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At least 1,063 records · Page 59Linked to original sources
PubMed2006

Magnetic resonance imaging of progressive cardiomyopathic changes in the db/db mouse.

The db/db mouse is a well-established model of diabetes. Previous reports have documented contractile dysfunction (i.e., cardiomyopathy) in these animals, although the extant literature provides limited insights into cardiac structure and function as they change over time. To better elucidate the natural history of cardiomyopathy in db/db mice, we performed cardiac magnetic resonance (CMR) scans on these animals. CMR imaging was conducted with a 4.7-T magnet on female db/db mice and control db/+ littermates at 5, 9, 13, 17, and 22 wk of age. Gated gradient echo sequences were used to obtain cineographic short-axis slices from apex to base. From these images left ventricular (LV) mass (LVM), wall thickness, end-diastolic volume (LVEDV), and ejection fraction (LVEF) were determined. Additionally, cardiac [(18)F]fluorodeoxyglucose ([(18)F]FDG) PET scanning, pressure-volume loops, and real-time quantitative PCR on db/db myocardium were performed. Relative to control, db/db mice developed significant increases in LVM and wall thickness as early as 9 wk of age. LVEDV diverged slightly later, at 13 wk. Interestingly, compared with the baseline level, LVEF in the db/db group did not decrease significantly until 22 wk. Additionally, [(18)F]FDG metabolic imaging showed a 40% decrease in glucose uptake in db/db mice. Furthermore, contractile dysfunction was observed in 15-wk db/db mice undergoing pressure-volume loops. Finally, real-time quantitative PCR revealed an age-dependent recapitulation of the fetal gene program, consistent with a myopathic process. In summary, as assessed by CMR, db/db mice develop characteristic structural and functional changes consistent with cardiomyopathy.

Animals↗
PubMed1997

Quantitative polymerase chain reaction for human herpesvirus diagnosis and measurement of Epstein-Barr virus burden in posttransplant lymphoproliferative disorder.

Human herpesviruses can cause acute diseases such as chicken pox or mononucleosis, but also may reactivate during immunosuppression and result in severe or life-threatening illnesses such as shingles or lymphoproliferative disorders. We report the development and validation of a quantitative PCR method to measure viral burden for all eight human herpesviruses (HSV1, HSV2, VZV, EBV, CMV, HHV6, HHV7, and KSHV) in patients' samples. The method uses an internal standard that is coamplified with the viral target, allowing quantification of viral genomes in absolute terms (e.g., viral targets/mL of blood) and ruling out false-negative results. We demonstrate that transplant patients with lymphoproliferative disorder carry an EBV viral burden 3 logs higher than nontransplant patients. EBV titers in transplant patients without a lymphoproliferative disorder are between these values. This quantitative PCR method may aid in differentiating clinically significant vs latent viral burden in immunosuppressed patients.

Herpesviridae Infections↗
PubMed2001

Quantitative fluorogenic PCR assay for measuring ovine herpesvirus 2 replication in sheep.

A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep.

Animals↗
PubMed2006

Expression of connexins 36, 43, and 45 during postnatal development of the mouse retina.

Gap junction channels formed by connexins (Cx) may play essential roles in some processes that occur during retinal development, such as apoptosis and calcium wave spread. The present study was undertaken to determine the distribution pattern of Cx36, Cx43, and Cx45 by immunofluorescence, as well as their gene expression levels by quantitative PCR during postnatal development of the mouse retina. Our results showed an increased expression of neuronal Cx36 from P1 until P10, when this Cx reached adult levels, and it was mainly distributed in the outer and inner plexiform layers. In turn, Cx43 was almost absent in retinal progenitor cells at P1, it became more prominent in glial cell processes about P10, and did not change until adulthood. Double-labeling studies in situ and in vitro with antivimentin, a Müller cell marker, confirmed that Cx43 was expressed by these cells. In addition, quantitative PCR showed that Cx43 and vimentin shared very similar temporal expression patterns. Finally, in contrast to Cx36 and Cx43, Cx45 mRNA was strongly down-regulated during development. In early postnatal days, Cx45 was seen ubiquitously distributed throughout the retina in cells undergoing proliferation and differentiation, as well in differentiated neurons. In adult retina, this protein had a more restricted distribution both in neurons and glial cells, as confirmed in situ and in vitro. In conclusion, we observed a distinct temporal expression pattern for Cx36, Cx43, and Cx45, which is probably related to particular roles in retinal function and maintenance of homeostasis during development of the mouse retina.

Age Factors↗
PubMed2006

Comparative in vivo activity of BAL4815, the active component of the prodrug BAL8557, in a neutropenic murine model of disseminated Aspergillus flavus.

BACKGROUND: BAL8557 (WSA) is the water-soluble prodrug of the triazole BAL4815 with in vitro anti-Aspergillus activity. We compared the activity of oral BAL8557 with oral itraconazole, oral voriconazole and intravenous caspofungin in a temporarily neutropenic murine model of disseminated Aspergillus flavus. METHODS: Mice were immunosuppressed using cyclophosphamide, then infected. Mice were treated either 2 h pre-infection (PRE), or 4 or 24 h post-infection (4POST and 24POST, respectively). Treatment was for 10 days followed by 4 days of observation. Surviving mice were killed and liver, kidneys, lungs and brain cultured. BAL8557 groups included doses corresponding to approximately 30, 15, 6 and 3 mg/kg of the active BAL4815; comparators included itraconazole 25 and 10 mg/kg/dose, voriconazole (plus oral grapefruit) 25 and 10 mg/kg/day or caspofungin 1 mg/kg/day. In a simultaneous tissue burden study mice were treated for 3 days, kidneys removed and homogenized and burden measured by quantitative culture and quantitative PCR using fluorescence resonance energy transfer (FRET). RESULTS: Control mice had 83-100% mortality. Over 66% of BAL8557-treated mice survived after >6 mg/kg PRE or >15 mg/kg POST. In the PRE models BAL8557 (6 mg/kg) and caspofungin were 100% protective and itraconazole 67% protective, but voriconazole 10 mg/kg had 100% mortality (P = 0.0016). In the 4POST and 24POST models survival was >66% with BAL8557 30 and 15 mg/kg/dose and similar to voriconazole or itraconazole. In the 24POST groups, sterilization of all organs was achieved in 11/16 survivors treated with BAL8557. The quantitative PCR correlated with kidney fungal burden (r2 = 0.59). Earlier treatment reduced burdens. CONCLUSIONS: BAL8557 demonstrated impressive antifungal activity against A. flavus in this model, in both survival and tissue burden.

Animals↗
PubMed2006

Eukaryotic picoplankton communities of the Mediterranean Sea in summer assessed by molecular approaches (DGGE, TTGE, QPCR).

The composition and abundance of eukaryotic picoplankton (defined here as cells smaller than 3 mum) was investigated in the Morocco upwelling and throughout the Mediterranean Sea in late summer using flow cytometry and molecular methods (gradient gel electrophoresis and quantitative PCR). The picoplankton displayed characteristics typical of oligotrophic oceanic areas with concentrations down to 1000 cells mL(-1) in the Eastern Basin. The most abundant eukaryotic sequences recovered by gradient gel electrophoresis were related to uncultivated marine groups: alveolates I (16%) and II (26%) and a newly discovered group (env Nansha, 17%) for which sequences have been recently obtained from the South China Sea and that could be related to Acantharians. Prasinophyceae (photosynthetic green algae) accounted for 10% of the sequences, whereas Cercozoa, Stramenopiles, Polycystinea, dinoflagellates and ciliates provided minor contributions. The use of quantitative PCR coupled with taxon-specific primers allowed us to estimate the relative abundance of several taxa belonging to the Prasinophyceae. Of the three genera assessed, Bathycoccus appeared as the most abundant, forming localized maxima at depth.

Animals↗
PubMed2024

Comprehensive analysis of the expression, prognostic, and immune infiltration for COL4s in stomach adenocarcinoma.

BACKGROUND: Collagen (COL) genes, play a key role in tumor invasion and metastasis, are involved in tumor extracellular matrix (ECM)-receptor interactions and focal adhesion pathways. However, studies focusing on the diagnostic value of the COL4 family in stomach adenocarcinoma (STAD) are currently lacking. METHODS: The TCGA database was employed to retrieve the clinical features and RNA sequencing expression profiles of patients with STAD. We conducted an investigation to examine the expression disparities between STAD and adjacent normal tissues. Kaplan-Meier survival analysis was utilized to assess their prognostic significance, while Spearman correlation analysis was employed to determine their association with immune checkpoint genes and immunomodulatory molecules. Furthermore, GO and KEGG analyses were performed on the COL4s-related genes, revealing potential biological pathways through gene set enrichment analysis (GSEA). Subsequently, we explored the extent of immune infiltration of the COL4 family in STAD using the TIMER database. Lastly, the expression levels of the COL4 family in STAD were further validated through quantitative PCR (qPCR) and western blot techniques. RESULTS: The expression levels of COL4A1/2 were significantly upregulated, while COL4A5/6 were conspicuously downregulated in STAD. The survival analysis revealed that the upregulated COL4s indicated poorer overall survival, first progression and post-progression survival outcomes. Additionally, our findings demonstrated a positive correlation between the expressions of COL4A1/2/3/4 and the infiltration of immune cells, including CD8 + T cells, dendritic cells, macrophages, neutrophils and CD4 + T cells. Further correlation analysis uncovered a favorable association between the expression of COL4A1/2/3/4 and various crucial immunomodulatory molecules, immunological checkpoint molecules, and chemokines. Quantitative PCR analysis confirmed that the expression patterns of COL4A1/3/4/6 genes aligned with the finding from the TCGA database. However, gastric cancer cells exhibited downregulation of COL4A2. Consistently, the protein level of COL4A1 was elevated, whereas the protein level of COL4A2 was reduced in the gastric cancer cell lines. CONCLUSION: COL4s could potentially serve as biomarkers for diagnosing and predicting the prognosis of STAD.

Stomach Neoplasms↗
PubMed2003

Higher susceptibility to Fas ligand induced apoptosis and altered modulation of cell death by tumor necrosis factor-alpha in periarticular tenocytes from patients with knee joint osteoarthritis.

The aim of the present study was to investigate the expression of Fas in periarticular tenocytes of patients with osteoarthritis (OA) and to study their susceptibility to Fas ligand-mediated apoptosis. Tendon samples were obtained from the quadriceps femoris muscle of patients with knee OA and used for histological evaluation, for immunohistochemical detection of Fas, and to establish tenocyte cultures. The expression of Fas mRNA was determined by quantitative PCR. Levels of soluble Fas and soluble tumour necrosis factor (TNF) receptor I were measured using ELISA. Apoptosis was induced with recombinant human Fas ligand and measured by a histone fragmentation assay and flow cytometry. The effects of TNF-alpha were studied by stimulation with TNF-alpha alone or 24 hours before the induction of apoptosis. Tendon samples from non-OA patients were used as controls. Histological evaluation revealed degenerative changes in the tendons of all OA patients but not in the controls. Fas was detected by immunohistochemistry in all specimens, but quantitative PCR revealed significantly higher levels of Fas mRNA in OA tenocytes. In contrast, lower levels of soluble Fas were found in OA tenocytes by ELISA. OA tenocytes were significantly more susceptible to Fas ligand induced apoptosis than were control cells. TNF-alpha reduced the Fas ligand induced apoptosis in OA tenocytes but had no effects on control tenocytes. These data suggest that knee OA is associated with higher susceptibility of periarticular tenocytes to Fas ligand induced apoptosis because of higher expression of Fas but lower levels of apoptosis-inhibiting soluble Fas. These changes may contribute to decreased cellularity in degenerative tendons and promote their rupturing. The antiapoptotic effects of TNF-alpha in OA tenocytes most likely reflect regenerative attempts and must be taken into account when anti-TNF strategies are considered for OA.

Apoptosis↗
PubMed2006

NASBA: a novel approach to assess hormonal receptors and ERBB2 status in breast cancer.

In human breast cancer, estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and human epidermal growth factor receptor (ERBB2) status are currently determined using different techniques. We propose to assess the mRNA expression of these three clinically relevant markers using a unique technique, real-time nucleic acid sequence-based amplification (NASBA). Gene expression of hormone receptors was analyzed and compared to the cytosolic functional protein content as determined with a ligand binding assay (LBA), while ERBB2 mRNA expression was compared to quantitative PCR and ELISA. We observed that the three markers are significantly overexpressed at the mRNA level in positive tumors, as measured by DNA- or protein-based techniques. Biostatistical analysis of the receiver operating characteristic (ROC) curve demonstrated high concordance between NASBA and LBA [area under the curve (AUC) for ROC of 0.899] and showed that ERalpha status could be predicted using the molecular assay with a sensitivity of 72.7% and a specificity of 93.5%. Similar results were obtained for PR (AUC ROC 0.938, sensitivity 75.3%, specificity 100%). Moreover, excellent concordance was observed between NASBA, quantitative PCR and ELISA with respect to ERBB2 (AUC ROC 0.92, sensitivity 90%, specificity 89.7%; and AUC ROC 0.98, sensitivity 100%, specificity 91.5%, respectively). These results suggest that NASBA is well suited for assessing ER, PR and ERBB2 status in breast tumor samples. This approach is rapid, highly sensitive and a standardized method that could be complementary to the existing techniques, especially for small tumors.

Adult↗
PubMed2003

Cloning and characterization of a gene expressed during terminal differentiation that encodes a novel inhibitor of growth.

We report here the cloning and initial characterization of a novel growth-related gene (EEG-1) that is located on the short arm of chromosome 12. Two spliced transcripts were cloned from human bone marrow and human erythroid progenitor cells: EEG-1L containing a 4350-nucleotide open reading frame encoding a putative protein of 1077 amino acids including a C1q-like globular domain, and an alternatively spliced transcript lacking exon 5 (EEG-1S) encodes a significantly smaller coding region and no C1q-like domain. Quantitative PCR revealed expression of both EEG-1 transcripts in all analyzed tissues. Plasmids encoding green fluorescent protein-tagged genes (GFP-EEG-1) were transfected into Chinese hamster ovary cells for localization and functional assays. In contrast to the diffuse cellular localization of the GFP control, GFP-EEG-1L was detected throughout the cytoplasm and excluded from the nucleus, and GFP-EEG-1S co-localized with aggregated mitochondria. Transfection of both isoforms was associated with significantly increased levels of apoptosis. Stable transfection assays additionally demonstrated decreased growth in those cells expressing EEG-1 at higher levels. Quantitative PCR analyses of mRNA obtained from differentiating erythroid cells from blood donors were performed to determine the transcriptional pattern of EEG-1 during erythropoiesis. EEG-1 expression was highly regulated with increased expression at the stage of differentiation associated with the onset of global nuclear condensation and reduced cell proliferation. We propose that the regulated expression of EEG-1 is involved in the orchestrated regulation of growth that occurs as erythroblasts shift from a highly proliferative state toward their terminal phase of differentiation.

Animals↗
PubMed2006

Hypoxia-inducible factors in the first trimester human lung.

Lung development takes place in a relatively low-oxygen environment, which is beneficial for lung organogenesis, including vascular development. Hypoxia-inducible factor (HIF)-1 plays an important role in mediating oxygen-regulated events. HIF-1 is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau (VHL) tumor suppressor protein leads to rapid degradation of the HIF-1alpha subunit. Interaction with VHL requires hydroxylation of HIF-1alpha proline residues by prolyl hydroxylases (PHDs). We investigated the expression of the various components regulating HIF-1alpha stability in first trimester (8-14 weeks) human lungs. Spatial expression was assessed by immunohistochemistry and temporal expression by quantitative PCR. Immunoreactivity for PHD1, PHD3, and seven in absentia homolog (SIAH)1 was noted in the pulmonary epithelium. PHD2 was not expressed in the airway epithelium, but in the lung parenchyma. HIF-1alpha and vascular endothelial growth factor (VEGF) immunoreactivity were primarily detected in the branching epithelium. HIF-2alpha and ARNT proteins localized to the developing epithelium as well as mesenchymal, most likely vascular, structures in the parenchyma. VEGF receptor 2 (VEGFR2) was found in the subepithelium as well as in vascular structures of the mesenchyme. All components of the VEC complex (VHL, NEDD8, and Cullin2) were found in the epithelium. Quantitative PCR analysis demonstrated that VEGF, VEGFR1, HIF-1alpha, HIF-2alpha, ARNT, PHD1, PHD2, PHD3, and SIAH1 gene expression was constant during early pulmonary organogenesis. Cumulatively, the data suggest that the lung develops in a low-oxygen environment that allows for proper vascular development through HIF-regulated pathways.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed2005

Expression profiling reveals differences in metabolic gene expression between exercise-induced cardiac effects and maladaptive cardiac hypertrophy.

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise-induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise-induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in beta-oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise-induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise-induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.

Animals↗
PubMed2003

Detection of fetal Rhesus D gene in whole blood of women booking for routine antenatal care.

OBJECTIVE: To determine if the RhD status of the fetus can be detected in an unselected group of RhD-negative women with ongoing pregnancies booking for routine antenatal care. MATERIAL AND METHODS: We obtained 2.5 ml of whole blood from 202 unselected women with a normal ongoing pregnancy who booked for antenatal care before 20 weeks' gestation. DNA was extracted and real time quantitative PCR performed. Primers and a specific probe were targeted at the Rhesus D gene. RESULTS: Of 194 women, 31 were RhD-negative with no evidence of prior isoimmunisation. They delivered 17 RhD-positive and 14 RhD-negative babies. Of the 17 RhD-negative women carrying a RhD-positive fetus, the RhD gene was detected in the maternal blood in 14 (82%). There were no false positives. The quantity of RhD gene detected in the maternal blood of RhD-negative mothers ranged from 2 x 10(2) to 4 x 10(6) copies/ml of whole blood. CONCLUSION: Using real time quantitative PCR assays to amplify free fetal DNA in the maternal circulation, identification of RhD-positive fetuses in RhD-negative mothers is feasible when they book for antenatal care. Before such RhD genotyping can be introduced into clinical practice, however, further studies are required to show that false negative results can be eliminated and to show that this diagnostic test is reliable outside a research setting.

DNA↗
PubMed2004

Growth and survival mechanisms associated with perineural invasion in prostate cancer.

Perineural invasion (PNI) is the major mechanism of prostate cancer spread outside the prostate. Apoptotic and proliferation indices were determined in PNI cells using the PNI in vitro model and human PNI in tissue microarrays. RNA was extracted from the PNI model and controls and evaluated by cDNA microarray analysis. Differential expression of candidate genes was confirmed by real-time quantitative PCR, fluorescence, and immunohistochemistry using tissue microarrays. Genistein and BAY 11-7085 were added to the supernatant of cocultures and controls in microchamber cultures. The significance of nuclear factor kappaB (NFkappaB) nuclear translocation in human PNI was analyzed using Kaplan-Meier analysis. An increase in proliferation and a decrease in apoptosis were observed in human PNI cells and the PNI model as compared with controls. Three of 15 genes up-regulated in the cDNA microarray were involved in the apoptosis signaling pathway (NFkappaB), and its downstream targets defender against cell death 1 and PIM-2. The increase was corroborated by real-time quantitative PCR and immunofluorescence. NFkappaB nuclear translocation was seen in the in vitro model and human tissues, where strong nuclear expression was associated with a decrease in recurrence-free survival. Addition of genistein and BAY 11-7085 resulted in a decrease in NFkappaB, PIM-2 and defender against cell death 1 as well as a reversal of the inhibition of apoptosis. This is the first description of a biological mechanism and functional significance of PNI. Cancer cells in a perineural location acquire a survival and growth advantage using a NFkappaB survival pathway. Targeting PNI might help detain local spread of the tumor and influence survival.

Anti-Infective Agents↗
PubMed2007

Gene dosage and mutational analyses of EGFR in oligodendrogliomas.

We have studied amplification/gene-dosage and sequence variations of the EGFR gene in 41 oligodendroglial tumours graded according to the WHO classification (21 oligodendrogliomas grade II, 13 oligodendrogliomas grade III and 6 oligoastrocytomas grade II-III), using multiplex ligation-dependent probe amplification (MLPA), real-time quantitative PCR, and PCR/SSCP techniques. To determine gene-dose we studied exons 11 (extracellular domain) and 25 (intracellular domain) in the EGFR gene. Overdose (1- to 5-fold increase) was present in exon 11 in 21 of 41 samples (52.5% of cases) and in exon 25, in 7 of 41 samples (17.5% of cases). Gene amplification > 5-fold increase) was present in exon 11, in 17 of 41 samples (42.5% of cases), and in exon 25 in 6 of 41 samples (15% of cases). Three tumours (two grade II oligodendrogliglioma, one mixed oligoastrocytoma) displayed high level amplifications: > 100 gene copies were identified by both real-time quantitative PCR and MLPA analyses. Gene sequence alterations were identified by PCR/SSCP and sequencing in four cases: two missense mutations: G1051A (Ala351Thr) and G2216A (Arg739Hys); one nonsense mutation: C2934T (Asp978Asp); and an 18 bp deletion in position 2423-2441 of E19. These changes were present only in tumoral DNA, not in the corresponding constitutional patients' DNA. We also found four previously unidentified polymorphic variants: G2025A (Ala675Ala), C2233T (Leu745Leu), C2895T (Treo965Treo) and C3168T (Asp1056Asp), and three previously described polymorphic changes: E12+22 Tright curved arrow A, G1748A (Arg583Lys) and A2547T (Glu849Glu). Our findings demonstrate that mutations and amplification/overdose in the EGFR gene are present in low-grade oligodendroglial tumours, and may contribute to the development of these brain neoplasms.

Amino Acid Substitution↗
PubMed2001

HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset.

BACKGROUND: T cells from HIV+ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8+ CD28- T cells. In order to compare cytokine production from T cells from these two states, CD4+ and CD8+ T cells from HIV+ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8+ T cell subsets CD28+ and CD28- from the HIV+ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls. RESULTS: Flow cytometric analysis indicated that CD8+ T cells from both HIV+ and aged donors showed an increase of approximately 2-3 fold over controls in percentage of cells producing inflammatory cytokines IFN-gamma and TNF-alpha. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1-2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4-5 fold) in IFN-gamma and TNF-alpha mRNA from HIV+ and aged CD8+ T cells, as did ELISA for secreted IFN-gamma and TNF-alpha (2.3-4 fold). Flow cytometric analysis showed that the CD8+ CD28- T cell subset accounts for approximately 80-86% of the IFN-gamma and TNF-alpha production from the CD8+ subset in the aged and HIV+ states. The CD4+ T cell, while not significantly changed in the HIV+ or aged states in terms of IFN-gamma production, showed a small but significant increase in TNF-alpha production in both states. CONCLUSIONS: Our data appear compatible with physiologic conditions existing in HIV+ and aged individuals, i.e. elevated serum levels and elevated CD8+ T cell production of IFN-gamma and TNF-alpha. Thus, the capacity for increased production of cytokines IFN-gamma and TNF-alpha in the aged individual by the dominant CD8+ CD28- subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8+ CD28- T cell subsets in both the HIV+ and the aged states may reflect a natural "endpoint" in CD8+ T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual.

Adult↗
PubMed2002

Protamine-1 and -2 mRNA in round spermatids is associated with RNA-binding proteins.

RNA-binding proteins in round spermatids have previously been assigned to the coding sequence of Prm1- and Prm2-mRNA. To further characterize this protein-RNA interaction, prior to cDNA synthesis, microdissected cell profiles were digested with different proteases exhibiting a specific cleavage site followed by both conventional and real-time quantitative PCR. Best results were obtained with proteinase K and A followed by factor Xa protease, genenase I, and proteases V8. While enterokinase revealed PCR signals solely for Prm2, no amplification signal was obtained using chymotrypsin. These data suggest a protein segment rich in basic amino acids to be important for the binding to Prm1- and Prm2-mRNA. The fact that phenanthroline treatment instead of protease digestion also resulted in amplification signals suggests the involvement of zinc-finger-like protein-RNA interactions. Employing different primer pairs, RNA-binding proteins were shown to be localized at the 5' end of Prm1- and Prm2-mRNA. Since protein-RNA interactions are a common principle of posttranscriptional regulation of gene expression, the combination of microdissection, protease digestion, and real-time quantitative PCR provides a suitable tool for its investigation in a cell type-specific manner. Furthermore, the presence of RNA-binding proteins within the coding sequence of mRNAs demands proteinase K treatment prior to cDNA synthesis, a compelling necessity for the study of gene expression.

Cell Separation↗
PubMed2005

Phase I-II trial of ONYX-015 in combination with MAP chemotherapy in patients with advanced sarcomas.

ONYX-015 is a provisionally replication competent adenovirus with oncolytic activity in cells with malfunctioning p53. Sarcomas represent a rational target for this approach given the high frequency of p53 mutations (40-75%) and MDM-2 amplification (10-30%). We, therefore, undertook a phase I/II study of ONYX-015, days 1-5 every month administered intratumorally under radiographic guidance, in combination with MAP (mitomycin-C, doxorubicin, cisplatin) chemotherapy in patients with advanced sarcoma. Six patients were treated. Injected lesions included liver metastases in four patients and chest wall metastases in two patients. Sarcoma histologies were gastrointestinal stromal tumors (GIST, two patients), leiomyosarcoma (two patients), liposarcoma (one patient), and malignant peripheral nerve sheath tumor (1 patient). Dose escalation was performed from 10(9) plaque forming units (PFU)/dose (total dose of 5 x 10(9) PFU/cycle) to 10(10) PFU/dose (total dose of 5 x 10(10) PFU/cycle) without dose-limiting toxicity being encountered. Immunohistochemistry of the metastatic lesions prior to treatment showed that five out of six patients were positive for p53, while two patients also had mdm-2 overexpression. Adenoviral replication was detected in two out of six patient biopsies on day 5 of the first cycle, by in situ hybridization (ISH). Both patients were treated at the highest dose level. ONYX-015 viral DNA was detected by quantitative PCR in the plasma of 5/6 patients on day 5 of the first cycle, and up to day 12 (7 days after the last viral dose) in one patient who had extended sampling for viral kinetics performed, suggesting viral replication in sarcoma tissue. One patient with p53 mutation and MDM-2 amplification achieved a partial response to treatment that lasted 11 months. In conclusion, intratumoral administration of ONYX-015 in combination with MAP chemotherapy is well tolerated with no significant toxicity due to ONYX-015 being encountered. Detection of viral DNA in post treatment tumor specimens by ISH and detection of the ONYX-015 genome in the peripheral blood by quantitative PCR, up to 7 days after the last viral dose provide evidence for adenoviral replication. There was evidence of antitumor activity in one out of six patients. Further investigation of this approach in patients with recurrent sarcomas is warranted.

Adenoviridae↗