Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors.

INTRODUCTION: Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. METHODS: PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. RESULTS: Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 10(6) to 1.9 × 10(6) platelets/µl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-ß3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF. CONCLUSIONS: Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.

Pulmonary histoplasmosis: unusual histopathologic findings.

Four patients with clinical diagnosis of interstitial lung disease (ILD) are presented. In these patients, lung biopsies revealed bronchocentric granulomatosis (BG), pulmonary alveolar proteinosis (PAP), diffuse alveolar damage (DAD), and in one biopsy, the clinical manifestations suggested tuberculous primo-infection with systemic dissemination. Three patients died without diagnosis. In all four cases, specific histological stains found Histoplasma capsulatum. Histoplasmosis may mimic other infectious or non-infectious pulmonary diseases, such as interstitial and granulomatous pulmonary disease. Therefore, the absolute need for identification of the organism by culture or special stains cannot be over-emphasized and may lead to a proper mycological diagnosis. This highlights the importance of differential diagnosis with systemic infectious diseases, especially in areas where deep-seated mycosis are endemic.

Identificación ultraestructural de Ehrlichia sp en un perro de Venezuela infectado experimentalmente / Ultraestructural identification of Ehrlichia spïin experimental infected dog from Venezuela

El presente estudio constituye el primer reporte en Venezuela de las características ultraestructurales de Ehrlichia sp en células mononucleares de sangre periférica en un perro infectado experimentalmente, utilizándose para ello microscopía electrónica de transmisión. El animal desarrolló manifestaciones clínicas características de la infección y al realizar los extendidos sanguíneos coloreados con Giemsa modificado, se observaron al microscopio de luz las inclusiones intracitoplasmáticas características en células mononucleares. Al microscopio electrónico se evidenciaron microorganismo extremadamente pleomórficos que se corresponden con los cuerpos elementales dentro de vacuolas citoplasmáticas, rodeados por una doble membrana, cada uno constituido por gránulos electrodensos, características ultraestructurales que identifican al género Ehrlichia

La vellosidad placentaria en caso de disritmia cerebral o epilepsia / Placental villus in case of cerebral dysrhythmia or epilepsy

Con el propósito de conocer el estado de la barrera placentaria en caso de una paciente con epilepsia, muestras de placenta fueron tomadas inmediatamente posparto y observadas con las técnicas convencionales de microscopia de luz y electrónica. Especímenes con los de embarazo sin epilepsia, con técnicas de fijación por inmersión controles apareados de la misma edad y muestreo sistemático del placentoma revelaron: vellosidades de aspecto similares a las observaciones en caso de hipermadurez o pre-término, preeclapsia y ocasionales figuras de vellosidades intermedias maduras con deficiencia de la vellosidad terminal. Se observó hipercapilaridad, edema del estroma y vacuolas en el sincitio y estroma. A pesar de que no hubo resultado fetal adverso las imágenes son indicadoras e ilustrativas de hipoxia temporal y ligero sufrimiento fetal de interés clínico para el manejo de este tipo de paciente de alto riesgo