSCs. iPSCs emerge as a promising cell source for treating cartilage defects and have the potential to be used in the clinical field. For this purpose, robust protocols to induce chondrogenesis, both in vitro an in vivo, are required. This review summarises the recent progress in iPSC technology and its applications for cartilage repair.

In vitro repair model of focal articular cartilage defects in humans.

Articular cartilage lesions, which do not affect the integrity of subchondral bone, are not able to be repaired spontaneously, thus inducing cartilage degeneration and developing an arthrosic process. To avoid the need for prosthetic replacement, different cell treatments were developed with the aim of generating a repaired tissue with structure, biochemistry composition, and functional behavior equal or similar to those of natural articular cartilage.The following protocols describe the methods for harvesting articular cartilage explants both from pig and human specimens and isolating and culturing pig chondrocytes. Moreover, the methodology for an in vitro model of xenoimplant of pig chondrocytes in focal defects of human articular cartilage is described.

[Short duration of initial intravenous treatment in 70 pediatric patients with osteoarticular infections]. / Tratamiento intravenoso inicial abreviado en 70 pacientes pediátricos con infecciones osteo-articulares.

UNLABELLED: Osteoarticular infections (OAI) are infrequent in pediatrics and there is controversy on the need for prolonged use of intravenous antimicrobials. OBJECTIVE: To characterize and describe evolution and complications of a regimen of 7 days initial intravenous antibiotic treatment for OAI in children, completing 4-6 weeks of total treatment. PATIENTS AND METHODS: In a large pediatric hospital, 70 children younger than 15 years of age were diagnosed with OAI between March 2003 and December 2004. Children received 7 days of intravenous antibiotics followed by 3 to 5 weeks of oral treatment. RESULTS: Incidence of OAI in this hospital was 1.8:10000. Patients mean age was 6.4 +/-4.4 years and 60% presented with septic arthritis, 36% osteomyelitis and 4% osteoarthritis. In 80% of cases, the infection was located in the lower extremity. Positive cultures were obtained in 59% predominating Staphylococcus aureus (46.5%). Seven patients had prolonged pain or persistently high or increasing serum C reactive protein levels and were maintained on prolonged intravenous therapy. None of the 63 children with 7 day intravenous antimicrobials nor the 7 children with prolonged intravenous use developed a complication in the short-term follow up. CONCLUSIONS: Seven days of intravenous antibiotic for the initial phase of OAI treatment was effective in a majority of children and may be recommended.

Tratamiento intravenoso inicial abreviado en 70 pacientes pediátricos con infecciones osteo-articulares / Short duration of initial intravenous treatment in 70 pediatric patients with osteoarticular infections

Osteoarticular infections (OAI) are infrequent in pediatrics and there is controversy on the need for prolonged use of intravenous antimicrobials. Objective: To characterize and describe evolution and complications of a regimen of 7 days initial intravenous antibiotic treatment for OAI in children, completing 4-6 weeks of total treatment. Patients and methods: In a large pediatric hospital, 70 children younger than 15 years of age were diagnosed with OAI between March 2003 and December 2004. Children received 7 days of intravenous antibiotics followed by 3 to 5 weeks of oral treatment. Results: Incidence of OAI in this hospital was 1.8:10000. Patients mean age was 6.4 ±4.4 years and 60 percent presented with septic arthritis, 36 percent osteomyelitis and 4 percent osteoarthritis. In 80 percent of cases, the infection was located in the lower extremity. Positive cultures were obtained in 59 percent predominating Staphylococcus aureus (46.5 percent). Seven patients had prolonged pain or persistantly high or increasing serum C reactive protein levels and were maintained on prolonged intravenous therapy. None of the 63 children with 7 day intravenous antimicrobials nor the 7 children with prolonged intravenous use developed a complication in the short-term follow up. Conclusions: Seven days of intravenous antibiotic for the initial phase of OAI treatment was effective in a majority of children and may be recommended.

Las infecciones osteoarticulares (IOA) son poco frecuentes en pediatría. Existe controversia acerca de la óptima duración y la vía de administración de la terapia antimicrobiana. Objetivo: Caracterizar y describir la evolución y complicaciones en niños con IOA que recibieron 7 días iniciales de terapia endovenosa, completando 4 a 6 semanas de terapia total. Pacientes y Métodos: Estudio descriptivo, que incluyó a 70 niños con diagnóstico de IOA, entre marzo de 2003 y diciembre de 2004 en un hospital pediátrico, quienes recibieron tratamiento endovenoso abreviado a 7 días, seguido de terapia oral por 3 a 5 semanas. Resultados: La incidencia de IOA fue de 1,8: 10.000 niños bajo 15 años de edad. El promedio de edad fue 6,4 ± 4,4 años. Sesenta por ciento presentaba artritis séptica, 36 por ciento osteomielitis y 4 por ciento osteoartritis. En 80 por ciento la infección se localizó en extremidades inferiores. Los cultivos fueron positivos en 59 por ciento. En 46,5 por ciento se aisló Staphylococcus aureus. Siete niños evolucionaron con dolor persistente o elevación significativa de PCR y requirieron prolongar la terapia endovenosa y/o total, sin presentar complicaciones a corto plazo. Conclusiones: Siete días de terapia antimicrobiana intravenosa parece ser efectivo y recomendable para el tratamiento, en su fase inicial, de las infecciones osteo-articulares.

[Human metapneumovirus as hospitalization cause in children under 3 years old with acute respiratory infections during 2004]. / Metapneumovirus humano como causa de hospitalización en niños bajo 3 años de edad, con infección respiratoria aguda, durante el año 2004.

Human metapneumovirus was detected in 15 of 123 children (12%) younger than 3 years of age hospitalized for treatment of acute respiratory infection between July and November 2004. The virus was detected by RT-PCR directly from nasopharyngeal swabs and/or from supernatants after cell culture. Children infected with hMPV were mostly younger than one year of age (67%), all presenting with fever and cough. The main cause for hospitalization was the need for oxygen therapy (73%). Four hMPV positive children had an identifiable co-morbid condition but had a similar clinical evolution when compared to previously healthy infants. Chest radiography showed an increase in interstitial infiltrates with focal consolidation in 6 children. Obstructive bronchial syndrome and bronchiolitis, with or without pneumonia, were the most frequent diagnosis associated with hMPV positivity. A rapid and sensitive diagnostic method is required to improve diagnosis and treatment of these patients.

Metapneumovirus humano como causa de hospitalización en niños bajo 3 años de edad, con infección respiratoria aguda, durante el año 2004 / Human metapneumovirus as hospitalization cause in children under 3 years old with acute respiratory infections during 2004

Metapneumovirus humano (MPVh) fue detectado entre julio y noviembre en 15 de 123 niños bajo 3 años de edad hospitalizados por infección respiratoria aguda (12 por ciento). Las muestras fueron estudiadas mediante técnicas de biología molecular (RPC-TR de muestra de hisopado nasofaríngeo y/o de sobrenadante de cultivo). El 67 por ciento de los niños hospitalizados con MPVh tenían menos de 1 año de edad, todos ellos presentaron tos y fiebre y el principal motivo de hospitalización fue el requerimiento de oxígeno en 73 por ciento de los casos. Si bien un tercio de los pacientes tenía patología previa, su evolución clínica no fue diferente respecto de los niños previamente sanos. El patrón radiológico mostró aumento de la trama intersticial, con focos de consolidación en 6 casos (40 por ciento). El diagnóstico más frecuente fue síndrome bronquial obstructivo o bronquiolitis, asociado o no a neumonía. Destaca la necesidad de un método de diagnóstico rápido para optimizar el diagnóstico diferencial, manejo y control de infecciones en estos pacientes.

Human metapneumovirus was detected in 15 of 123 children (12 percent) younger than 3 years of age hospitalized for treatment of acute respiratory infection between July and November 2004. The virus was detected by RT-PCR directly from nasopharyngeal swabs and/or from supernatants after cell culture. Children infected with hMPV were mostly younger than one year of age (67 percent), all presenting with fever and cough. The main cause for hospitalization was the need for oxygen therapy (73 percent). Four hMPV positive children had an identifiable co-morbid condition but had a similar clinical evolution when compared to previously healthy infants. Chest radiography showed an increase in interstitial infiltrates with focal consolidation in 6 children. Obstructive bronchial syndrome and bronchiolitis, with or without pneumonia, were the most frequent diagnosis associated with hMPV positivity. A rapid and sensitive diagnostic method is required to improve diagnosis and treatment of these patients.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test.

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Endocarditis por Streptococcus pneumoniae en niños: presentación de un caso clínico y revisión de la literatura / Endocarditis caused by Streptococcus pneumoniae in children: case report and review

La endocarditis causada por Streptococcus pneumoniae es una patología muy poco frecuente en niños, correspondiendo sólo a 3 - 7 por ciento de los casos. Sin embargo, su importancia radica en que se puede presentar de forma muy agresiva, con complicaciones como destrucción valvular y abscesos, y con una mortalidad reportada hasta 61 por ciento, de no mediar tratamiento antimicrobiano precoz y muchas veces cardiocirugía. En más del 50 por ciento se puede asociar a otros focos infecciosos, como meningitis, neumonía, sinusitis o mastoiditis. Se describe el caso de una lactante de 10 meses que presentó una meningitis asociada a endocarditis debidas a S. pneumoniae, con grave compromiso cardíaco, y que requirió reemplazo valvular. Se realizó una revisión de la literatura médica acerca de endocarditis por S. pneumoniae en niños.

Effect of medium composition on the production of tetanus toxin by Clostridium tetani

The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G0) and N-Z Case TT (NZ0) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 Lf/mL), achieved at G0= 9.7 g/L and NZ0= 43.5 g/L, was 80% higher than that obtained with standard cultivations (G0= 8.0 g/L and NZ0= 25.0 g/L).

Use of sephacryl high resolution (HR) in tetanus anatoxin purification.

The tetanus purified anatoxin is used in the preparation of the tetanus toxoid and multiple vaccines (dT, DT and DTP), all of them strictly following specifications established by the WHO with a minimum antigenic purity equal to 1,000 Lf/mgPN. Aiming to establish more sensitive and accurate methods for purification, samples from four different lots of tetanus anatoxin were submitted to gel filtration in twenty independent trials using the Sephacryl S-100 HR and S-200 HR resins. The Authors were careful to optimize their parameters of performance as to sample volume, elution and selectivity flow for tetanus anatoxin purification, allowing their use in industrial scale. The Sephacryl S-100 HR resin presented the best selectivity, that is, the best separation, allowing a greater linear-flow and, consequently, the best purity index. Satisfactory results were also achieved with the Sephacryl S-200 HR resin after optimization of chromatographic parameters for elution flow and volume of the sample applied. The good results of purification obtained, as well as the high chemical stability, have pointed out both the Sephacryl S-100 HR and S-200 HR resins as equally efficient for industrial production.

Development and validation study for the chromatographic purification process for tetanus anatoxin on Sephacryl S-200 High Resolution.

The tetanus purified anatoxin is used in the preparation of multiple immunoprophylactics. WHO (World Health Organization) specifies that the tetanus anatoxin must exhibit a degree of purity greater than or equal to 1,000 Lf/mg protein nitrogen (PN). Today liquid chromatography is a well established technique for the purification of tetanus anatoxin and several different methods are used in production scale. On a small scale, we purified tetanus anatoxin on Sephacryl S-200 High Resolution (gel filtration) and we obtained a successful high-yield purification. On the basis of these results, by combining conventional tangential flow filtration (TFF) at 50,000 N.M.W.L. (Nominal Molecular Weight Limit) ultrafiltration membrane with gel filtration on Sephacryl S-200 High Resolution, we have been able to purify 14 lots of tetanus anatoxin using the Bioprocess System (Amersham Pharmacia Biotech) to a large scale operation. Using this method, 77,401,332 doses of tetanus toxoid were prepared in 14 consecutive lots, supporting the reproducibility and reliability of the method presented here.

Production of an effective anti-Bothrops-tetanus mixed hyperimmune serum of equine origin

The present investigation reveals the possibility of simultaneous immunization of horses with Bothrops or Crotalus snake venoms and Tetanus antigens for the production of anti-Bothrops-Tetanus or anti-Crotalus-Tetanus mixed serum, with high titers of the respective specific antibodies. Bothrops antivenoms with an average neutralizing titer of 4.16 mg venom/ml were obtained from plasma of horses with titers lower than 0.5 mg venom/ml when Tetanus antigens were not used. This suggests the existence of a synergism between Bothrops venoms and Tetanus antigens in the stimulation of the antibody response. The pooled plasma of the animal had a neutralizing titer of 21.0 mg/ml reference Bothrops venoms and 3,300 IU/ml to Tetanus antigens after purification by enzymatic digestion and ammonium sulphate precipitation. These experiments lead us to conclude that Bothrops envenomation therapy can be successfully performed using Anti-Bothrops-Tetanus serum also serving as Tetanus prophylaxis. anti-Crotalus-Tetanus serum can also be produced, although it is not of medical interest as Crotalus envenomation rarely results in local necrotizing lesions.

Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin.

Casein pancreatic digest is the basic bacterial growth medium used for diphtheria, botulinum and tetanus toxin vaccine production. It is known that the variation in the peptide content of the casein digest directly affects final toxin yields. In this study, the identification and sequences of eight peptides, four to eight amino acids in length, of casein pancreatic digestion, which seem to be involved in the enhancement of tetanus toxin production, are described. They all contain one or two residues of proline/molecule and a predominance of hydrophobic amino acid residues. The most active peptides show a general structure of Pro-aromatic-Pro, and this pattern resembled the motif displayed by bradykinin-potentiating peptides found in snake venoms. By analogy with the mechanism of bradykinin potentiation through inhibition of the proteolytic degradation of bradykinin, it is suggested that the six peptides identified here could protect the tetanus toxin from proteolysis, once secreted by the bacteria.

An effective toxoid from tetanus toxin prepared by stepwise iodination of purified toxin.

Purified samples of tetanus toxin were gradually iodinated by stepwise addition of iodine, up to saturation. The residual capacity of each sample to provoke tetanus was tested by injecting sc 25 Lf (Limit of flocculation) in mice. Toxicity diminished in relation to iodine incorporated, and with iodine-saturated samples, doses of up to 100 Lf in mice, or 500 Lf in guinea-pigs, proved innocuous. Mice immunized with two doses of this toxoid adsorbed on Al(OH)3, and challenged with standard lethal toxin, gained protection against 10 MLD (Minimum Lethal Doses). Guinea-pigs were immunized by a single sc dose of 27.5 Lf with fluid toxoid, and all resisted challenge against 10 MLD applied 30 days later. The mice sera gave strong immunoprecipitation lines against the native toxin. The findings indicated that by controlled iodination of tetanus toxin an effective and inexpensive toxoid can be prepared.