Avoiding leukocyte contamination and early platelet activation in platelet-rich plasma.

The objective of this study was to describe a new platelet-rich plasma (PRP) protocol with a reduced concentration of leukocytes and intact platelets. We collected 8 mL of venous blood (VB) from marginal ear veins of 10 male New Zealand white rabbits in acid dextrose citrate Vacutainer tubes. Tubes were centrifuged at 302g for 10 minutes. All plasma was collected in plastic tubes to avoid buffy-coat contamination and centrifuged at 2862g for 5 minutes. A 10% calcium chloride activator (10 PRP:2 CaCl2) was added to the lower third of this plasma (PRP), and the PRP gel was obtained. Mean platelet count was 317.7 x 10(3) +/- 39.9/microL in VB and 1344.9 x 10(3) +/- 347.5/microL in PRP. Leukocyte counts were 3.96 x 10(3) +/- 2.01/microL and 0.46 x 10(3) +/- 0.45/microL in VB and PRP, respectively. Mean platelet enrichment was 327.4 +/- 97.8%. All differences were statistically significant (P > .05). This protocol is practical and reproducible, resulting in a high concentration of intact platelets to help tissue repair and low levels of leukocytes.

Ophthalmic patterns of captive brown brocket deer (Mazama gouazoubira)

Captive brown brocket deer (Mazama gouazoubira) were manually restrained to assess tear production by the Schirmer tear test I to measure intraocular pressure by applanation tonometry, to examine ocular conjunctival epithelial cells via cytologic and histologic samples, and to survey ocular conjunctival microflora by microbiologic culture. The mean value for the Schirmer tear test I was 8.9 ± 1.8 mm/min, and the mean intraocular pressure was 15.3 ± 3.1 mm Hg. Conjunctival epithelium contained stratified pavimentous layers of cells, and the microflora consisted of predominantly gram-positive bacteria. Copyright 2007 by American Association of Zoo Veterinarians.

Aspirado traqueal de cavalos clinicamente sadios da raça quarto de milha após prova de três tambores

The aim of the present study was to evaluate through endoscopy the tracheal aspiration cytology in twenty seven Quarter Horses from Curitiba and surroundings, following the Three Barrel Competittion. Upper respiratory tract secretion was obtained by tracheal aspiration using a polyethylene catheter introduced through the endoscopic fiberoptic working channel, at the level of tracheal bifurcation. Cytologic slides were prepared by smear and stained by diff-quick technique and the differential was performed in 500 cells counting by 1,000X optic microscopy. None of the horses presented abnormality, including epixtasis, at the clinical examination. However, hemosiderophages were detected at cytology in three animals, suggesting that some may be suffering of subclinical pulmonary hemorrhage. Differential cell counting of tracheal aspiration results were, in average: 44.09 ± 35.68% of epithelial cells; 1.10 ± 2.18% of Globet cells; 23.10 ± 35.93% of neutrophils; 0.13 ± 0.37% of lymphocytes; 0.91 ± 2.81% of eosinophils; 30.57 ± 23.62% of macrophages and 0.13 + 0.93% of hemosiderophages. In conclusion, based in the present study, the evaluation of cellular populations with the tracheal aspiration may offer important additional information to the clinician, particularly about the inflammatory processes of lower respiratory tract and pulmonary bleeding.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Background: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods: Eosinophils were purified using a percoll gradient followed byimmunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion. © 2008 Lintomen et al; licensee BioMed Central Ltd.

MTA-induced neutrophil recruitment: a mechanism dependent on IL-1β, MIP-2, and LTB4

Objective: The objective of this study was to investigate the mediators and the resident peritoneal cells involved in the neutrophil migration (NM) induced by mineral trioxide aggregate (MTA) in mice. Study design: MTA (25 mg/cavity) was injected into normal and pretreated peritoneal cavities (PC) with indomethacin (IND), dexamethasone (DEX), BWA4C, U75302, antimacrophage inflammatory protein-2 (MIP-2), and anti-interleukin-1β (IL-1β) antibodies and the NM was determined. The role of macrophage (MO) and mast cells (MAST) was determined by administration of thioglycollate 3% or 48/80 compound, respectively. The concentration of IL-1β and MIP-2 exudates was measured by ELISA. Results: MTA induced dose- and time-dependent NM into mice PC, with the participation of MO and MAST. NM was inhibited by DEX, BWA4C, and U75302, as well as anti-MIP-2 and anti-IL-1β antibodies. In the exudates, IL-1β and MIP-2 were detected. Conclusions: This study suggests that MTA induces NM via a mechanism dependent on MAST and MO mediated by IL-1β, MIP-2, and LTB4. © 2008 Mosby, Inc. All rights reserved.

Comparison of exfoliative pap stain and AgNOR counts of the tongue in smokers and nonsmokers

Objective To compare exfoliative cytology from the oral mucosa of smokers and nonsmokers, with emphasis on proliferative activity. Methods Exfoliative cytology specimens were obtained from clinical normal mucosa from the lateral border of the tongue in 30 nonsmokers and 30 smokers ranging in age from 40 to 70 years of age, who were seen at the Heart Institute's Patient Center and the Smoking Cessation Program of the University Hospital, University of São Paulo Medical School (InCor-HCFMUSP). The cytologic specimens were evaluated by Papanicolaou staining and AgNOR quantification in order to evaluate the presence of cytological alterations suggestive of inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. Results Only Papanicolaou Class I and Class II smears were observed. Inflammatory alterations were found in 90% of smokers and in 87% of nonsmokers. The number of AgNORs/nucleus differed significantly between smokers and nonsmokers (3.372 ± 0.375 versus 2.732 ± 0.236). Conclusions Within the limitations of this research, the results indicate higher proliferative activity in smoking patients compared to nonsmoking patients, even in the absence of clinical lesions. © The Author(s) 2008.

Avaliação hematológica de cães naturalmente infectados por Leishmania (Leishmania) chagasi submetidos a tratamento com antimoniato de meglumina

The present research was carried out aiming to assess the hematological response of dogs with visceral leishmaniasis submitted to treatment. For this, seven animals naturally infected by Leishmania sp. were submitted to a treatment with 75 mg/kg meglumine antimoniate subcutaneously, 12-12h/3 weeks. In all animals, a complete blood count and bone marrow aspiration biopsy were carried out for a descriptive evaluation at up to seven moments: before the treatment, 30, 60, 90, 120, 150 and 180 days after the start of the treatment. Before the beginning of the experiment hematological alterations were observed in four of the seven dogs (57.1%), among them, nonregenerative anemia, lymphopenia, lymphocytosis and monocytosis. During the course of the experiment the occurrence of leukocytoses, such as left shift neutrophilia and eosinophilia, were observed in some of the animals. Before the beginning of the treatment (M1), the occurrence of erythrocytic hypoplasia was detected by bone marrow cytology in two of the dogs (28.6%). This was reversed through an increase in the amount of erythroid progenitor cells after the administration of meglumine antimoniate. Thus, it can be concluded that the treatment led to normalization of the hematological alterations and recovery of the bone marrow.

Identification of ellagic acid derivatives in methanolic extracts from Qualea species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell junctions in fish seminiferous epithelium

Similar to mammals, in fish the cellular interactions between Sertoli cells (SC) and germ cells (GC) in the seminiferous epithelium have important structural and functional roles. In this review, we give a brief summary of these interactions, in particular those on the cell junctions. Despite the scarcity of detailed empirical data, it appears that both basic types of adhesive junctions (actin- and intermediate filaments-related) are present between SC. However, the actin-related multifunctional junction known as the ectoplasmic specialization is seemingly present only in some cartilaginous fish. Conversely, SC in other fish species are joined by actin-related junctions similar to typical zonulae or puncta adherens found in other epithelia. Adhesive junctions are also found between SC and GC and between GC and GC, and due to their particular characteristics these junctions are known as desmosome-like junctions. In terms of intercellular communication, connexins and gap junctions have been shown to occur between SC in fish, and they may be involved in the coordination of the synchronous development of GC within the cysts. It is also possible that gap junctions may form an interconnected network between SC and GC within a cyst. Concerning the SC barrier, tight junctions between fish SC apparently form a functional barrier only in cysts containing haploid GC, and different from mammals, meiotic GC in fish are not shielded from the vascular system. In summary, although still not well investigated, cell-cell interactions in the seminiferous epithelium of fish seem to be crucial for GC development, and their disturbance, for example by changing environmental conditions, will probably affect GC survival and fertility. © Springer Science+Business Media B.V. 2008.

Indirect cytotoxicity of a 35% hydrogen peroxide bleaching gel on cultured odontoblast-like cells

The aim of this study was to evaluate the trans-enamel and trans-dentinal effects of a 35% hydrogen peroxide (H2O2) bleaching gel on odontoblast-like cells. Enamel/dentin discs obtained from bovine incisors were mounted in artificial pulp chambers (APCs). Three groups were formed: G1- 35% H2O2; G2- 35% H2O2 + halogen light application; G3- control. The treatments were repeated 5 times and the APCs were incubated for 12 h. Then, the extract was collected and applied for 24 h on the cells. Cell metabolism, total protein dosage and cell morphology were evaluated. Cell metabolism decreased by 62.09% and 61.83% in G1 and G2, respectively. The depression of cell metabolism was statistically significant when G1 and G2 were compared to G3. Total protein dosage decreased by 93.13% and 91.80% in G1 and G2, respectively. The cells in G1 and G2 exhibited significant morphological alterations after contact with the extracts. Regardless of halogen light application, the extracts caused significantly more intense cytopathic effects compared to the control group. After 5 consecutive applications of a 35% H2O2 bleaching agent, either catalyzed or not by halogen light, products of gel degradation were capable to diffuse through enamel and dentin causing toxic effects to the cells.

Padronização dos critérios de seleção em estudos sobre medicações nasais

Clinical studies on nasal topical medications require the standardization of nasosinusal normality in order to establish control groups through a specific evaluation of the upper airways. Aim: to standardize the evaluation of candidates for control groups in clinical studies on nasal topical medications. Material and Methods: healthy male volunteers of 18 to 50 years of age, asymptomatic from the nasosinusal standpoint were subjected to a sequential and excluding assessment made up of clinical evaluation, immediate hypersensitivity skin test, saccharin test, flexible nasofibroscopy and nasal cytology. Study design: Crosssectional contemporary cohort. Results: Of the 33 people originally enrolled, 14 (42.4%) were excluded for clinical reasons. Of the 19 remaining, 2 (10.5%) had atopy diagnosed in the skin test and were excluded. 17 were tested with saccharin and presented normal mucociliary clearance. Evaluation by nasal endoscopy showed abnormality in 2 cases (11.8%) and these were excluded. The remaining 15 were submitted to nasal cytology, which proved normal, representing 45.5% of those initially included. Conclusion: The proposed protocol for sequential and excluding evaluation was effective in defining candidates for the establishment of control groups in clinical studies on nasal topical medications. © Revista Brasileira de Otorrinolaringologia. All Rights reserved.

Characterization and in vitro cytocompatibility of an acid-etched titanium surface

The aims of this study were to characterize the microstructure of a commercially pure titanium (cpTi) surface etched with HCl/H 2SO 4 (AE-cpTi) and to investigate its in vitro cytocompatibility compared to turned cpTi (T-cpTi). T-cpTi showed a grooved surface and AE-cpTi revealed a surface characterized by the presence of micropits. Surface parameters indicated that the AE-cpTi surface is more isotropic and present a greater area compared to T-cpTi. The oxide film thickness was similar between both surfaces; however, AE-cpTi presented more Ti and O and less C. Osteoblastic cell proliferation, alkaline phosphatase activity, and bone-like nodule formation were greater on T-cpTi than on AE-cpTi. These results show that acid etching treatment produced a surface with different topographical and chemical features compared to the turned one, and such surface modification affected negatively the in vitro cytocompatibility of cpTi as demonstrated by decreasing culture growth and expression of osteoblastic phenotype.

Spermatogenesis and karyotypes of three species of water striders (Gerridae, Heteroptera).

Although they are of economic importance, there have been few cytogenetic studies of the Gerridae (Heteroptera) in Brazil. We examined spermatogenesis (meiosis and spermiogenesis) and nucleolar behavior in three species of the family Gerridae. Brachymetra albinerva and Halobatopsis platensis were found to have a chromosome complement of 2n = 25 (24A + X0) and Cylindrostethus palmaris 2n = 29 (28A + X0) chromosomes. Fifteen individuals of these species were collected from the reservoir of São José do Rio Preto, SP, using screens and were transported in pots containing water to the laboratory, where cytogenetic preparations were made. The polyploidy nuclei are formed by several heteropyknotic regions; cells in meiotic prophase have a heteropyknotic region that is probably the sex chromosome, and the chromosomes from chiasmata. The spermatids are rounded and have a heteropyknotic region at the periphery of the nucleus; the sperm head is small, with a long tail. Silver impregnation of meiotic cells showed one or more disorganized bodies around the perichromosomal sheath. The round spermatids had two bodies next to each other, but these were elongated; one of the bodies remained in the head and the other migrated to the initial part of the tail at the end of spermagenesis, when the staining was no longer evident. The meiotic cells appear during spermatogenesis and have very similar silver-impregnation patterns in different species of Heteroptera.

Cytoplasmic and nuclear morphometric parameters in cytologic preparations of canine cutaneous mast cell tumors

Thirty fine-needle biopsy (FNB) samples from 28 dogs subjected to surgical resection of cutaneous mast cell tumors (MCTs) were stained with Giemsa. At least 100 neoplastic cells from each cytology slide were evaluated by morphometric analysis. The parameters were: area, perimeter of the cell, cytoplasm, nucleus and circumference factor. MCTs of grade III had a mean cellular area of 231.70 μm2 ± 57.1, and grade II had a mean of 252.30 μm2 ± 55.0. Cellular perimeter was 61.20 ± 7.1 in grade II and 59.1 ± 8.6 in grade III. Cellular parameters were not statistically different between grades (p>.05). Mean nuclear area was 88.90 μm2 ± 19 in grade III and 72.30 μm2 ± 13.9 in grade II, with statistical difference between grades (P =.011). Mean nuclear perimeter was 32.40 ìm ± 3.0 in grade II and 35.70 ìm ± 4.0 in grade III, with statistical difference between grades (P =.018). Mean nuclear circumference factor was 1.0 ± 0.33 in grade II and 1.1 ± 0.28 in grade III, with no statistical difference between grades (P = 0.78). Nuclear-tocytoplasmic ratio in grade II was 0.29 ±.07 and 0.39 ±.08 in grade III, with statistical difference (P =.02). The number of binucleated and multinucleated cells and mitotic figures was significantly increased in grade III MCTs (P <.001). In conclusion, the number of mitotic figures, presence of binucleation and multinucleation, and nuclear-to-cytoplasmic ratio can help to guide a profile of MCT aggressiveness in cytologic preparations.

Cytogenetic evidence for de novo synthesis of rRNA and involvement of nucleolar material in the organization of cell structures during spermiogenesis of Chariesterus armatus (Heteroptera, Coreidae).

The nucleolar material of Chariesterus armatus was analyzed during spermiogenesis in cell preparations impregnated with silver nitrate. Nucleolar corpuscles were observed in spermatids at the beginning of the process, showing that this organoid is also maintained after meiosis. In addition, nucleoli were seen in the round spermatids connected to the X-chromosome (bearer of the nucleolar organizer in C. armatus), indicating de novo synthesis of nucleolar material. This differs from the reorganization of ribosomal granules, transported from meiotic spermatocytes to round spermatids, where they would support protein synthesis, which is reported for other species. We also observed connections of nucleolar corpuscles to the nuclear membrane regions where the tail and the acrosome will be formed, suggesting close involvement of the nucleolar material in the formation of these structures. In addition to the nucleolar bodies, we detected silver-positive structures, which will require new approaches to clarify their role. One of these structures, observed in the cytoplasm, appears to correspond to the chromatoid body, which has been found in several organisms, but is still poorly understood; another is a complex structure to which the tail appears to be connected. We conclude that C. armatus is an appropriate model for understanding not only the synthesis of rRNA in the spermiogenesis, but also the functional meaning of the close relationship of nucleolar material with other structures during this process.