Human Osteoarthritic Cartilage Shows Reduced In Vivo Expression of IL-4, a Chondroprotective Cytokine that Differentially Modulates IL-1β-Stimulated Production of Chemokines and Matrix-Degrading Enzymes In Vitro

Background

In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β.

Methodology/Principal Findings

The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13.

Conclusions/Significance

Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.     

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.     

High Rates of HIV Seroconversion in Pregnant Women and Low Reported Levels of HIV Testing among Male Partners in Southern Mozambique: Results from a Mixed Methods Study

Introduction

Prevention of acute HIV infections in pregnancy is required to achieve elimination of pediatric HIV. Identification and support for HIV negative pregnant women and their partners, particularly serodiscordant couples, are critical. A mixed method study done in Southern Mozambique estimated HIV incidence during pregnancy, associated risk factors and factors influencing partner''s HIV testing.

Methods

Between April 2008 and November 2011, a prospective cohort of 1230 HIV negative pregnant women was followed during pregnancy. A structured questionnaire, HIV testing, and collection of dried blood spots were done at 2–3 scheduled visits. HIV incidence rates were calculated by repeat HIV testing and risk factors assessed by Poisson regression. A qualitative study including 37 individual interviews with men, women, and nurses and 11 focus group discussions (n = 94) with men, women and grandmothers explored motivators and barriers to uptake of male HIV testing.

Results

HIV incidence rate was estimated at 4.28/100 women-years (95%CI: 2.33–7.16). Significant risk factors for HIV acquisition were early sexual debut (RR 3.79, 95%CI: 1.04–13.78, p = 0.04) and living in Maputo Province (RR 4.35, 95%CI: 0.97–19.45, p = 0.05). Nineteen percent of women reported that their partner had tested for HIV (93% knew the result with 8/213 indicating an HIV positive partner), 56% said their partner had not tested and 19% did not know their partner test status. Of the 14 seroconversions, only one reported being in a serodiscordant relationship. Fear of discrimination or stigma was reported as a key barrier to male HIV testing, while knowing the importance of getting tested and receiving care was the main motivator.

Conclusions

HIV incidence during pregnancy is high in Southern Mozambique, but knowledge of partners'' HIV status remains low. Knowledge of both partners'' HIV status is critical for maximal effectiveness of prevention and treatment services to reach elimination of pediatric HIV/AIDS.     

Agrobacterium-mediated transformation of African violet

A method for genetic transformation of Saintpaulia ionantha by co-cultivation of in vitro-grown leaves and petioles with Agrobacterium tumefaciens is described. Two bacterial strains, EHA105 and A281 both harbouring the binary plasmid pKIWI105 carrying the genes uidA and nptII, were used in the experiments. Regenerants were not obtained using the disarmed strain EHA105. The oncogenic strain A281 resulted in efficient transient and stable expression of the transferred traits for petiole explants only. After transformation and regeneration, the integration of the transgenes in the plant genome was confirmed by PCR analysis and Southern hybridization.     

Phylogeography of Agathistoma (Turbinidae,Tegulinae) snails in tropical and southwestern Atlantic

The rocky intertidal gastropods Agathistoma viridulum and A. hotessierianum occur from the Caribbean to southern Brazil, with a gap in the equatorial region, giving them an anti-tropical distribution. We used sequences from mitochondrial genes to elucidate the phylogeography of A. viridulum and A. hotessierianum and to infer their relationships to other species of Agathistoma. For A. hotessierianum, haplotype networks and phylogenetic analyses split samples into two distinct groups: one (A. hotessierianum) in the Caribbean region (Greater and Lesser Antilles; Venezuela: Sucre and Isla Margarita) and a new species that we describe from northeastern Brazil. For A. viridulum, genetic analyses split the samples into three groups (Caribbean, northeastern Brazil and southeastern Brazil), but genetic divergence among these was too low for them to be considered species, and morphological differences were not significant. The mtDNA tree identified two clades of eastern Pacific Agathistoma, but many lower-level relationships within Agathistoma were not well resolved, suggesting that more complete taxon sampling and additional genetic data will be needed to establish more robust relationships among Tegulinae.