Macrophages and Dendritic Cells in Antigen-Induced Arthritis

Two non-lymphoid cell types play an important role in the pathogenesis of arthritis, i.e. the 'classical' macrophage and the antigen-presenting dendritic cell. In the present study, the roles of both cell types are studied in antigen-induced arthritis of the rat knee joint. Cryostat sections of whole, unfixed, undecalcified knee joints were used for immunohistochemical staining of non-lymphoid cells and lymphocyte subsets. For the demonstration of the different types of non-lymphoid cells, monoclonal antibodies against rat macrophages (ED1, ED2, and ED3) and against Ia antigen were used with an immunoperoxidase method. The results show in an overall view of the arthritic joint the different sites of action of the classical macrophages on the one hand and the Ia-positive dendritic cells on the other. Classical macrophages were mainly found in the superficial layers of the synovium bordering joint space and articular cartilage. Dendritic cells and T cells of the helper phenotype were mainly found in clusters surrounding small blood vessels within the synovium. These clusters express the immunological background of the antigen-induced arthritis and may well be responsible for the continuation of the arthritic process.     

Effect of disruption versus continuation of gonadotrophin-releasing agonist after human chorionic gonadotrophin administration on corpus luteum function in patients undergoing ovulation induction for in-vitro fertilization

Previous studies have described the luteolytic effect of gonadotrophin- releasing hormone agonist (GnRHa) administered in the early luteal phase. The present work was undertaken to compare in a prospective and randomized design the effect of disruption versus continuation of daily GnRHa after human chorionic gonadotrophin (HCG) administration on corpus luteum function in patients undergoing ovulation induction for in-vitro fertilization (IVF). Two different studies were designed and a total of 38 ovum donors, aged 23-30 years, were included. In the first study, the effect of GnRHa on the early luteal phase of IVF-stimulated cycles was investigated (n = 27); the patients were divided into two groups, according to whether they stopped (n = 13) or continued with daily GnRHa injections (n = 14) for an additional period of 15 days after HCG administration. Blood was drawn from luteal phase days 2 to 6 (day 0 = day of HCG administration) and oestradiol and progesterone concentrations were analysed. The second study focused on the effects of continuation versus disruption of GnRHa administration in the mid- late luteal phase. A similar design was employed including six patients who stopped GnRH on day 0 and five other women who continued GnRHa for 15 days after HCG administration. In this second study, blood was drawn from days 5 to 11 and oestradiol, progesterone and luteinizing hormone (LH) concentrations were analysed. IVF parameters were similar in both groups. The results indicate that continuous GnRHa administration, after HCG injection, does not produce changes in oestradiol, progesterone and LH concentrations in the early, mid- and late luteal phases compared to those patients in whom GnRHa is discontinued at the day of HCG administration. The present work demonstrates that, when ovulation induction is performed, the corpus luteum is driven primarily by the HCG, regardless of the administration or disruption of GnRHa in the luteal phase. This suggests that the lack of differences between continuation versus disruption of GnRHa may be due to the accumulation of the product over the previous 2-3 weeks of treatment.      

Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.     

Regulation of hematopoiesis II: the role of polyamine inhibition on helper or suppressor influences of the thymus

We have previously suggested in murine model systems, that two cell subpopulations with differing proliferative capacity, from the thymus, modify the growth of erythroid progenitor cells in vitro. In order to further characterize these populations, we have specifically inhibited polyamine biosynthesis; this pathway is essential for the process of cell replication. Thus, alpha-difluoromethyl ornithine (DFMO) was used to block the conversion of ornithine to putrescine, the first and rate- limiting step in polyamine biosynthesis. We observed a threefold increase in hematopoietic progenitors (CFU-S and CFU-E) from bone marrow in animals treated with DFMO. We further examined the effect of DFMO on accessory "helper" and "suppressor" cells from the thymus and observed an increase in helper activity with an elimination of suppressor activity. All of these effects of DFMO were specific for inhibition of polyamine biosynthesis, since simultaneous addition of the depleted biosynthetic product, putrescine, restored suppressor activity. We conclude that polyamine biosynthesis is required acutely for accessory cell regulation of hematopoiesis.     

Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11- dUTP

Blood donations are routinely screened by multiple serologic assays for antigens/antibodies associated with infection by blood-borne viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1 and HIV-2), and human T-cell lymphotropic virus (HTLV-I and HTLV-II). A direct detection of these viruses would be more effective for the prevention of transfusion- transmitted infections than the indirect measurement of the variable host immune response to these agents. Because the polymerase chain reaction (PCR) for viral gene amplification offers the most sensitive and direct means of detecting viruses in blood, we have developed a nonisotopic PCR procedure for the detection of HBV, chosen as a prototype. The problems, common to previously described PCR methods, of nucleic acid extraction and inhibition of the PCR by plasma proteins were overcome by isolation of HBV from plasma by means of 450-microns polystyrene beads covalently coated with monoclonal antibody to the Pre- S1 region of the viral envelope protein. Detergent lysis and proteinase K digestion of the immunocaptured virions isolated from plasma released the HBV DNA. A modified PCR-amplification protocol, incorporating digoxigenin-labeled dUTP in the amplified gene products followed by hybridization with a specific biotinylated oligonucleotide probe bound to streptavidin-coated 2.8-microns magnetic beads, allowed flow cytometric analyses of HBV-specific PCR products by means of antibodies to digoxigenin labeled with fluorescein isothiocyanate. The endpoint serial dilutions of pedigreed human plasma samples containing chimpanzee infectious dose (CID50) of 10(7) for adw and CID50 of 10(7.5) for the ayw subtypes were compared in repeated testing of PCR products by our immunoreactive bead (PCR-IRB) assay. HBV DNA was consistently detected in a 5 x 10(-10) dilution of each sample. In testing 20 coded specimens of blood donors, with or without serologic markers of HBV infection, the PCR-IRB was specific and more sensitive than the PCR analyses by slot blot hybridization with radioactive probe. The PCR-IRB assay can be adapted for simultaneous detection of multiple blood-borne viruses by an automated flow cytometric analysis system.     

Indications for vascular grafts as hemodialysis access: consensus from experience in Italy

Introduction: In Italy, the use of arteriovenous grafts (AVG) is limited (1-5 %) due to different approaches to vascular access (VA) management as compared to other Countries, where guidelines (which may not apply to the Italian setting) have been produced. Therefore, the Study Group (GdS) on VA of the Italian Society of Nephrology produced this position paper, providing a list of 8 recommendations built upon current guidelines. Methods: The most controversial and innovative issues of existing guidelines have been summed up in 12 different statements. We selected 60 Italian dialysis graft experts, nephrologists and vascular surgeons (PP1SIN Study Investigators). They were asked to express their agreement/disagreement on each issue, thus creating a new method to share and exchange information. Results: Most of them agreed (consensus > 90%) on specific criteria set to choose AVG over native AVF (nAVF) and tunnelled venous catheter (tVC) and on the necessary conditions to implant them. They did not fully agree on the use of AVG in obese patients, in patients at risk of developing ischemia, on the priority of AVG as an alternative to brachial-basilic fistula with vein transposition, and in case of a poorly organized setting regarding graft maintenance. Keeping in mind that the nAVF should be preferred whenever is feasible, AVGs are indicated when superficial veins are unavailable or to repair a nAVF (bridge graft). An AVG is an alternative to tVC if the expected patient survival is long enough to guarantee its clinical benefits.     

Physiological roles of dopamine and neuropeptides in the retina

Summary— The retina is a highly complex nervous tissue that converts light into patterns of electrical action potentials in order to process visual information. To carry out its function as a transducer and processor of visual information, the retina must be structurally and biochemically organized to send a coherent signal to the visual areas of the brain. In recent years, a number of biologically active substances have been demonstrated to be located within neurons in the retina. Most of them are thought to be involved in the modulation of the signal and its transmission to the brain through the optic nerve. The present paper attempts to summarize the immunocytochemical distribution and physiology of some neuronally localized substances in the mammalian retina, namely dopamine and neuropeptides.     

The role of the gut in migraine: the oral 51-Cr EDTA test in recurrent abdominal pain

Sixteen children with recurrent abdominal pain (or: "recurrent syndrome"), regarded as migraine equivalent in childhood, were submitted to the 51-Cr EDTA gut permeability test. The results were compared with those obtained in 10 healthy young adults and in 11 control children. The gut permeability in the recurrent syndrome was significantly higher than in healthy adults and control children (p less than 0.0006): The following results were obtained: 4.83 +/- 0.40 (mean +/- SEM) in the children with recurrent abdominal pain, and 2.35 +/- 0.24 2.51 +/- 0.21 in the healthy young adults and control children, respectively. The implications of these findings as far as migraine is concerned, are discussed.     

A glucose-stimulated BOLD fMRI study of hypothalamic dysfunction in mice fed a high-fat and high-sucrose diet

The hypothalamus is the central regulator of energy homeostasis. Hypothalamic neuronal circuits are disrupted upon overfeeding, and play a role in the development of metabolic disorders. While mouse models have been extensively employed for understanding the mechanisms of hypothalamic dysfunction, functional magnetic resonance imaging (fMRI) on hypothalamic nuclei has been challenging. We implemented a robust glucose-induced fMRI paradigm that allows to repeatedly investigate hypothalamic responses to glucose. This approach was used to test the hypothesis that hypothalamic nuclei functioning is impaired in mice exposed to a high-fat and high-sucrose diet (HFHSD) for seven days. The blood oxygen level-dependent (BOLD) fMRI signal was measured from brains of mice under light isoflurane anaesthesia, during which a 2.6 g/kg glucose load was administered. The mouse hypothalamus responded to glucose but not saline administration with a biphasic BOLD fMRI signal reduction. Relative to controls, HFHSD-fed mice showed attenuated or blunted responses in arcuate nucleus, lateral hypothalamus, ventromedial nucleus and dorsomedial nucleus, but not in paraventricular nucleus. In sum, we have developed an fMRI paradigm that is able to determine dysfunction of glucose-sensing neuronal circuits within the mouse hypothalamus in a non-invasive manner.