Population structure and admixture in Cerro Largo, Uruguay, based on blood markers and mitochondrial DNA polymorphisms.

Recent studies of the Uruguayan population revealed different amounts of Amerindian and African genetic contributions. Our previous analysis of Afro-Uruguayans from the capital city of the Department of Cerro Largo showed a high proportion of African genes, and the effects of directional mating involving Amerindian women. In this paper, we extended the analysis to a sample of more than 100 individuals representing a random sample of the population of the whole Department. Based on 18 autosomal markers and one X-linked marker, we estimated 82% European, 8% Amerindian, and 10% African contributions to their ancestry, while from seven mitochondrial DNA site-specific polymorphic markers and sequences of hypervariable segment I, we determined 49% European, 30% Amerindian, and 21% African maternal contributions. Directional matings between Amerindian women and European men were detected, but differences involving Africans were not significant. Data about the specific origins of maternal lineages were also provided, and placed in a historical context.     

HIV-induced IL-6/IL-10 dysregulation of CD4 cells is associated with defective B cell help and autoantibody formation against CD4 cells

To analyse CD4 cell cytokine secretion and helper/suppressor function at a clonal level we established 446 CD4⁺ T cell clones (TCC) in four healthy controls, three HIV^? haemophilia patients, four CDC II,III and four CDC IV patients. Spontaneous TCC secretion of Th1 cytokines (IL-2, interferon-gamma (IFN-γ)) and Th2 cytokines (IL-4, IL-6, IL-10) was determined by ELISA. TCC helper and suppressor functions were tested in a pokeweed mitogen (PWM)-stimulated allogeneic co-culture system using a reverse haemolytic plaque assay for assessment of B cell responses. There was a significant association of TCC surface marker expression (Leu-8, CD45RA) with TCC IL-6 secretion in healthy controls (P < 0.01), HIV^? patients (P 0.001) and CDC II,III patients (P 0.01) but not in CDC IV patients. Likewise, TCC expression of Leu-8 and CD45RA was significantly associated with TCC suppressor function in healthy controls (P 0.0005) but not in HIV-infected patients. A reduced TCC helper frequency (10% of TCC) and an enhanced TCC suppressor frequency (> 80% of TCC) were detected only in those HIV-infected patients who showed an excessively increased TCC IL-6 secretion (> 70% of TCC) together with a significantly diminished TCC IL-10 secretion (10% of TCC). CD4 cell autoantibodies also were found only in patients with this type of cytokine dysregulation. These data indicate that CD4 cell surface markers lose their functional relevance in HIV-infected patients. HIV-induced IL-6/IL-10 dysregulation of CD4⁺ T cells, i.e. the up-regulation of spontaneous IL-6 and down-regulation of spontaneous IL-10 secretion, appears to be involved in inducing CD4 helper defects and may promote autoantibody formation against CD4 cells.     

A current appreciation of sites for pharmacological intervention in allergic conjunctivitis: effects of new topical ocular drugs.

Two important realizations about pathophysiological mechanisms involved in allergic conjunctivitis have led to novel drug discovery efforts and new topical ocular medications for prevention and treatment of this prevent allergic disease. The first of these, interspecies and intraspecies mast cell heterogeneity, was established in the mid-1980's by investigators working in the field of asthma. It is now appreciated that secretory responses as well as effects of pharmacological agents differ depending upon the mast cell population studied. Two types of human mast cells, the tryptase containing (T) and the tryptase/chymase containing (TC) mast cells, have been characterized in a variety of tissues. Significantly, Irani et al. (1) demonstrated by immunohistochemical staining that the mast cells present in conjunctival tissues from patients with allergic conjunctivitis were 100% TC. Functional responses of human conjunctival mast cells to a variety of secretagogues (2) were consistent with their classification as TC or connective tissue type mast cells. Importantly, the studies by Miller et al. mentioned above allowed the harvesting and preparation of human conjunctival mast cells for use in drug screening studies. Utilization of these cells has led to the identification of Patanol, the most effective human conjunctival mast cell stabilizer available for topical use in allergic conjunctivitis (3). These same studies demonstrated the lack of mast cell stabilizing activity for cromolyn and nedocromil in these connective tissue type, TC containing, human conjunctival mast cells. Similar lack of effect was noted with these drugs on human skin mast cell degranulation (4). The second important discovery in the area of allergic conjunctivitis has been the demonstration that conjunctival epithelial cells may contribute to the perpetuation of the allergic response. A report from Gamache et al. (5) identified cytokines produced by human conjunctival epithelial cells following treatment with a number of stimuli. Significantly, Sharif et al. (6) subsequently identified functional histamine H1 receptors on these same cell types. Recently, Weimer et al. (7) have shown that exposure of human conjunctival epithelial cells to histamine leads to the production of pro-inflammatory cytokines IL-6 and IL-8. Importantly, treatment of the epithelial cells with drugs that possess histamine H1 antagonist properties prevents cytokine production. It is noteworthy that first generation anti-histamines antazoline and pheniramine are not potent inhibitors of histamine-stimulated cytokine synthesis in intact epithelial cells, while newer anti-histamines Emadine and levocabastine are more potent. Surprisingly, Patanol was more potent as an inhibitor of histamine-stimulated cytokine production by the epithelial cells than would be predicted from its histamine H1 antagonist affinity. These inhibitory effects on conjunctival epithelial cell production of pro-inflammatory cytokines may contribute to enhanced clinical activity noted with these recently approved drugs.     

Sequential treatment of patients with advanced renal cell carcinoma with autologous tumor vaccine and subcutaneous administration of recombinant interleukin-2 and interferon α2b

Summary Thirty patients with advanced renal cell carcinoma (RCC), 23 of whom had distant metastases in at least one organ, were entered after nephrectomy into a protocol involving vaccination with Newcastle disease virus (NDV)-modified autologous tumour material, with a subsequent induction week and repetitive bi-weekly cycles of interleukin-2 (rIL-2) and interferon _2b/rIFN-_2b at a lower s. c. dose (1.5 million Cetus units m^–2 day^–1 every 12 h on 2 days and 3 million IU/m² once a day on days 1, 3 and 5). The inpatient treatment was followed by a maintenance phase during which 0.3 million Cetus units/m² rIL-2 was given s. c. every 12 h on days 1–5 and 3 million IU m^–2 day^–1 rIFN-_2b was administered on days 1, 3, and 5 on an outpatient basis. All but 3 patients completed the induction week and 6 weeks of outpatient therapy. No grade 3 or 4 toxicities occurred during the therapy. Therapy was discontinued for 3 patients because of rapid tumour progression. Of the 23 evaluable RCC patients, 3 exhibited a complete response and 4 displayed partial remission, 7 showed stable disease during 1–18 months (median = 5 months) of therapy, and progression was seen in 9. We conclude that vaccination with autologous tumour material combined with s. c. rIL-2 and rIFN-_2b administration can induce regressions in patients with advanced RCC and that even in non-responding patients a more favourable course of the disease can be achieved.     

Breakpoint characterization of the der(19)t(11;19)(q13;p13) in the ovarian cancer cell line SKOV‐3

About 20% of ovarian carcinomas show alterations of 19p13 and/or 19q13 in the form of added extra material whose origin often is from chromosome 11. Based on earlier spectral karyotype analysis of the ovarian cancer cell line SKOV‐3, which shows an unbalanced translocation der(19)t(11;19), the aim of this study was to determine the precise breakpoints of that derivative chromosome. After rough delimitation of the breakpoints of microdissected derivative chromosomes by array analysis, we designed a matrix of primers spanning 11q13.2 and 19p13.2 detecting multiple amplicons on genomic and cDNA. Sequencing the amplicons, accurate localization of both breakpoints on both chromosomes was possible and we found that exon 14 of HOOK2 from chromosome 19 and exon 2 of ACTN3 from chromosome 11 were fused in the derivative chromosome. The breakpoint in the HOOK2 gene was in an intrinsic triplet of nucleic acids leading to a shift in the ACTN3 reading frame in the derivative chromosome. This frameshift alteration should give rise to an early stop codon causing a loss of function of ACTN3. Signals in two‐dimensional Western blotting exactly match to calculated molecular mass and the isoelectric point of the fusion protein. © 2013 Wiley Periodicals, Inc.     

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain,a novel recombinant single-chain factor VIII

Introduction

rVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII.

Materials and Methods

Binding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate^®) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl₃-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200–250 IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice.

Results

rVIII-SingleChain displayed a high affinity for von Willebrand factor (K_D = 44 pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl₃-induced arterial occlusion model.

Conclusions

rVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.     

Placebo responses and placebo effects in functional bowel disorders

The nature and determinants of the placebo response are widely unknown, as are the underlying psychological and biological mechanisms. Placebo response rates in functional bowel disorders (functional dyspepsia, irritable bowel syndrome) trials are similar to those in nonintestinal pain conditions and are comparable with other organic gastrointestinal diseases (duodenal ulcer, inflammatory bowel diseases). In this narrative review, different methodologies (meta-analyses, reanalyses, and experimental setups) are discussed that have been applied to the study of the placebo response in functional dyspepsia and the irritable bowel syndrome.