El biomodelo porcino en la investigación médica traslacional: del biomodelo al humano en trasplante pulmonar / The porcine biomodel in translational medical research: From biomodel to human lung transplantation

Resumen Introducción. La anatomía humana y porcina son comparables. En consecuencia, el biomodelo porcino tiene el potencial de ser implementado para entrenar al profesional quirúrgico en áreas como el trasplante de órganos sólidos. Objetivo. Describir los procedimientos y hallazgos obtenidos mediante experimentos de medicina respiratoria traslacional con biomodelos porcinos realizados en un laboratorio de experimentación animal, y hacer una revisión comparativa entre el pulmón humano y el porcino. Materiales y métodos. El experimento se llevó a cabo en nueve cerdos de raza híbrida en un laboratorio de cirugía experimental. Se estudiaron la anatomía y la histología de las vías respiratorias mediante fibrobroncoscopia, biopsia bronquial y lavado broncoalveolar. El lavado broncoalveolar se estudió con citología en base líquida y se evaluó con las coloraciones de Papanicolau y hematoxilina y eosina. Se utilizaron técnicas de patología molecular, como inmunohistoquímica, citometría de flujo y microscopía electrónica. Los cerdos se sometieron a neumonectomía izquierda con posterior implante del injerto en otro cerdo experimental. Resultados. Los estudios histopatológicos y moleculares evidenciaron un predominio de macrófagos alveolares (98 %) y linfocitos T (2 %) en el lavado broncoalveolar porcino. En los estudios del parénquima pulmonar porcino se encontró tejido linfoide hiperplásico asociado a las paredes bronquiales. La microscopía electrónica evidenció linfocitos T dentro del epitelio y el diámetro de las cilias porcinas fue similar al de las humanas. Conclusiones. El biomodelo porcino es viable en la investigación traslacional para el entendimiento de la anatomía del sistema respiratorio y el entrenamiento en trasplante pulmonar. La implementación de este modelo experimental podría fortalecer los grupos que planean implementar un programa institucional de trasplante pulmonar en humanos.

Abstract Introduction: Human and porcine anatomy are comparable. In consequence, the porcine biomodel has the potential to be implemented in the training of surgical professionals in areas such as solid organ transplantation. Objectives: We described the procedures and findings obtained in the experiments of translational respiratory medicine with the porcine biomodel, within an experimentation animal laboratory, and we present a comparative review between human and porcine lung. Materials and methods: The experiment was done in nine pigs of hybrid race within a laboratory of experimental surgery. The anatomy and histology of the respiratory tract were studied with fibrobronchoscopy, bronchial biopsy and bronchoalveolar lavage. The bronchoalveolar lavage was studied with liquid-based cytology and assessed with Papanicolau and hematoxylin-eosin staining. Molecular pathology techniques such as immunohistochemistry, flow cytometry, and electronic microscopy were implemented. The pigs were subjected to left pneumonectomy with posterior implantation of the graft into another experimental pig. Results: Histopathologic and molecular studies evidenced predominance of alveolar macrophages (98%) and T-lymphocytes (2%) in the porcine bronchoalveolar lavage. Studies on the porcine lung parenchyma revealed hyperplasic lymphoid tissue associated with the bronchial walls. Electronic microscopy evidenced the presence of T-lymphocytes within the epithelium and the cilia diameter was similar to the human. Conclusions: The porcine biomodel is a viable tool in translational research applied to the understanding of the respiratory system anatomy and the training in lung transplantation. The implementation of this experimental model has the potential to strength the groups who plan to implement an institutional program of lung transplantation in humans.

Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis

The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34+ HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34+ cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34+ cells, but not CD34- cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80+ macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

Estudio del polimorfismo genético de las células de la médula ósea y del sistema nervioso central de ratas mediante la técnica de RAPD / Study of genetic polymorphism of the bone marrow and the central nervous system of rats cells by RAPD technique

Los modelos experimentales en rata han sido de gran utilidad en las evaluaciones terapéuticas o de reemplazo de células en enfermedades neurodegenerativas. Se ha comprobado que las células de la médula ósea (CMO) de ratas pueden diferenciarse en células que no forman parte de sus linajes normales. Hay evidencias de estos procesos de trans-diferenciación, pero aún no se conocen los mecanismos moleculares que activan estos procesos. El propósito de nuestro trabajo fue estudiar el polimorfismo genético del ADN de los tipos celulares que conforman las CMO y las células del sistema nervioso central (SNC), estríatales y de la corteza de ratas mediante la técnica de RAPD. Las CMO, las células mononucleares (CMMO), las células estromales (CEMO) y las del SNC fueron obtenidas de ratas, y su ADN genómico fue purificado y amplificado mediante la técnica de RAPD, utilizando 15 cebadores al azar. Se construyó un dendograma de las bandas de amplificación generadas utilizando el método de UPGMA. Las células estudiadas según el análisis del RAPD quedaron en 2 grupos bien definidos, pudiéndose diferenciar las CEMO del resto de las células estudiadas. Los cebadores OPA-6, 7 y 12, mostraron el polimorfismo genético de los linajes de células estudiadas. Mediante la técnica de RAPD se demostró la variabilidad genética entre las CEMO y las CMMO, células estriadas y de corteza que mostraron una homogeneidad genética, proponiéndose marcadores específicos de RAPD para cada grupo de células. Este es el primer estudio del polimorfismo genético de las CMO y del SNC de ratas.

Experimental models have been of grate usefulness in the therapeutic or replacement cells in neurodegenerative diseases. It has been demonstrated that bone marrow cells (BMC), can be difefferentiated in cells that do not form part of their normal lineage. There is evidence of these trans-differentiation processes in these cells, but nevertheless, molecular mechanisms that activate these differentiation process still not known. The purpose of our work was to study the genetic polymorphism of those cellular types; that conform the rat bone marrow cells (BMC) as well as those of the central nervous system (CNS), striatum cells and cortex ones, trough RAPD technique. BM, mononuclear cells (BMMC), estromal cells (BMSC) and the CNS cells were obtained from rats and genomic ADN was purified and amplified through RAPD technique, using 15 random primers. A dendogram was constructed according to UPGMA method of the amplifying RAPD bands. Studied cells as- according to the RAPD analysis- were grouped into 2 well- defined groups, as CEMO coud be differentiated from the rest of studied cells. OPA-6, 7 and 12 primers showed the genetic polymorphism of the studied lineages cells. Also will be proposed specific RAPD genetic markers. Through RAPD technique permitted the genetic variability was demonstrated betwen BMEC and BMMC of striated cells and of cortex, which demonstratd a genetic homogeneity through RAPD technique so specific genetic markers of RAPD were thus propose for each group of cells. These constitute the first study on genetic polymorphism of BMC and CNS.

Conventional and fluorescence in situ hybridization analysis of three-way complex BCR-ABL rearrangement in a chronic myeloid leukemia patient.

Chromosomal analysis was carried out in bone marrow sample of an 11-year-old girl suspected of myeloproliferative disorder. Conventional G-banding study detected a complex three-way translocation involving 7, 9 and 22, which has resulted in the formation of a variant Philadelphia chromosome causing rearrangement of abl and bcr genes in 87% cells. Fluorescence in situ hybridization (FISH) confirmed the fusion of bcr-abl oncogene. Thus the bone marrow karyotype was observed as 46,XX (13%)/46,XX,t(7;9;22)(q11;q34;q11) (87%). Hyperdiploidy was present in two cells. In this study, both conventional cytogenetic and FISH diagnosis proved to be significant to identify the variant nature of the Philadelphia chromosome and hyperdiploid condition for introduction of a suitable treatment regimen and estimation of life expectancy of the young girl.

Correlative study between immunodulatory action of propolis on cultured PBMNCs and it's schistosomicidal activity

Propolis, a beehive product widely used in folk medicine as an anti-nflammatory agent, have heen attracting researchers attention to scientifically elucidate us biological properties and therapeutic activities. This study aimed to spot light on the value of propolis as an immune-stimulant and to evaluate the influence on schistosome hematobium infection cure rate. To achieve this goal we estimated the effect of propolis on cultured peripheral blood mononuclear cells activation in-vitro by IL-2 and NO determination. We also evaluated the effect of in-vivo treatment with propolis on Schistosoma hematobium worm and bone marrow by parasitological and ultrastructural studies. Twenty S. haematobium infected golden hamsters were included in the study, subdivided into two groups each of 10 animals Group 1: Infected Control with 300 +/- 10 cercariae of S. haematobium by abdominal skin exposure. Group 2: Animals were treated with propolis three months post the infection. Our in-vitro results revealed that propolis induces a discreet elevation in IL-2 and NO release in PBMNCs cultures supernatant of S. hematobium infected hamsters. Mean level of IL-2 was 16.17 +/- 1.67 pg/ml in the presence of propolis and 3.31 +/- 0.76 in its absence with highly statistically significant difference [p < 0.001]. Regarding NO, Mean level of NO was 7. 76 +/- 1.30 U/ml in the presence of propolis and 2.6 +/- 0.42 in its absence with. highly statistically significant difference [p < 0.001]. Also, propolis caused observed activation and absence of apoptotic changes at the ultrastructural level of cultured PBMNCs revealed. In-viva results, revealed significant reductions in mature worm loads [either male or female], tissue egg loads [either intestinal or hepatic] 21.00 and 19.79% respectively and Percentage reductions of egg developmental stages was 68.07% with statistically significant difference compared with infected control group [P < 0.05]. Ultrastructural study of S. hematobium women revealed implantation and degeneration of the spines within vesiculated tegument and for the bone marrow it revealed evidence of lymphocyte and promonocyts activation in addition to remarkable increase in the number of the activated natural killer cell. Data suggest that propolis acts on host immunity by PBMNCs activation. This information would provide new insights in considering propolis to have a potential therapeutic benefit if used in conjunction with antischistosomal drug in treatment of schistosome infection

protective effect of black seed [Nigella sativa] against carbon tetrachloride-induced chromosomal aberrations and ultrastructural changes of bone marrow cells

The effect of the oral administration of aqueous suspension of Nigella sativa [50 mg/kg b.wt.] against chromosomal aberrations and ultrastructural changes of the bone marrow cells in mice treated with carbon tetrachloride CCl[4] was studied. CCl[4] was administered in two dose levels equivalent to[1/4] [1.9 ml/kg b.wt.] and [1/2] [3.8 ml/kg b.wt.] of the oral LD[50] in mice. The data indicated a significant dose-dependent decrease in the mitotic activity of bone marrow cells in animals treated with CCl[4]. Also a significant dose-dependent increase in the number of bone marrow cells with different types of chromosomal aberrations was recorded in these animals. Ultrastructural changes were also dose-dependent including both nucleus and cytoplasm of erythroid and myeloid elements of the bone marrow cells. Treatment of the animals with N. sativa improved both genotoxicity and ultrastructural changes induced by the two dose levels of CCl[4]

Development of granulocytes and monocytes in adult rabbit bone marrow ultrastructural aspects

The hematopoietic tissue of bone marrow constitutes a heterogeneous family of indistinguishable pleuripotential stem cells; they subsequently pass through a series of developmental stages that are microscopically identifiable. The aim of the present work was to throw mor light on the development of blood granulocytes in the red bone marrow of adult rabbit using the electron microscope.in bone marrow, developing granulocytopoietic series appeared to be grouped into clusters. Among these cells, myeloblasts were characterized by a relatively large nucleus with fully granular chromatin. The cytoplasm contained much free eibosomes. During the prom yelocyte stage, Iry granules being to be formed, while the maturation sequence was evidenced by change in the nuclear configuration and size and by the appearance 2ry granules which later on dominated over the Iry type. In this study it was observed that the myelocytes nuclei were rounded or oval in shape. However, gradual elongation and indentation of the nucleus was the initial sign of maturation from myelocyte to metamyelocyte. After that, nucear remodeling continued forming the staff granulocyte, and later on constrictions appeared to segment the nucleus into two or more lobes and thus a muture segmented granulocyte was formed. A characteristic feature in eosinophilic granulocytes was their 2ry specific granules which contained electron dense crystals. Monoblasts had similarities with their mature forms, but the chromatin was homogeneously fine and granular and the cytoplasm was entirely devoid of granules, while promonocytes contained small clear vesicles. The maturation of the latter cell into monocyte was associated with development of lysosomal granules and an increase of condensed chromatin with a progressive decrease in the number of ribosomes

Chronic meningitis in acute promyelocytic leukemia [a case report]

The occurrence of extramedullary disease [EMD] at presentation or at relapse has long been considered a rare event in Acute Promyelocytic Leukemia [APL]. Our purpose from recent reports of EMD at presenting in APL have raised increasing concern about one of causes of chronic meningitis. This study describe a case of acute promyelocytic leukemia [APL] in 25 years old female patient admitted in Emam Reza hospital, presenting with severe headache and vomiting and showed signs of meningeal irritation as well as papilledema. Cytocentrifuge examination of CSF showed an excess of promylocytes, but peripheral blood didn't show any abnormal cell or blasts where as BMA showed increase of promyelocyte with multiple auer rod. After 2 weeks of aleukemic phase, this patient developed hematologic picture of APL diagnosed myeloid origin cells with myelopperoxidase stain. She was diagnosed as having leukemic meningitis and after 3 weeks in spite of chemotherapy, she dead Leukemic relapse or first presentation of leukemia may be the etiology of aseptic meningitis. Rarely central nervous. system leukemia and leukemic meningitis are associated with normal bone marrow and sometimes patients at Aleukemic phase present with meningitis. therefore we suggested that in patients with aseptic meningitis cerebrospinal fluid cytospine smear should be evaluated for neoplasic and leukemic cells

possible role of vitamin E on bone marrow cytogenic toxicity of cisplatin in adult male albino rat

Cisplatin; myelotoxicity is a major limiting factor in tile treatment of several neoplasms. Vitamin E, a slow acting free radical scavenger, has been shown to ameliorate nephro, oto and neurotoxicities in animals receiving cisplatin. The purpose of this study is a trial to evaluate the effectiveness of vitamin E as a myeloprotectant. Twenty five adult male albino wistar rats weight [200-250 gm] were used in this experiment. The experimental animals were divided into three groups [A1B1C]. Group [A]: was kept as control. Group [B]: treated with cisplatinum twice weekly for one month by intraperitoneal injection in a dose of 4 mg/kg. Group [C]: treated with vitamin E in a dose of 100 mg/kg by intramuscular injection concomitant with cisplatin twice weekly for one month. Samples were taken from bone marrow and processed for light and electron microscopic studies. The light microscopic examination of cisplatin treated bone marrow speciemens showed accumulation of large number of both fat and mast cells while in vitamin E treated group, there was a reduction in the percentage of both type of cells but not significantly. By electron microscopic examination, the most striking effect of cisplatin on bone marrow was evidenced by the marked increase in the number of dark degenerated cells in comparison to that of control. On the other hand, a maximal reduction in the number of these degenerated cells was observed in vitamin E treated bone marrow speciemens in comparison to group [B]. The percentage of eosinophil was increased significant in vitamin E treated group. By this work, vitamin E, a free radical scavenger, appears to have a protective role against cytogenic toxicity of cisplatin on bone marrow

Local and systemic effects of ketoprofen [a light and ultrastructural study]

Ketoprofen is one of the non steroidal anti-inflammatory drugs widely used in our population. Due to its easy access, drug abuse is very common leading to verious side effects. The effect of ketoprofen was seen on gastric mucosa and bone marrow of adult albino rats. Two regimen of the treatment were followed; either six regular interrupted doses, one week interval, given to starved rats or daily administration of the drug for six weeks. Samples from the gastric mucosa were examined both by light microscope and scanning electron microscope. Samples of the bone marrow were prepared for semithin sections, stained with toluidine blue, and examined by light microscope. Quantitative study was done using image analyser computer system. The data obtained was statistically analysed. Gastric lesions were demonstrated in both methods of treatment, although lesions were severer in chronic course. Bone marrow affection in the form of decreased granulocytic and increased erythrocytic precursors occurred only in chronically treated rats

Histological study of the bone marrow mast cells in ovariectomized adult albino rats

Ten 90 days old female albino rats were utilized in this research aiming to study the bone marrow mast cells in ovariectomized adult albino rats in an attempt to clarify one of the possible underlying cellular causes of postmenopausal osteoporosis. These rats were equally classified into 2 groups [a control group and an experimental one]. Bilateral ovariectomy was performed to the rats of the experimental group. After 30 days of the ovariectomy, all the rats of the two groups were sacrificed. Their tibiae were dissected out carefully and their proximal parts were obtained and processed for light and electron microscope examinations. The spongy bone at the proximal tibial metaphysis of the ovariectomized rats appeared with few bone trabeculae. Numerous osteoclasts were observed in the examined sections. These cells appeared with multiple euchromatic nuclei and prominent nucleoli and their cytoplasm contained numerous mitochondria. The surrounding bone marrow contained numerous mast cells. Some of these cells were seen near the bone trabeculae. In conclusion, the concomitance between increased bone marrow mast cells number and osteoclasts number and activity with reduced bone trabeculae of the spongy bone of the proximal tibial metaphysis of the estrogen- deficient rats may suggest a role for mast cells in postmenopausal osteoporosis. So, regulation of mast cells activation may be a critical issue and a promising therapeutic approach in the treatment of this disease

Ultrastructural comparison between the effect of secretory / exceretory products of lung stage and the mechanical S. mansoni schistosomula on each of S. mansoni worm and lymphocyte activation

Ultrastructural observations were made to evaluate the effect of secretory/excretory product [SEP] of 7-days old lung stage S. mansoni schistosomula and the mechanical schistosomula on each of S. mansoni worm, lymphocyte activation and humoral immune response in an attempt to develop a protective vaccine with potent efficiency. Multiple doses [100 micro g followed by two booster doses 50 micro g each] at three weeks intervals were injected intraperitoneally into CFW1 SPE albino mice one week prior to expossure to 100 S. mansoni cercariae, mice were sacrified 6 weeks post infection. Separated worms and lymphocytes in peripheral blood, bone marrow and spleen were prepared for electron microscopic [EM] examination. Serum specific anti-schistosomula SEP IgG was measured. EM examination revealed that immunization with lung stage S. mansoni schistosomula [Group I] induced more damage to the S. mansoni worm represented by extend of the degeneration to subtegumental longitudinal muscle layer, some degeneration in the spermatocyts and vacuolation within the testis of male reproductive organ while degeneration was only whithin the tegument in worm treated with mechanical schistosomula [Group II]. Peripheral blood lymphocytes were activated in both groups while bone marrow lymphocytes were activated only in mice treated with SEP of lung stage S. mansoni schistosomula in which more activation of splenic lymphocytes could be detected in the form of increase number of plasma cells. These results confirmed by the highly significant elevation [p<0.01] in the serum level of anti-SEP IgG in group II. In conclusion, our findings showed that multiple intra-peritoneal administration of low doses of SEP released from 7 days-lung schistosomula induced more damage of the S. mansoni worms, activation of lymphocytes and a significant increase serum level of anti-schistosomula SEP IgG than that of mechanical schistosomula

In vivo induction of sister chromatid exchanges analysis of mice bone-marrow chromosomes, primary spermatocytes and micronucleus test: accurate cenotoxic bioassays for DNA damage induced by the drug alexoprine

Accurate genotoxic bioassays for measuring DNA damage [in vivo induction of sister chromatid exchanges, analysis of mice bone marrow chromosomes, analysis of mice primary spermatocytes, and micronucleus test] were employed and used. Analysis of sister chromatid exchange frequencies revealed that Alexoprine is capable of inducing primary DNA damage. The results showed that the tested drug has clastogenic activity upon mice bone marrow chromosomes. Analysis of mice primary spermatocytes revealed that Alexoprine is capable of affecting the germinal cells. Analysis of micronucleated polychromatic erythrocytes presented an evidence that Alexoprine is a clastogenic agent. The tested drug Alexoprine is capable of inducing primary DNA damage, as SCEs revealed it has clastogenic activity upon somatic and germinal cells as well

Bone marrow changes in human malaria: a retrospective study

The bone marrow reports of 1966 patients admitted to a provincial teaching hospital between January, 1992 to April, 1995 were retrospectively analyzed. Twenty-six [1.3%] bone marrows showed the presence of malarial parasites. Sixteen [62%] patients had Plasmodium falciparum 9 [34%] Vivax malaria and one [4%] mixed infection. All these patients gave a history of prolonged illness and had low parasite counts. Plasmodium vivax malaria was not associated with any significant pathology in the bone marrow, except iron deficiency anaemia. The bone marrows with Plasmodium falciparum malaria showed myeloid hyperplasia, erythroid hyperplasia, megaloblastosis and hypoplasia in different proportions. No evidence of dyserythropoiesis was found in this series. The possible mechanisms producing these changes and the factors responsible for the discrepancy in bone marrow findings in different geographical areas are discussed

Effect of acute lead intoxication on the ultrastructure of the peripheral blood and bone marrow of male albino rats

Forty adult male albino rats were used in the current study [control rats n =20, 10 rats per each experimental group, group 1 - experimental "after 3 hoursof injection", and group 2 - experimental" after 6 hours of injection" n =10]. The experimental groups were injected intraperitoneally with a singledose of lead acetate, 150 mg/kg body weight dissolved in distilled water. Retro-orbital blood was sampled for the determination of blood lead level,complete blood count, and differential leucocytic count. In addition, bloodand bone marrow samples were obtained for transmission electron microscopicstudies from ten randomly chosen animals. Blood lead levels showed asignificant increase in both group 1 and group 2. Red blood cells countshowed a nonsignificant decrease in both experimental groups, while the totalleucocytic count showed a nonsignificant increase. Hemoglobin concentrationand platelet count decreased in both experimental groups, but this decreasewas significant only in group 2. Hypersegmentation of the neutrophilicnucleus was observed in both experimental groups. The ultrastructuralalterations such as irregular nuclei, occasional nuclear pockets, the presenceof cytoplasmic vacuoles containing inclusions, dilatation of rough endoplasmicreticulum cisternae were most clearly expressed in some neutrophils of group 2. Furthermore, electron dense nuclear inclusions, with a densecenter and outer fibrilly zone, together with dilated nuclear membrane werepresented among the erythroid cell series. Besides, bizarre shaped ordeformed red cells were also seen. Platelets showed hypo-granularity withdilated open canalicular system [OCS] and satellitism

Estudio citogenético en 22 adultos y tres niños con leucemia linfoblástica aguda / Cytogenetic study in 22 adults and therr children with acute lymphoblastic leukemia

Se presentan los resultados del estudio citogenético realizado en sangre periférica y/o médula ósea de 25 pacientes (22 adultos y 3 niños) con LLA estudiados entre 1987 a 1990. Todos los casos presentaron alteraciones cromosómicas (25/25), aunque en 23 pacientes (23/25) estaba presente una línea celular normal. Entre las aberriguaciones estructurales encontradas están t(17;19) (q11;p13), t(2;9;22)(q34;q34;q11), t(1;7)(p13;q33), t(6;11)(q26;p16), t(3;4)(q24-25;q26), t(1;12)(q23;q34), t(2;18)(q15;p12), t(2;4;)(q23;35) y t(4;11)(q21ñq23). Estos rearreglos fueron indicadores de riesgo y se correlacionaron con la respuesta al tratamiento y la supervivencia de los pacientes. Algunas de estas anomalías no habían sido previamente descritas en LLA; sin embargo, los puntos de ruptura coinciden con los observados en otras neoplasias hematológicas, lo cual suguiere que esas regiones son críticas en la patogénesis de estos desórdenes.