Report on the Iowa Department of Human Services for the year ended June 30, 2013

Report on the Iowa Department of Human Services for the year ended June 30, 2013

Comparative assessement of intimal hyperplasia development after 14 days in two different experimental settings : tissue culture versus ex vivo continuous perfusion of human saphenous vein

Résumé de l'article : L'hyperplasie intimale est un processus de remodelage vasculaire ubiquitaire après une lésion, pouvant menacer la perméabilité de tout type de reconstruction vasculaire. Les mécanismes physiopathologiques impliqués dans le développement de l'hyperplasie intimale ne sont que partiellement élucidés. Il est par conséquent nécessaire d'effectuer des recherches complémentaires afin d'en améliorer la compréhension et ainsi permettre l'élaboration de nouvelles stratégies thérapeutiques médicamenteuses. La culture de veines en milieu statique permet le développement de l'hyperplasie intimale. Ce modèle maintient la viabilité tissulaire, comme décrit précédemment dans d'autres études, mais empêche l'analyse des paramètres hémodynamiques. La mise au point d'un modèle de perfusion in vitro permettant la perfusion de segments vasculaires représente une approche expérimentale intégrant les différents facteurs hémodynamiques. Le système de perfusion (Ex Vivo Vein Support System) que nous avons élaboré conserve l'intégrité pariétale ainsi que les propriétés vasomotrices des veines pour une durée de 14 jours. Cette étude démontre que les deux modèles permettent le développement de l'hyperplasie intimale. Toutefois, les propriétés vasomotrices ainsi que l'influence des paramètres hémodynamiques ne peuvent être analysées que par l'utilisation du système de perfusion. Ce dernier a permis de perfuser des vaisseaux humains sans contamination bactérienne tout en maintenant l'intégrité cellulaire. Ce modèle de perfusion se rapproche plus des conditions hémodynamiques rencontrées in vivo que le modèle statique. Abstract : Background. Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise con-trolled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. Materials and methods. Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14 days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, α-actin, and PAI-1 were determined before and after 14 days of either experimental conditions. Results, Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell ( SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII and α-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. Conclusion. This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

In vitro combination of human and bovine free secretory component with IgA of various species.

The free form of the secretory component usually associated with secretory IgA can be isolated from human and bovine milk. These free secretory components of different origin combine in vitro with human polymeric myeloma IgA, with mouse myeloma IgA, and with the serum IgA of nine different mammalian species.

Combined report on the institutions under the control of the Iowa Department of Human Services for the five years ended June 30, 2013

Combined report on the institutions under the control of the Iowa Department of Human Services for the five years ended June 30, 2013

Fluorodeoxyuridine improves imaging of human glioblastoma xenografts with radiolabeled iododeoxyuridine.

Use of radiolabeled nucleotides for tumor imaging is hampered by rapid in vivo degradation and low DNA-incorporation rates. We evaluated whether blocking of thymidine (dThd) synthesis by 5-fluoro-2'-deoxyuridine (FdUrd) could improve scintigraphy with radio-dThd analogues, such as 5-iodo-2'-deoxyuridine (IdUrd). We first show in vitro that coincubation with FdUrd substantially increased incorporation of [125I]IdUrd and [3H]dThd in the three tested human glioblastoma lines. Flow cytometry analysis showed that a short coincubation with FdUrd (1 h) produces a signal increase per labeled cell. We then measured biodistribution 24 h after i.v. injection of [125I]IdUrd in nude mice s.c. xenografted with the three glioblastoma lines. Compared with animals given [125I]IdUrd alone, i.v. preadministration for 1 h of 10 mg/kg FdUrd increased the uptake of [125I]IdUrd in the three tumors 4.8-6.8-fold. Compatible with previous reports, there were no side effects in mice observed for 2 months after receiving such a treatment. The tumor uptake of [125I]IdUrd was increased < or =13.6-fold when FdUrd preadministration was stepwise reduced to 1.1 mg/kg. Uptake increases remained lower (between 1.7- and 5.8-fold) in normal proliferating tissues (i.e., bone marrow, spleen, and intestine) and negligible in quiescent tissues. DNA extraction showed that 72-80% of radioactivity in tumor and intestine was bound to DNA. Scintigraphy of xenografted mice was performed at different times after i.v. injection of 3.7 MBq [125I]IdUrd. Tumor detection was significantly improved after FdUrd preadministration while still equivocal after 24 h in mice given [125I]IdUrd alone. Furthermore, background activity could be greatly reduced by p.o. administration of KClO4 in addition to potassium iodide. We conclude that FdUrd preadministration may improve positron or single photon emission tomography with cell division tracers, such as radio-IdUrd and possibly other dThd analogues.

Conformational changes modulate the activity of human RAD51 protein.

Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homologue, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Antibody-fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice.

To improve the detectability of tumors by light-induced fluorescence, the use of monoclonal antibodies (MoAb) as carriers of fluorescent molecules was studied. As a model for this approach, the biodistribution of an anticarcinoembryonic antigen (CEA) MoAb coupled to fluorescein was studied in mice bearing a human colon carcinoma xenograft. In vitro, such conjugates with fluorescein-MoAb molar ratios ranging from four to 19, doubly labeled with 125I, showed more than 82% binding to immobilized CEA. In vivo, conjugates with a fluorescein-MoAb molar ratio of ten or less resulted in a tumor uptake of more than 30% of the injected dose of radioactivity per gram tumor at 24 hours. Tumor to liver, kidney, and muscle ratios of 20, 30 and 72, respectively, were obtained 48 hours after injection of the 125I-MoAb-(fluorescein)10 conjugate. The highest fluorescence intensity was always obtained for the tumor with the anti-CEA MoAb conjugate; whereas in control mice injected with fluoresceinated control immunoglobulin G1, no detectable increase in tumor fluorescence was observed. To compare these results with a classically used dye, mice bearing the same xenografts received 60 micrograms of Photofrin II. The intensity of the fluorescence signal of the tumor with this amount of Photofrin II was eight times lower than that obtained after an injection of 442 ng of fluorescein coupled with 20 micrograms of MoAb, which gave an absolute amount of fluorescein localized in the tumor of up to 125 ng/g of tumor. These results illustrate the possibility of improving the specificity of in vivo tumor localization of dyes for laser-induced fluorescence photodetection and phototherapy by coupling them to MoAb directed against tumor markers.

A microRNA profile of human CD8.

BackgroundRecently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8+CD25+ Treg cells and the impact of microRNAs on molecules associated with immune regulation.MethodsWe purified human natural CD8+ Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA `signature¿ for CD8+CD25+FOXP3+CTLA-4+ natural Treg cells. We used the `TargetScan¿ and `miRBase¿ bioinformatics programs to identify potential target sites for these microRNAs in the 3¿-UTR of important Treg cell-associated genes.ResultsThe human CD8+CD25+ natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.ConclusionsWe are examining the biological relevance of this `signature¿ by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

Sensitive gene expression profiling of human T cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages.

The differentiation of CD4(+) or CD8(+) T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4(+) or CD8(+) T cell differentiation. Surprisingly, our data revealed that while CD4(+) and CD8(+) T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4(+) and CD8(+) T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.

Examination of the Iowa Medical Assistance Disproportionate Share Hospital Payments Program and the accompanying schedule identified as Schedule 1 for the Iowa Department of Human Services for the year ended June 30, 2011

Examination of the Iowa Medical Assistance Disproportionate Share Hospital Payments Program and the accompanying schedule identified as Schedule 1 for the Iowa Department of Human Services for the year ended June 30, 2011

Interleukin-1 receptor antagonist gene polymorphism and circulating levels of human immunodeficiency virus type 1 RNA in Brazilian women.

Interleukin-1 receptor antagonist (IL-1ra) gene polymorphisms in 83 human immunodeficiency virus (HIV)-seropositive women were evaluated. Fourteen of the subjects (16.9%) were homozygous for IL-1ra allele 2 (IL-1RN*2). These women had a lower median level of HIV RNA than did women homozygous for allele 1 (IL-1RN*1) (P = 0.01) or heterozygous for both alleles (P = 0.04). Among 46 subjects not receiving antiretroviral treatment, HIV levels were also reduced in IL-1RN*2 homozygous individuals (P < 0.05). There was no relation between IL-1ra alleles and CD4 levels.

Determinants of vaccine immunity in the cohort of human immunodeficiency virus-infected children living in Switzerland.

BACKGROUND: Human immunodeficiency virus (HIV)-infected children are at increased risk of infections caused by vaccine preventable pathogens, and specific immunization recommendations have been issued. METHODS: A prospective national multicenter study assessed how these recommendations are followed in Switzerland and how immunization history correlates with vaccine immunity. RESULTS: Among 87 HIV-infected children (mean age: 11.1 years) followed in the 5 Swiss university hospitals and 1 regional hospital, most (76%) had CD4 T cells >25%, were receiving highly active antiretroviral treatment (79%) and had undetectable viral load (60%). Immunization coverage was lower than in the general population and many lacked serum antibodies to vaccine-preventable pathogens, including measles (54%), varicella (39%), and hepatitis B (65%). The presence of vaccine antibodies correlated most significantly with having an up-to-date immunization history (P<0.05). An up-to-date immunization history was not related to age, immunologic stage, or viremia but to the referral medical center. CONCLUSIONS: All pediatricians in charge of HIV-infected children are urged to identify missing immunizations in this high-risk population.

Influence of multiple injections of human chorionic gonadotropin (hCG) on urine and serum endogenous steroids concentrations.

Since it is established that human chorionic gonadotropin (hCG) affects testosterone production and release in the human body, the use of this hormone as a performance enhancing drug has been prohibited by the World Anti-Doping Agency. Nowadays, the only validated biomarker of a hCG doping is its direct quantification in urine. However, this specific parameter is subjected to large inter-individual variability and its determination is directly dependent on the reliability of hCG immunoassays used. In order to counteract these weaknesses, new biomarkers need to be evidenced. To address this issue, a pilot clinical study was performed on 10 volunteers submitted to 3 subsequent hCG injections. Blood and urine samples were collected during two weeks in order to follow the physiological effects on related compounds such as the steroid profile or hormones involved in the hypothalamo-pituitary axis. The hCG pharmacokinetic observed in all subjects was, as expected, prone to important inter-individual variations. Using ROC plots, level of testosterone and testosterone on luteinizing hormone ratio in both blood and urine were found to be the most relevant biomarker of a hCG abuse, regardless of inter-individual variations. In conclusion, this study showed the crucial importance of reliable quantification methods to assess low differences in hormonal patterns. In regard to these results and to anti-doping requirements and constraints, blood together with urine matrix should be included in the anti-doping testing program. Together with a longitudinal follow-up approach it could constitute a new strategy to detect a hCG abuse, applicable to further forms of steroid or other forbidden drug manipulation.