Immunohistochemical and morphological features of a small bowel leiomyoma in a black crested macaque (Macaca nigra)

ABSTRACT: BACKGROUND: Spontaneous gastrointestinal neoplasms in non-human primates are commonly seen in aged individuals. Due to genetic similarities between human and non-human primates, scientists have shown increasing interest in terms of comparative oncology studies. CASE PRESENTATION: The present study is related to a case of an intestinal leiomyoma in a black crested macaque (Macaca nigra), kept on captivity by Matecana Zoo, Pereira City, Colombia. The animal had abdominal distension, anorexia, vomiting, diarrhea and behavioral changes. Clinical examination showed an increased volume in the upper right abdominal quadrant caused by a neoplastic mass. The patient died during the surgical procedure. Necropsy revealed several small nodules in the peritoneum with adhesion to different portions of the small and large intestines, liver, stomach and diaphragm. Tissue samples were collected, routinely processed and stained by H&E. Microscopic examination revealed a mesenchymal tumor limited to tunica muscularis, resembling normal smooth muscle cells. Neoplastic cells were positive for alpha-smooth muscle actin and vimentin, and negative for cytokeratin AE1/AE3 by immunohistochemistry. Those morphological and immunohistochemical findings allowed to diagnose the intestinal leiomyoma referred above. CONCLUSION: Neoplastic diseases in primates have multifaceted causes. Their manifestations are understudied, leading to a greater difficulty in detection and measurement of the real impact provides by this disease.     

Synthesis and radiosynthesis of [(18)F]FPhEP, a novel alpha(4)beta(2)-selective, epibatidine-based antagonist for PET imaging of nicotinic acetylcholine receptors

FPhEP (1, (+/-)-2-exo-(2'-fluoro-3'-phenyl-pyridin-5'-yl)-7-azabicyclo[2.2.1]heptane) belongs to a recently described novel series of 3'-phenyl analogues of epibatidine, which not only possess subnanomolar affinity and high selectivity for brain alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs), but also were reported as functional antagonists of low toxicity (up to 15 mg/kg in mice). FPhEP (1, K(i) of 0.24 nM against [(3)H]epibatidine) as reference as well as the corresponding N-Boc-protected chloro- and bromo derivatives (3a,b) as precursors for labelling with fluorine-18 were synthesized in eight and nine steps, respectively, from commercially available N-Boc-pyrrole (overall yields=17% for 1, 9% for 3a and 8% for 3b). FPhEP (1) was labelled with fluorine-18 using the following two-step radiochemical process: (1) no-carrier-added nucleophilic heteroaromatic ortho-radiofluorination from the corresponding N-Boc-protected chloro- or bromo derivatives (3 a,b-1mg) and the activated K[(18)F]F-Kryptofix(222) complex in DMSO using microwave activation at 250 W for 1.5 min, followed by (2) quantitative TFA-induced removal of the N-Boc-protective group. Radiochemically pure (>99%) [(18)F]FPhEP ([(18)F]-1, 2.22-3.33 GBq, 66-137 GBq/micromol) was obtained after semi-preparative HPLC (Symmetry C18, eluent aq 0.05 M NaH(2)PO(4)/CH(3)CN, 80:20 (v:v)) in 75-80 min starting from a 18.5 GBq aliquot of a cyclotron-produced [(18)F]fluoride production batch (10-20% nondecay-corrected overall yield). In vitro binding studies on rat whole-brain membranes demonstrated a subnanomolar affinity (K(D) 660 pM) of [(18)F]FPhEP ([(18)F]-1) for nAChRs. In vitro autoradiographic studies also showed a good contrast between nAChR-rich and -poor regions with a low non-specific binding. Comparison of in vivo Positron Emission Tomography (PET) kinetics of [(18)F]FPhEP ([(18)F]-1) and [(18)F]F-A-85380 in baboons demonstrated faster brain kinetics of the former compound (with a peak uptake at 20 min post injection only). Taken together, the preliminary data obtained confirm that [(18)F]FPhEP ([(18)F]-1) has potential for in vivo imaging nAChRs in the brain with PET.     

FLUKA Monte Carlo simulation for the Leksell Gamma Knife Perfexion radiosurgery system: Homogeneous media

The purpose of this work is to investigate the capability of the FLUKA Monte Carlo (MC) code to simulate the Elekta Leksell Gamma Knife Perfexion (LGK-PFX) and reproduce the Treatment Planning System (TPS) Leksell GammaPlan version 8.2 (LGP) dose calculations for the case of a water equivalent phantom target. Thanks to the collaboration with Elekta Instruments AB, the collimation system geometry, the source positions and all the involved material have been simulated in detail. The relative linear dose distribution along the three coordinate axes, for each collimator size, and the Relative Output Factors (ROF) have been investigated. The simulation has been validated comparing simulated linear dose profiles with measurements performed with EBT radiochromic films. The acceptance criterion between experimental data and FLUKA results is based on the gamma index (GI) method. The FLUKA MC calculation for the ROF provided the values of 0.920 for the 8 mm collimators and 0.800 for the 4 mm collimators. These values are in good agreement with the Elekta reference data of 0.924 and 0.805 respectively. The percentage difference between calculated and reference values for the ROF is under 1% and within the FLUKA uncertainty. Also the simulated relative dose profiles show a good agreement with the LGP calculation expressed by means of the gamma index method. This established accuracy proves that FLUKA is a suitable and powerful tool in order to reproduce successfully the LGP calculations for the homogeneous media.     

A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.     

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 μM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.     

BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm

The medical device-related infections are frequently a consequence of Staphylococcus biofilm, a lifestyle enhancing bacterial resistance to antibiotics. Antibiotic susceptibility tests are usually performed on planktonic forms of clinical isolates. Some methods have been developed to perform antibiotic susceptibility tests on biofilm. However, none of them counts bacterial inoculum. As antibiotic susceptibility is related to bacterial inoculum, the test results could be mistaken. Here, a new method, BioTimer Assay (BTA), able to count bacteria in biofilm without any manipulation of samples, is presented. Moreover, the BTA method is applied to analyze antibiotic susceptibility of six Staphylococcus strains in biofilm and to determine the number of viable bacteria in the presence of sub-inhibitory doses of four different antibiotics. To validate BTA, the new method was compared to reference methods both for counting and antibiotic susceptibility tests. A high agreement between BTA and reference methods is found on planktonic forms. Therefore, BTA was employed to count bacteria in biofilm and to analyze biofilm antibiotic susceptibility. Results confirm the high resistance to antibiotics of Staphylococcus biofilm. Moreover, BTA counts the number of viable bacteria in the presence of sub-inhibitory doses of antibiotics. The results show that the number of viable bacteria depends on sub-inhibitory doses, age of biofilm and type of antibiotic. In particular, differently to gentamicin and ampicillin, sub-inhibitory doses of ofloxacin and azithromycin reduce the number of viable bacteria at lower extent in young than in old biofilm. In conclusion, BTA is a reliable, rapid, easy-to-perform, and versatile method, and it can be considered a useful tool to analyze antibiotic susceptibility of Staphylococcus spp. in biofilm.     

Autologous and Heterologous Neutralization Analyses of Primary Feline Immunodeficiency Virus Isolates

Feline immunodeficiency virus (FIV) provides a model system with which the significance of neutralizing antibody (NA) in immunosuppressive lentivirus infections may be studied. To date, no detailed analysis of the neutralization properties of primary FIV isolates has been reported. In this study, we have conducted the first comprehensive study of the sensitivity to autologous and heterologous neutralization in a lymphoid cell-based assay of 15 primary FIV isolates and, for comparison, of one tissue culture-adapted strain. Primary isolates in general proved highly NA resistant, although there was considerable individual variation. Variation was also observed in the capacity of immune sera to neutralize heterologous FIV isolates. The ability of sera to neutralize isolates or for isolates to be neutralized by sera did not correlate with epidemiological and genetic relatedness or with the quasispecies complexity of the isolates. From the study of specific-pathogen-free cats experimentally infected with viral isolates associated with NA of different breadths, it appears that the development of FIV vaccines cannot rely on the existence of viral strains inherently capable of inducing especially broad NA responses.Feline immunodeficiency virus (FIV) is a lentivirus that is regarded as the feline counterpart of human immunodeficiency virus (HIV) because it produces persistent infections of domestic cats which, after an incubation period of several years, progress to clinical manifestations of immunodeficiency and neurological damage that closely resemble those observed in HIV-infected humans. FIV is therefore a valuable model for investigating many aspects of AIDS pathobiology and control, including vaccination (4, 11, 39, 56).Based on DNA phylogenesis, FIV isolates worldwide have been classified into at least five distinct genetic subtypes, designated A to E, with uneven geographical distributions (2). While there is little hope of developing a monovalent vaccine capable of protecting across different FIV subtypes, a more reasonable goal is the development of one or several protective immunogens for each subtype and subsequent selection of the immunogens on the basis of the subtypes prevalent in the area where the vaccine is to be used (56). However, because genetic diversity is also high within a subtype, especially in the env region (2, 42), successful vaccines will have to induce immune responses effective against a wide range of antigenically diverse strains. Mapping the immunological relatedness of FIV strains belonging to the same genetic subtype therefore represents a prerequisite for identifying shared critical protective epitopes and an essential step for ongoing vaccine development efforts. Similar problems exist for HIV vaccine development (33).Although the humoral and cell-mediated immune responses that will eventually prove important for vaccine-induced protection against lentiviruses are unresolved (3, 7, 17), the ability to evoke a broadly reactive neutralizing-antibody (NA) response would seem to be an advantageous feature of candidate immunogens because it would at least contrast the dissemination of the initial viral inoculum from the site of entry (8, 9). In previous studies, we found that cats immunized with a fixed-cell vaccine were protected against FIV challenge in the apparent absence of NA (27, 28), but it is possible that a detectable NA response could be elicited with improved vaccines, adjuvants, and immunization regimens.FIV vaccines must be designed to protect against strains of FIV as they circulate in nature. For this reason, it is important to learn more about the immunobiological properties of fresh clinical isolates, including their ability to evoke and interact with NA and their neutralizing determinant(s). Here we report on the sensitivity of 15 FIV isolates subjected to minimal passage in culture to neutralization by autologous and heterologous immune sera. Primary FIV isolates proved only slightly prone to inhibition by immune sera. However, certain isolates were more neutralizable by heterologous sera than others and certain infected cat sera neutralized fairly large numbers of primary isolates. A relationship was also sought between neutralization properties of the isolates and immune sera and a number of factors that theoretically might influence the induction or the activity of cross-reactive NA, including epidemiological and genetic relatedness and quasispecies complexity of the isolates. Finally, to ascertain whether the cross-neutralizing potency of anti-FIV antibody was dependent on properties of the viruses that had induced their formation, we studied the NA response of specific-pathogen-free (SPF) cats inoculated with selected FIV isolates.     

Changes in the surface morphology of the rabbit endometrium related to the estrous and progestational stages of the reproductive cycle

Summary Changes occurring on the surface of the uterine luminal epithelium of the rabbit during the estrous and progestational stages of the reproductive cycle were examined by scanning and transmission electron microscopy. The findings demonstrate that the uterine epithelium, or endometrium, contains two cell types: ciliated cells and nonciliated, microvillous cells. In estrous animals, ciliated cells, although not very numerous, were usually observed in small groups. However, at increasing intervals of time following mating, ciliated cells progressively disappeared from the endometrium until approximately eight to ten days post coitum, when they became scare. From several hours to four to five days following mating, extensive changes occurred on the surfaces of microvillous cells. When observed by TEM, these elements contained organelles typical of cells involved in the synthesis and secretion of glycoproteins. Furthermore, microvillous cells during this period displayed numerous apical protrusions of different sizes and shapes and containing material of varying electron density. Parallel SEM examinations of the same material confirmed the presence of these protrusions. Some of the protrusions appeared as spheroidal masses attached to the cytoplasm by means of a cytoplasmic strand. Other surface masses were clearly unattached to the cell surface and were distributed (1) on the surface of microvillous cells, (2) on the cilia of adjacent ciliated cells, and (3) on the surface of spermatozoa.Changes occurring on the luminal surface during the early postcoital period are interpreted as an expression of morphodynamic processes likely involving coupled secretion (exocytosis) and resorption (endocytosis) of luminal material. The observations presented here also demonstrate that between six and ten days post coitum, the rabbit endometrium contained increasing numbers of enlarged, nonciliated cells that probably arose by the fusion of smaller, microvillous elements.The work reported here was supported by C.N.R. contracts No. CT 760128809 and CT 77014239 (to P.M.) and NIH. Grant HD-04274 (to J.V.B.)     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches.Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.