Decellularization of placentas: establishing a protocol

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Índice de massa corporal, circunferência da cintura e hipertensão arterial em estudantes / Índice de masa corporal, circunferencia de la cintura y hipertensión arterial en estudiantes / Body mass index, waist circumference, and arterial hypertension in students

RESUMO Objetivo: investigar qual o melhor preditor antropométrico de hipertensão arterial em alunos de escolas privadas. Método: estudo transversal, com amostra composta por 286 alunos com idade de 10 a 14 anos de duas escolas privadas de Paranavaí-Paraná. As variáveis analisadas foram: índice de massa corporal, circunferência de cintura e pressão arterial. Na análise estatística foram utilizados os testes de correlação parcial de Pearson e a regressão logística multivariada, considerando-se p<0,05. Resultados: os dois indicadores antropométricos demonstraram fracas correlações com os níveis sistólicos e diastólicos, com coeficientes (r) variando de 0,27 à 0,36 (p< 0,001). Na análise multivariada, o único indicador antropométrico associado ao risco de hipertensão arterial foi a circunferência de cintura (OR= 2,3; IC 95%: 1,1-4,5) independente da idade e gênero. Conclusão: nesta faixa etária, a circunferência de cintura parece ser melhor do que índice de massa corporal como preditor de hipertensão arterial. .

RESUMEN Objetivo: investigar cuál es el mejor predictor antropométrico de la hipertensión arterial en los alumnos de escuelas particulares. Métodos: estudio transversal con muestra compuestas por 286 alumnos con edad de 10 a 14 años de dos escuelas privadas de Paranavaí-Paraná. Las variables analizadas fueron: índice de masa corporal, circunferencia de la cintura y la presión arterial sistólica y diastólica. En el análisis de estadísticas fueron utilizadas las pruebas de correlación parcial de pearson y regresión logística multivariada considerándose p<0.05. Resultados: los dos indicadores antropométricos han mostrado débiles correlaciones con los niveles sistólicos y diastólicos, con Coeficientes (r) variando de 0,27 a 0,36 (p<0,001). En el análisis multivariado el único indicador antropométrico asociado al riesgo de hipertensión arterial fue la circunferencia de la cintura (OR=2,3; IC 95%: 1,1- 4,5) independiente de la edad y el género. Conclusión: en este grupo de edad, la circunferencia de la cintura parece ser mejor de que el índice de masa corporal como predictor de la hipertensión arterial. .

ABSTRACT Objective: to investigate what is the best anthropometric predictor of arterial hypertension among private school students. Method: this was a cross-sectional study with 286 students between the ages of 10 and 14 from two private schools in the city of Paranavaí, Paraná, Brazil. The following variables were analyzed: body mass index, waist circumference and blood pressure. Statistical analysis was conducted with Pearson’s partial correlation test and multivariate logistic regression, with p<0.05. Results: both anthropometric indicators displayed weak correlation with systolic and diastolic levels, with coeffi cients (r) ranging from 0.27 to 0.36 (p < 0.001). Multivariate analysis showed that the only anthropometric indicator associated with arterial hypertension was waist circumference (OR= 2.3; 95% CI: 1.1-4.5), regardless of age or gender. Conclusion: this age group, waist circumference appeared to be a better predictor for arterial hypertension than body mass index. .

Amyloid-beta oligomers regulate the properties of human neural stem cells through GSK-3beta signaling

Alzheimer's disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-beta peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-beta oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-beta on NSCs derived from various brain regions at different developmental stages. However, the Abeta effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-beta oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-beta oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-beta oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-beta oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3beta-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Abeta oligomers on human NSCs.

The lesser occipital nerve in fetuses / El nervio occipital menor en fetos

The lesser occipital nerve (LON) is an ascending superficial branch of the cervical plexus that has a variable origin either from the ventral ramus of the second cervical nerve or second and third cervical nerves and is purely sensory. Forty fetuses (right side: 40/80; left: 40/80) with gestational ages between 15 to 28 weeks were microdissected to document the anatomy of the LON. Results: a) Incidence and Morphometry: LON present in 100 percent specimens, with average length on the right and left sides of 23.59 +/- 2.32 mm and 23.45 +/- 2.27 mm, respectively; b) Course: In its ascent towards the occipital region, the LON was located on the splenius capitus muscle in 85 percent of specimens and in 15 percent of the specimens, it ascended vertically on the sternocleidomastoid muscle towards the ear, innervating its superior third; c) Branching pattern: LON displayed (i) single: 70 percent; (ii) duplicate: 26 percent and (iii) triplicate: 4 percent patterns; d) Variation in the course of LON was observed in 6 percent of the specimens. Knowledge of the anatomy and variations of the LON may assist in the understanding of cervicogenic headaches and may be of assistance to anesthetists performing regional anesthesia for surgical procedures in the neck.

El nervio occipital menor (NOM) es una rama ascendente superficial del plexo cervical que tiene un origen variable ya sea del ramo ventral del segundo nervio cervical o de los nervios cervicales segundo y tercero, y es solamente sensitivo. Cuarenta fetos (lado derecho: 40/80; izquierdo: 40/80), con edades gestacionales de 15 a 28 semanas fueron microdisecados para documentar la anatomía del NOM. a) Incidencia y morfometría: el NOM estuvo presente en el 100 por ciento de los especímenes, con una longitud media de los lados derecho e izquierdo de 23,59 +/- 2,32 mm y 23,45 +/- 2,27 mm, respectivamente; b) Curso: en su ascenso hacia la región occipital, el NOM se localiza en el músculo esplenio de la cabeza en el 85 por ciento de las muestras y en el 15 por ciento de las muestras, ascendió verticalmente sobre el músculo esternocleidomastoideo hacia el oído, inervando el tercio superior, c) Patrón de ramificación: el NOM se observa (i) individual: 70 por ciento, (ii) duplicado: 26 por ciento y (iii) triplicado: 4 por ciento de los patrones; d) Variación en el curso de NOM se observó en el 6 por ciento de las muestras. El conocimiento de la anatomía y las variaciones del NOM puede ayudar en la comprensión de los dolores de cabeza cervical y puede ser de ayuda a los anestesiólogos a realizar la anestesia regional para procedimientos quirúrgicos en el cuello.

SRY sequence in maternal plasma: Implications for non-invasive prenatal diagnosis: First report from India.

AIM: The presence of circulatory cell-free fetal DNA in maternal plasma has found new applications in non-invasive risk-free prenatal diagnosis. MATERIALS AND METHODS: We made use of a size separation approach along with real time polymerase chain reaction (PCR) to evaluate the use of fetal DNA in the detection of the sex of the fetus. Cell-free fetal DNA was isolated from the plasma of 30 women (10–20 weeks gestation) using a size separation approach. We made use of Taq Man Chemistry and real time PCR using primers and probes for GAPDH and SRY. RESULTS: Only 24 cases could be studied as there was no amplification in six cases. Fetal sex was accurately determined in all of the 24 cases wherein 19 women were carrying male fetuses and five women were carrying female fetuses. An increase in the amount of fetal DNA was observed with an increase in the gestational age. CONCLUSIONS: Real time PCR analysis is a highly sensitive and accurate tool for non-invasive prenatal diagnosis, allowing detection of the sex of the fetus as early as 10 weeks of gestation. Non-invasive prenatal diagnosis eliminates the risk of fetal loss associated with the invasive procedure.

Dopplervelocimetria fetoplacentária em gestantes hipertensas e resultados perinatais segundo a idade gestacional / Fetal and placental Doppler velocimetry in hypertensive pregnant women and perinatal outcomes according to gestational age

OBJETIVO: Avaliar índices de pulsatilidade das artérias umbilical (IPAU) e cerebral média (IPACM) e relação do índice de pulsatilidade umbilico-cerebral (IPAU/IPACM) em fetos de gestantes hipertensas e presença de resultados perinatais adversos. MATERIAIS E MÉTODOS: Analisamos IPAU, IPACM e IPAU/IPACM de 289 fetos de gestantes hipertensas quanto à previsão dos resultados perinatais adversos. Os resultados foram comparados sem e com ajuste pela idade gestacional. RESULTADOS: O índice de Apgar < 7 no 5º minuto foi associado com resultados alterados após o ajuste por idade gestacional. O risco para recém-nascidos pequenos para a idade gestacional aumentou em três vezes após o ajuste, com significância estatística em todos os parâmetros do Doppler. Na síndrome da hipóxia neonatal o aumento do risco ajustado pela idade gestacional foi estatisticamente significante no IPAU e IPAU/IPACM. Não houve aumento no risco de síndrome do desconforto respiratório na análise ajustada. A mortalidade perinatal e o IPAU alterado apresentaram um risco três vezes maior e foram estatisticamente significantes após o ajuste. CONCLUSÃO: Em gestantes hipertensas, o IPAU apresentou melhor correlação com os resultados perinatais do que o IPACM ou relação IPAU/IPACM. O risco de resultados adversos deve considerar a idade gestacional.

OBJECTIVE: To evaluate the pulsatility index of umbilical artery (UAPI) and middle cerebral artery (MCAPI), as well as the umbilical-cerebral pulsatility (UAPI/MCAPI) ratio in fetuses of hypertensive pregnant women and associated adverse perinatal outcomes. MATERIALS AND METHODS: The authors have analyzed UAPI, MCAPI and UAPI/MCAPI ratio in 289 fetuses of hypertensive women, correlating the results with the presence of adverse perinatal outcomes. Results were compared with and without adjustment for gestational age. RESULTS: Apgar score < 7 at the 5th minute was associated with altered outcomes after adjustment for gestational age. The risk for small-for-gestational-age infant increased three times after such adjustment, with statistical significance for all the Doppler parameters. The increase in risk for neonatal hypoxia after adjustment for gestational age was statistically significant for UAPI and UAPI/MCAPI ratio. No increase was observed in the risk for respiratory distress syndrome in the adjusted analysis. A three-time higher risk for perinatal mortality and altered UAPI with statistical significance was observed after adjustment. CONCLUSION: In fetuses of hypertensive pregnant women, UAPI demonstrated better correlations with perinatal outcomes than MCAPI and UAPI/MCAPI ratio. The risk for adverse gestational outcome should be evaluated taking the gestational age into consideration.

Human telomerase catalytic subunit (hTERT) suppresses p53-mediated anti-apoptotic response via induction of basic fibroblast growth factor

Although human telomerase catalytic subunit (TERT) has several cellular functions including telomere homeostasis, genomic stability, cell proliferation, and tumorigenesis, the molecular mechanism underlying anti-apoptosis regulated by TERT remains to be elucidated. Here, we show that ectopic expression of TERT in spontaneously immortalized human fetal fibroblast (HFFS) cells, which are a telomerase- and p53-positive, leads to increases of cell proliferation and transformation, as well as a resistance to DNA damage response and inactivation of p53 function. We found that TERT and a mutant TERT (no telomerase activity) induce expression of basic fibroblast growth factor (bFGF), and ectopic expression of bFGF also allows cells to be resistant to DNA-damaging response and to suppress activation of p53 function under DNA-damaging induction. Furthermore, loss of TERT or bFGF markedly increases a p53 activity and DNA-damage sensitivity in HFFS, HeLa and U87MG cells. Therefore, our findings indicate that a novel TERT-bFGF axis accelerates the inactivation of p53 and consequent increase of resistance to DNA-damage response.

Irrigación de las cápsulas adrenales en humanos / Irrigation of the human adrenal capsules

Las cápsulas adrenales también conocidas como glándulas adrenales o suprarrenales, son 2 pequeñas estructuras, una derecha y otra izquierda, ubicadas en la región suprahiliar del borde interno de cada riñón. A pesar de su pequeño tamaño, su función dentro del sistema endocrino es indispensable para el equilibrio metabólico de los seres humanos, de allí que su aporte vascular viene a jugar un papel fundamental en su funcionamiento. Los diferentes textos de anatomía han descrito por años un patrón vascular clásico, sin embargo, éste presenta importantes variaciones en el origen y distribución de sus ramas. Trabajamos con fetos frescos, cuidadosamente disecados, perfundidos con vinil rojo en el sistema arterial, extrayendo un bloque visceral y resecando 66 glándulas adrenales. En nuestros resultados el patrón vascular clásico descrito por los libros de anatomía sólo se observó en el 15,38 por ciento de los casos. El origen de la arteria suprarrenal superior fue detectado en la arteria Aorta (46,15 por ciento), la arteria media también en la Aorta (53,85 por ciento) y la rama inferior igualmente en la Aorta abdominal en tres cuartas partes de los casos (61,54 por ciento sola y en un 12,31 por ciento junto a la arteria renal). La distribución vascular de la glándula derecha difiere de la izquierda en 36 (56,25 por ciento) de los casos

The adrenal glands also known as suprarenal glands, are two small structures located on the suprahiliar border of the kidney. They belong to the endocrine system. The Anatomical books describe for them a classic vascular pattern, nevertheless they really have important variations in origin and branching pattern. In this work we employed human fetuses, carefully dissected, vinyl perfused. The sample was constituted by 66 adrenal glands. After a very careful observation of the serie we found that the classic pattern was seen in 15.30 percent of the sample. Superior suprarenal artery was localized at the abdominal Aorta in 46.15 percent, the medial artery was arising from Aorta in 53.85. percent, and the inferior suprarenal artery was also found taking origin at abdominal Aorta,on its own in 61.54 percent , and in 12.31 percent together with the renal artery. We also notice that the vascular approach to right adrenal gland is different from that for the left gland in 56.25 percent

Consideraciones científicas y éticas en las perspectivas diagnósticas y terapéuticas en medicina fetal / Scientific and ethical perspectives of perinatal and fetal medicine

This review emphasizes the importance of recent developments and knowledge on cell biology and human genetics than have integrated, through a basic-clinical concept to an emerging branch of medicine, called Perinatal and Fetal Medicine. We discuss the possible role of fetal cells and DNA in the diagnosis and treatment of diseases in the intrauterine environment. The associated bioethical issues associated to these medical actions are discussed, considering the imminent use ofthese agents in the human species.

Regeneration of kidney tissue using in vitro cultured fetal kidney cells

Transplanting fetal kidney cells (FKCs) can regenerate kidney. This requires in vitro expansion in cell number to acquire enough cells for transplantation. However, FKCs may change their cellular characteristics during expansion and, thus, may not regenerate kidney tissue upon transplantation. We investigated how cell culture period affects cellular characteristics and in vivo regenerative potential of FKCs. As the passage number increased, cell growth rate and colony forming ability decreased while senescence and apoptosis increased. To examine in vivo regenerative potential, FKCs cultured through different numbers of passages were implanted into the parenchyma of kidneys of immunodeficient mice using fibrin gel for 4 wk. Histological analyses showed passage-dependent kidney tissue regeneration, and the regeneration was better when cells from lower number of passages were implanted. This result shows that in vitro culture of FKCs significantly affects the cell characteristics and in vivo tissue regenerative potential.

Avaliação da densidade folicular em ovários de fetos humanos / Follicular density evaluation in ovaries of human fetuses

OBJETIVO: avaliar a variação da população de folículos ovarianos ao longo do desenvolvimento fetal e acrescentar dados aos escassos, incompletos e, algumas vezes, divergentes dados descritos na literatura. MÉTODOS: doze ovários de fetos necropsiados foram estudados, sendo nove de fetos e três de neonatos. As idades dos fetos foram determinadas pela cronologia e por ultra-sonografia, enquanto os neonatos nascidos na 39ª semana de gravidez faleceram nas primeiras horas de vida. As peças foram fixadas com formaldeído e incluídas em parafina. Foram realizados cortes seriados com espessura de 7 mm e a cada 50 cortes, o material foi corado com HE e analisado com microscópio com aumento de 400 vezes. Foram contados os folículos em dez diferentes regiões do córtex ovariano, cada região com uma área de 625 mm². O número total de folículos em 1 mm³ foi calculado usando-se a fórmula: Nt=(No x St x t)/do, onde Nt é o número de folículos, No é a média de folículos observados em 1 mm², St é o total de cortes em 1 mm³ do ovário, t é a espessura do corte e do é o diâmetro médio do núcleo. RESULTADOS: a idade dos fetos variou de 24 a 39 semanas. O número de folículos por 0,25 mm² variou de 10,9 ± 4,8 em um neonato até 34,7 ± 10,6 também em um neonato. Entre os fetos, tivemos o menor valor com 36 semanas (11,1 ± 6,2) e o maior valor com 28 semanas (25,3 ± 9,6). O número de cortes observados por ovário variou de seis a 13, correspondendo à contagem de folículos em áreas que variaram de 15 a 32,5 mm². O total de folículos estimado variou de 500.000, na idade de 22 semanas, a mais de 1.000.000, na idade de 39 semanas. CONCLUSÕES: nossos resultados demonstram as diferentes densidades de folículos ovarianos durante o período gestacional, contribuindo para o escasso conhecimento existente na literatura até o momento.

PURPOSE: to determine the variation of the number of ovarian follicles during fetal life. METHODS: twelve ovaries donated for research were included in our study, nine from fetuses and three from newborn babies who died in the first hour after being delivered with 39 weeks of pregnancy. Fetal age was confirmed both by the last menstrual period of the woman and by ultrasonography. Ovaries were fixed in formaldehyde, included in paraffin and serially sliced at 7 mm. At every 50 cuts, the obtained material was haematoxilin-eosin stained and evaluated with an optical microscope (400 X). The follicles were counted in ten different regions of the ovarian cortex, each region with an area of 625 mm². The presence of a nucleus was considered the parameter for counting. Follicular density, per 1 mm³ was calculated using the formula Nt=(No x St x t)/do, where Nt is the number of follicles; No is the mean number of follicles in 1 mm²; St is the total number of slices in 1 mm³; t is the slice thickness and do is the nuclei mean diameter. RESULTS: the gestational age of fetuses ranged from 24 to 39 weeks. The number of follicles per 0.25 mm² ranged from 10.9 ± 4.8 in a newborn to 34.7 ± 10.6 in another newborn. Among the fetuses, the least value was obtained in a 36 week-old fetus (11.1 ± 6.2) and the highest in a 28 week-old fetus (25.3 ± 9.6). The total number of slices per ovary ranged from six to 13, corresponding to follicles counted in areas from 15 to 32.5 mm². The total number of follicles ranged from 500,000 at the age of 22 weeks to > 1,000,000 at the age of 39 weeks. CONCLUSIONS: our results demonstrate different (increasing) densities of ovarian follicles along the gestational period, providing more knowledge about this still not well-known subject.

Development of fetal rat pancreatic islet A cells. A quantitative and immunocytochemical study

To perform a detailed quantitative immunocytochemical study of the development of fetal rat pancreatic islet A cells. Pancreases were obtained from 19 and 21-day- old fetal rats. Ten rats were used per each group. Non-fasting blood glucose levels were measured to confirm that the animals were normoglycemic. The pregnant rats were anesthetized by ether inhalation, and the fetuses were removed from their uteruses. They were fixed in buffered neutral formalin, dehydrated and embedded in paraplast and serially sectioned [5 um]. We examined 32-48 islets [8-12 per section] for each fetus. Sections were stained by avidin biotin complex technique. A quantitative study was performed on the pancreatic islet A cells. Carl Zeiss software from Zeiss was used in this study. This study was carried out at the Department of Anatomy, King Abdul-Aziz University, Jeddah, Kingdom of Saudi Arabia during the period January to December 2005. The volume density and the number of A cells showed a significant increase during the last days of gestation. All other parameters showed a significant increase during the last days of gestation. The A cell nuclear diameter and volume did not increase significantly during the last days of pregnancy. The A cells were well stained and occupied the peripheral part of the islets. The present study represented a detailed quantitative immunohistochemical study and demonstrated that the size of the endocrine tissue and the islet A cells increased significantly during the last days of gestation

Differential Expression, Shedding, Cytokine Regulation and Function of TNFR1 and TNFR2 in Human Fetal Astrocytes

Tumor necrosis factor (TNF) -alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF- receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF-alpha receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.

Noninvasive prenatal diagnosis of fetal sex by single-cell PEP-PCR method / 华中科技大学学报（医学）（英德文版）

A new method for noninvasive prenatal diagnosis of fetal sex was developed by using single-cell PEP-PCR techniques. Micromamipulation techniques were used to obtain single fetal cells from 273 maternal blood samples. The genome of single cells was preamplified by PEP and SRY genes were analyzed by PCR method. The SRY genes of 149 samples were detected by the new method among 153 samples carrying male fetus, while 119 out of 120 samples carrying female fetus were proved negative for SRY genes. The sensitivity and specificity of the new method were 97.39% and 99.17% respectively and the correct rate was 98.17%. The new method has the advantage of high sensitivity and specificity in noninvasive prenatal diagnosis of fetal sex and provides the basis of other researches such as sex-linked inherited diseases.

Bio-artificial liver from cultured human foetal hepatocytes: feasibility and prospects.

BACKGROUND: Conventional therapy for acute liver failure has not been able to improve survival beyond 40%. Apart from liver transplantation, the most promising development in this field is the utilization of cultured hepatocytes to make 'bio-artificial liver support systems' as a 'bridge to transplantation' or ideally as a 'bridge to total recovery'. This study examines the feasibility of culturing foetal hepatocytes without the use of growth factors and formulating a bio-artificial liver support device in our set-up. METHODS: Foetal hepatocytes were harvested from the liver obtained from mid trimester abortions at Armed Forces Medical College and Command Hospital (SC), Pune. The liver was perfused with Phosphate Buffered Saline (PBS) and collagenase type IV and was cut with a pair of sterile scissors into tiny pieces. Cells so separated, were washed with PBS plus foetal calf serum and stirred to disperse the cell aggregates. Filtered cell suspensions were inoculated in polystyrene flasks containing hepatocyte culture medium (MEM E: 75%, M199: 25%, BSA: 0.1%, Bovine Insulin 5 micrograms/ml, FCS: 10%, Penicillin: 10 i.u., Streptomycin 50 micrograms/ml, Hydrocortisone 5 micrograms/ml and incubated at 37 degrees C. The functional capabilities of the cultured hepatocytes were analyzed by studying production of albumin and a foetoprotein. Structural integrity of hepatocytes was assessed by light and electron microscopy. RESULTS: The hepatocyte yield varied from 2 to 60 x 10(6) cells/L with an average of 38 x 10(6) cells/L in the eight consecutive experiments. Initial hepatocyte viability varied from 25% to 90% with an average of 61%. The yield and the viability of hepatocytes were adversely affected by the condition of foetus at birth and use of intra-amniotic injections for inducing abortions. Hepatocyte monolayers and colonies formed in 75% experiments. The cultures could be maintained in incubation without the use of epidermal or hepatocyte growth factors for 2-25 days with a mean survival of 8.9 days. The cells in culture were found to be structurally normal and functionally active and could be cryo-preserved. These hepatocytes were inoculated into a hollow fiber module to formulate bio-artificial liver support device. The cultures ultimately developed either cellular disintegration or bacterial infections despite use of antibiotics in the culture medium. CONCLUSIONS: We conclude that it is feasible to maintain foetal hepatocyte cultures without the use of expensive growth factors for over 8 days. Bio-artificial liver formulated with cultured foetal hepatocytes is now a step closer to clinical trials.

Beta agonist regulation of sodium transport in fetal lung epithelium: roles of cell volume, cytosolic chloride and protein tyrosine kinase

1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.

Little role of anti-gB antibodies in neutralizing activity of patient's sera with human cytomegalovirus (HCMV) infection

Human cytomegalovirus (HCMV) gB is known to play important roles in cell surface attachment, virion penetration, spread of infection from cell to cell, and provocation of neutralizing antibody. This study was performed to determine the role of anti-HCMV gB antibody in overall neutralizing response in patients with HCMV infection and healthy control with past infection. HCMV gB was stably expressed in 293 cells. With the stable cell line expressing gB as a specific immunosorbent, anti-gB antibody was removed from the current and past HCMV-infected sera and the remaining neutralizing activity was measured by plaque assay. It was shown that 19-50% of the total virus-neutralizing activity of sera with past HCMV infections was derived from anti-gB antibody, but anti-gB antibody had little effect on the total serum virus-neutralizing activity in patients currently infected with HCMV. This result suggests that neutralizing antibody to HCMV gB may reflect disease status.

Cariotipo de células fetales en el diagnóstico prenatal en Costa Rica / Karyotyping of fetal cells in the prenatal diagnosis in Costa Rica

The results of 182 genetic amniocenteses between 14 and 37 weeks gestation, from 1986 to 1992, and of two cordocenteses in 1992, are reported. There were two main reasons for referral: maternal age 35 years and older and abnormal ultrasound assessment. Fetal cells were closed cultured and mass harvested. In 3.7 per cent of cases fetal chromosomes were defective. Turn around time was about three weeks up to and including 1991 and two weeks in 1992, culture failure rate was 7 per cent that year. No cytogenetic misdiagnosis and no complication or sequelae related to the amniocenteses were detected. We conclude this is a safe and reliable procedure to obtain fetal karyotypes and to improve obstetric management of high-risk pregnancies

Isolation & cryo-preservation of human foetal articular chondrocytes.

An attempt to determine the ideal temperature and duration of storage of human foetal chondrocytes yielded highly cellular preparations with no alteration in morphology or loss of viability. Initial digestion with activated papain was followed by incubation in 0.5 per cent collagenase. Trypan blue exclusion test revealed a viability count of 95-99 per cent and radioactive thymidine uptake a corresponding labelling index. On TEM no subcellular damage was evident. The isolated viable chondrocytes were further banked at varying temperatures of +4 degrees, -4 degrees, -30 degrees, -79 degrees and -196 degrees C, in Eagles MEM with 10 per cent dimethyl sulfoxide. Post storage morphology and viability of these cells, thawed after durations of 20 h, 1 wk, 1, 2, 3, 4 and 6 months, were compared with prestorage readings in an attempt to define the ideal temperature for banking. Storage in liquid nitrogen at -196 degrees C demonstrated excellent preservation even at the end of six months with minimal subcellular change. Electron microscopy and labelling index were found to be superior to Trypan blue exclusion test in assessing the stored chondrocytes for retention of their functions.