Effects of IFN-gamma and interleukin-1beta on major histocompatibility complex antigen and intercellular adhesion molecule-1 expression by 9L gliosarcoma: relevance to its cytolysis by alloreactive cytotoxic T lymphocytes.

To enhance the efficacy of cellular immunotherapy for gliomas, we tested the concept of using proinflammatory cytokine treatment with interferon-gamma (IFN-gamma) or interleukin-1beta (IL-1beta) or both to render glioma cells more susceptible to cytolysis by alloreactive cytotoxic T lymphocytes (aCTL). The cytokines, separately or in combination, were able to upregulate major histocompatibility complex (MHC) class I antigen or intercellular adhesion molecule-1 (ICAM-1) on Fischer rat 9L gliosarcoma cells. 9L cells were incubated in vitro for 24, 48, or 72 h with varying concentrations of rat IFN-gamma (0-2000 U/ml) or recombinant human IL-1 (rHUIL-1) (0-1000 U/ml) or both. By 48 h, IFN-gamma (500 U/ml) maximally induced the percentage of positive expressing cells and the relative antigen density of MHC class I and ICAM-1 on 9L cells, whereas IL-1 induced only ICAM-1 expression. Simultaneous incubation of IL-1 with IFN-gamma did not further affect the induction of class I on 9L cells more than that achieved with IFN-gamma alone. 9L cells with upregulated MHC class I and ICAM-1 expression were more sensitive to lysis by aCTL in in vitro cytotoxicity assays, regardless of whether the precursor aCTL came from naive or from 9L-immunized rats. Furthermore, inhibition of 9L cytotoxicity in assays that included blocking antibodies to MHC class I or to ICAM-1 revealed that T cell receptor (TCR) interactions with MHC class I and that ICAM-1 interactions with lymphocyte function-associated-1 (LFA-1) antigen account for a portion of the glioma lysis by aCTL.     

Quantitative (82)Rb PET/CT: development and validation of myocardial perfusion database.

The use of myocardial perfusion (82)Rb PET/CT studies continues to increase but its accuracy using database quantification methods for the diagnosis of coronary artery disease (CAD) has not been established. METHODS: A sex-independent normal database and criteria for abnormality for rest-stress (82)Rb PET/CT myocardial perfusion imaging were developed and validated by evaluation of 281 patients (136 females: mean age +/- SD, 63.3 +/- 13.3 y; 145 males: mean age +/- SD, 63.9 +/- 12.8 y) who underwent a rest-adenosine stress (82)Rb PET/CT study. These patients were divided into 3 groups: (a) healthy group: 30 patients, with <5% likelihood of CAD (low likelihood [LLK]) based on sequential Bayesian analysis; these patients were used to generate the normal distribution; (b) pilot group: 174 patients; these patients were used to determine the optimal criteria for detecting and localizing the perfusion abnormality; and (c) validation group: 76 patients (23 with LLK of CAD and 53 who underwent coronary angiography; these patients were used for prospective validation. RESULTS: Of the 53 patients who underwent coronary angiography, 8 had <50% stenosis and 45 patients had at least one stenosis > or =50% in one major artery. Fifteen patients had single-vessel disease, 17 had double-vessel disease, and 13 had triple-vessel disease. The prospective validation shows a normalcy rate of 78% (18/23) for global CAD. The analyses by individual arteries show a normalcy rate of 96% (22/23) for the left anterior descending coronary artery, 96% for the left circumflex coronary artery (22/23), and 100% for the right coronary artery (23/23). The overall sensitivity for detection of CAD (> or =50% stenosis) was 93% (42/45). The overall specificity for detection of the absence of CAD (< or =50% stenosis) was 75% (6/8). Also, the positive predictive value for global CAD was 95% (42/44), the negative predictive value was 67% (6/9), and the accuracy was 91% (48/53). CONCLUSION: The quantitative (82)Rb PET/CT database created and validated in this study is highly accurate for the detection and localization of CAD. Physicians should consider using the quantitative output of these algorithms as decision support tools to aid with image interpretation.     

Ontogenetic profile of glutamate uptake in brain structures slices from rats: sensitivity to guanosine

The excitotoxicity of the neurotransmitter glutamate has been shown to be connected with many acute and chronic diseases of the CNS. High affinity sodium-dependent glutamate transporters play a key role in maintaining adequate levels of extracellular glutamate. In the present study, we used slices of striatum, hippocampus and cortex from rat brain to describe the in vitro profile of glutamate uptake during development and ageing, and its sensitivity to guanosine. In all structures, glutamate uptake was higher in immature animals. There was a maximum decrease in glutamate uptake in striatum and hippocampus in 15-month-old rats, which later increased, while in cortex there was a significant decrease in rats aged 60 days old. The effect of guanosine seems to be age and structure dependent since the increase in basal glutamate uptake was only seen in slices of cortex from 10-day-old animals.     

Evaluation of stromal metalloproteinases and vascular endothelial growth factors in a spontaneous metastasis model

This study aims to investigate MMP2 and MT1-MMP protein as well as VEGF-C and VEGF-D mRNA expression in tumor cells and distant organs considered to be targets for metastasis in a tumor spontaneous metastasis model previously described. Cultured tumor cells, able to express pro-MMP2, MMP2, pro-MMP9, and MT1-MMP, develop tumor growth and metastasis, mainly in the liver and spleen, when they are injected in the mammary pad gland of Wistar rats. Immunohistochemical studies of tumor masses showed small groups of tumor cells staining for MT1-MMP but not for MMP2. In the liver, tumor metastatic foci and a stromal positive staining for both MMP2 and MT1-MMP were shown. The spleen and lymph nodes, with only scattered metastatic cells, did not show MMPs immunostaining. Using RT-PCR, a significantly higher VEGF-C and VEGF-D gene expression was shown in the liver of tumor-bearing rats respect to normal rats, whereas spleen and lymph nodes did not show significant differences in mRNA VEGF-C/D levels. Taken together, our results suggest that the stroma microenvironment of target organs for metastasis has the ability to produce MMPs and VEGFs that facilitate the anchorage of tumor cells and promote tumor cell growth and angiogenesis.     

Connective tissue growth factor expression and Smad signaling during mouse heart development and myocardial infarction.

Connective tissue growth factor (CTGF) is reported to be a target gene of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) in vitro. Its physiological role in angiogenesis and skeletogenesis during mouse development has been described recently. Here, we have mapped expression of CTGF mRNA during mouse heart development, postnatal adult life, and after experimental myocardial infarction. Furthermore, we investigated the relationship between CTGF and the BMP/TGFbeta signaling pathway in particular during heart development in mutant mice. Postnatally, CTGF expression in the heart became restricted to the atrium. Strikingly, 1 week after myocardial infarction, when myocytes have disappeared from the infarct zone, CTGF and TGFbeta expression as well as activated forms of TGFbeta but not BMP, Smad effector proteins are colocalized exclusively in the fibroblasts of the scar tissue, suggesting possible cooperation between CTGF and TGFbeta during the pathological fibrotic response.     

Microsatellite analysis in Turner syndrome: parental origin of X chromosomes and possible mechanism of formation of abnormal chromosomes.

Turner syndrome is a chromosomal disorder in which all or part of one X chromosome is missing. The meiotic or mitotic origin of most cases remains unknown due to the difficulty in detecting hidden mosaicism and to the lack of meiotic segregation studies. We analyzed 15 Turner patients, 10 with a 45,X whereas the rest had a second cell line with abnormal X-chromosomes: a pseudodicentric, an isochromosome, one large and one small ring, and the last with a long arm deletion. Our aims were: to detect X cryptic mosaicism in patients with a 45,X constitution; to determine the parental origin of the abnormality; to infer the zygotic origin of the karyotype and to suggest the timing and mechanism of the error(s) leading to the formation of abnormal X chromosomes from maternal origin. Molecular investigation did not revealed heterozygosity for any microsatellite, excluding X mosaicism in the 45,X cases. Parental origin of the single X chromosome was maternal in 90% of these patients. Three of the structurally abnormal Xs were maternally derived whereas the other two were paternal. These results allowed us to corroborate breakpoints in these abnormal X chromosomes and suggest that the pseudodicentric chromosome originated from post-zygotic sister chromatid exchange, whereas the Xq deleted chromosome probably arose after a recombination event during maternal meiosis.     

Helicobacter pylori gastritis in cats with long-term natural infection as a model of human disease

A natural infection with Helicobacter pylori (H. pylori) in domestic cats (Felis cattus) less than 2 years of age has been well described in a closed colony of animals. Six cats from this colony that were serially evaluated by culture, polymerase chain reaction, and light and electron microscopy for a period of 3 years demonstrated persistent gastric colonization with a single cag(-) vac(+) strain of H. pylori. In these cats, as well as five other 5- to 6-year-old cats that were examined, a long-term infection resulted in chronic diffuse lymphofollicular atrophic gastritis with areas of mucosal dysplasia in the antrum and predominantly midsuperficial gastritis in the body and cardia. Topographically, the distribution of lesions was similar in both young and older cats and closely resembled that found in humans, with the most severe changes occurring in the gastric antrum. Few granulocytes and no significant elevation in mast cells were seen in older H. pylori-infected cats compared with uninfected controls; however, marked increases in interepithelial globule leukocytes and numerous active mucosal lymphoid follicles were present in infected animals. Indices of gastritis were significantly greater in older infected cats when compared with uninfected controls and younger cats (P < 0.05). The antral cell proliferation index of infected older cats was significantly (P = 0.021) greater than that of uninfected controls. Apoptotic indices of the gastric antrum and body of infected cats were significantly (P = 0.01) increased versus controls. Chronic infection with H. pylori in cats shares many features of long-term H. pylori infection in humans, including the development of preneoplastic processes. This similarity provides useful, comparative insights into host-pathogen interactions.     

Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.     

Chain conformation and compatibility in polymer blends. A small-angle X-ray scattering study of the system poly(propylene glycol)-poly(methyl methacrylate)

The thermodynamic compatibility of dilute, solid solutions of poly(propylene glycol) (PPG) (mass-average molar mass M?_w = 3900 g/mol) in poly(methyl methacrylate) (PMMA) (M?_w = 550000 g/mol) was investigated by means of small-angle X-ray scattering. The results of the analysis show that PPG is molecularly dispersed in the solid, amorphous matrix of PMMA, and that the molecules display the typical statistical coil macroconformation of polymers in dilute, liquid solution, whereby the numerical value of the second virial coefficient seems to point to the unperturbed nature of the coils. This behaviour, consistent with the concept of “interpenetration of segments”, is in total agreement with the experimentally measured compositional variation of the glass transition temperature of the blends.