Cerebrospinal fluid is an efficient route for establishing brain infection with feline immunodeficiency virus and transfering infectious virus to the periphery

Like human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV) invades and infects the central nervous system (CNS) soon after peripheral infection. The appearance of viral RNA is particularly prominent in the cerebrospinal fluid (CSF), suggesting an efficient route of virus transfer across the blood-CSF barrier. This raises the concern whether this route can establish a stable viral reservoir and also be a source of virus capable of reseeding peripheral systems. To examine this possibility, 200 μl of cell-free NCSU1 FIV or FIV-infected choroid plexus macrophages (ChP-Mac) was directly injected into the right lateral ventricle of the brain. Negative controls were sham inoculated with uninfected ChP-Mac or virus-free culture supernatant and positive controls were infected systemically by intraperitoneal (i.p.) injection. Intracerebroventricular (i.c.v.) inoculation with cell-free FIV resulted in high levels of plasma FIV RNA detected as early as 1 to 2 weeks post inoculation in all cats. In each case, the plasma viremia preceded the detection of CSF viral RNA. Compared to i.p. cats, i.c.v. cats had 32-fold higher CSF viral loads, 8-fold higher ratios of CSF to plasma viral load, and a 23-fold greater content of FIV proviral DNA in the brain. No FIV RNA was detected in plasma or CSF from the cats inoculated with FIV-infected ChP-Mac but an acute inflammatory response and a slight suppression of the CD4+:CD8+ ratio were observed. These results indicate that free FIV circulating in the CSF promotes infection of the CNS and provides a highly efficient pathway for the transfer of infectious virus to the periphery

A tale of two lineages: how the strains of the earliest divergent symbiotic Frankia clade spread over the world

It is currently assumed that around 100 million years ago, the common ancestor to the Fabales, Fagales, Rosales and Cucurbitales in Gondwana, developed a root nodule symbiosis with a nitrogen-fixing bacterium. The symbiotic trait evolved first in Frankia cluster-2; thus, strains belonging to this cluster are the best extant representatives of this original symbiont. Most cluster-2 strains could not be cultured to date, except for Frankia coriariae, and therefore many aspects of the symbiosis are still elusive. Based on phylogenetics of cluster-2 metagenome-assembled genomes (MAGs), it has been shown that the genomes of strains originating in Eurasia are highly conserved. These MAGs are more closely related to Frankia cluster-2 in North America than to the single genome available thus far from the southern hemisphere, i.e., from Papua New Guinea. To unravel more biodiversity within Frankia cluster-2 and predict routes of dispersal from Gondwana, we sequenced and analysed the MAGs of Frankia cluster-2 from Coriaria japonica and Coriaria intermedia growing in Japan, Taiwan and the Philippines. Phylogenetic analyses indicate there is a clear split within Frankia cluster-2, separating a continental from an island lineage. Presumably, these lineages already diverged in Gondwana. Based on fossil data on the host plants, we propose that these two lineages dispersed via at least two routes. While the continental lineage reached Eurasia together with their host plants via the Indian subcontinent, the island lineage spread towards Japan with an unknown host plant

Scleroderma and related disorders: 223. Long Term Outcome in a Contemporary Systemic Sclerosis Cohort

Background: We have previously compared outcome in two groups of systemic sclerosis (SSc) patients with disease onset a decade apart and we reported data on 5 year survival and cumulative incidence of organ disease in a contemporary SSc cohort. The present study examines longer term outcome in an additional cohort of SSc followed for 10 years. Methods: We have examined patients with disease onset between years 1995 and 1999 allowing for at least 10 years of follow-up in a group that has characteristics representative for the patients we see in contemporary clinical practice. Results: Of the 398 patients included in the study, 252 (63.3%) had limited cutaneous (lc) SSc and 146 (36.7%) had diffuse cutaneous (dc) SSc. The proportion of male patients was higher among the dcSSc group (17.1% v 9.9%, p = 0.037) while the mean age of onset was significantly higher among lcSSc patients (50 ± 13 v 46 ± 13 years ± SD, p = 0.003). During a 10 year follow-up from disease onset, 45% of the dcSSc and 21% of the lcSSc subjects developed clinically significant pulmonary fibrosis, p < 0.001. Among them approximately half reached the endpoint within the first 3 years (23% of dcSSc and 10% of lcSSc) and over three quarters within the first 5 years (34% and 16% respectively). There was a similar incidence of pulmonary hypertension (PH) in the two subsets with a steady rate of increase over time. At 10 years 13% of dcSSc and 15% of lcSSc subjects had developed PH (p=0.558), with the earliest cases observed within the first 2 years of disease. Comparison between subjects who developed PH in the first and second 5 years from disease onset demonstrated no difference in demographic or clinical characteristics, but 5-year survival from PH onset was better among those who developed this complication later in their disease (49% v 24%), with a strong trend towards statistical significance (p = 0.058). Incidence of SSc renal crisis (SRC) was significantly higher among the dcSSc patients (12% v 4% in lcSSc, p = 0.002). As previously observed, the rate of development of SRC was highest in the first 3 years of disease- 10% in dcSSc and 3% in lcSSc. All incidences of clinically important cardiac disease developed in the first 5 years from disease onset (7% in dcSSc v 1% in lcSSc, p < 0.001) and remained unchanged at 10 years. As expected, 10-year survival among lcSSc subjects was significantly higher (81%) compared to that of dcSSc patients (70%, p = 0.006). Interestingly, although over the first 5 years the death rate was much higher in the dcSSc cohort (16% v 6% in lcSSc), over the following years it became very similar for both subsets (14% and 13% between years 5 and 10, and 18% and 17% between years 10 and 15 for dcSSc and lcSSc respectively). Conclusions: Even though dcSSc patients have higher incidence for most organ complications compared to lcSSc subjects, the worse survival among them is mainly due to higher early mortality rate. Mortality rate after first 5 years of disease becomes comparable in the two disease subsets. Disclosure statement: The authors have declared no conflicts of interes

HMGA1 Reprograms Somatic Cells into Pluripotent Stem Cells by Inducing Stem Cell Transcriptional Networks

PMC3499526BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.JH Libraries Open Access Fun

Regulatory function of a novel population of mouse autoantigen-specific Foxp3 regulatory T cells depends on IFN-gamma, NO, and contact with target cells.

BACKGROUND:Both naturally arising Foxp3(+) and antigen-induced Foxp3(-) regulatory T cells (Treg) play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy. METHODOLOGY/PRINCIPAL FINDINGS:Using a novel class II MHC tetramer, we have isolated a population of CD4(+) Foxp3(-) T cells specific for the autoantigen glutamic acid decarboxylase p286-300 peptide (NR286 T cells) from diabetes-resistant non-obese resistant (NOR) mice. These Foxp3(-) NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-gamma was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg. CONCLUSIONS/SIGNIFICANCE:These studies identified a unique population of autoantigen-specific Foxp3(-) Treg that can exert their regulatory function dependent on not only IFN-gamma and NO but also cell contact with target cells

NR286 T cells inhibit diabetes development.

<p>NOD/scid recipient mice were transferred with either NOD mouse splenocytes (Nspl)(1×10⁷ cells/mouse) alone, NR286 T cells alone (1×10⁷ cells/mouse), or co-transferred with an equal numbers of both NR286 T cells and Nspl. The difference of diabetes onset and development between co-transferred mice and Nspl single transferred mice was significant (p<0.002).NOD mouse splenocytes were isolated from 7–8 wk old female NOD mice. At least 8 mice were studied in each group.</p

NR286 T cells suppress proliferation of BDC2.5 T cells.

<p>(<b>A</b>) Inhibition of CFSE-labeled BDC2.5 T cell proliferation co-cultured with NR286 T cells. CD4⁺ BDC2.5 T cells were isolated from BDC2.5 TCR transgenic mice and labeled with CFSE (2×10⁵/well), then cultured under eight different conditions (indicated as the numbers in parenthesis shown in each histogram). In control cultures, BDC2.5 T cells were cultured alone either (1) without p79 or (2) with p79 (0.1 µg/ml) plus CD4⁺ cell-depleted irradiated APCs from NOD mice. Cells also were co-cultured in the presence of either NR286 T cells (conditions 3–5) or with NR286 T cells plus p286 (conditions 6–8) for 4 days. NR286 T cells were added to cell cultures with a 2-fold serial dilution starting at a 1∶1 ratio (BDC2.5 T cells: NR286 T cells). The numbers shown in each histogram represent the percentage of cells that underwent <2 rounds of cell division. Data are representative from at least n = 3. (<b>B</b>) Cell culture supernatant was harvested from 4-day cell cultures under the same eight different conditions as shown in (<b>A</b>). Levels of IL-10 detected in conditions 3, 4, and 6–8 were significantly reduced compared to condition 2 (p<0.01). NO production in conditions 3–8 was significantly increased compared to conditions 1 and 2.</p

Cytokine production and phenotype analyses of NR286 T cells.

<p>(<b>A</b>) Analysis of cytokine production by NR286 T cells (2×10⁵/well) in response to various concentrations of p286 plus APCs. Cell culture supernatant was harvested after 24 h cell culture for ELISA (lower detection limit, 8 pg/ml for IL-4 and 30 pg/ml for IL-10 and IFN-γ). Data represent average ± SEM, n = 3. (<b>B</b>) Phenotypic analysis of NR286 T cells. NR286 T cells were co-stained with anti-CD4 plus antibodies against indicated markers. Numbers in each quadrant represent the percentage of cells positively detected by antibodies.</p

Blockade of IFN-γ and NO production abolished NR286 T cell regulatory function.

<p>(<b>A</b>) CFSE-labeled CD4⁺ BDC2.5 T cells (2×10⁵/well) were cultured under seven different culture conditions. In control cell cultures, BDC2.5 T cells were cultured in conditions of either (1) alone or (2) with p79 plus CD4⁺ cell-depleted irradiated APCs for activation. BDC2.5 T cells were also cultured with (3) an equal number of NR286 T cells. In some experiments, antibodies against (4) IFN-γ or (5) IL-10 (24 µg/ml), and NO inhibitors, either (6) L-NMMA (active) or (7) D-NMMA (inactive) (0.2 µM), were added to the cultures to determine their effect on blocking the regulatory function of NR286 T cells. The number shown in each histogram represents the percentage of BDC2.5 T cells that underwent <2 rounds of cell division. Data are from at least 2 experiments. (<b>B</b>) CFSE-labeled CD4⁺ BDC2.5 T cells (2×10⁵/well) were cultured under three conditions. Similar to those in (A), in control cell cultures, BDC2.5 T cells were cultured in conditions of either (1) alone or (2) with p79 plus CD4⁺ cell-depleted irradiated APCs for activation. BDC2.5 T cells were also cultured with (3) antibody against IFN-γ (24 µg/ml), in the absence of NR286 T cells. <b>(C)</b> Cell culture supernatants from (A) were harvested for ELISA to detect the presence of IFN-γ, IL-10, and NO.</p