Human blood contains at least 2 subpopulations of antigen-presenting dendritic cells (DCs) that can be differentiated by their expression of CD11c. Myeloid DCs (myDCs), which are CD11c ؉ , trap invading pathogens in the tissues and then migrate to lymphoid tissues where they stimulate pathogenspecific T-cell responses. Plasmacytoid DCs (pcDCs), which are CD11c ؊ , secrete interferon-␣ in response to viral infections. This study reports that in HIV-1 infection there is a progressive depletion of both these DC populations and that this correlates with an increasing HIV-1 plasma virus load. The median numbers of myDCs and pcDCs were 6978/mL and 9299/mL, respectively, in healthy male controls and 2298/mL and 1640/mL, respectively, in patients with more than 10 5 HIV-1 RNA copies/mL. Both DC populations expressed CD4, CCR5, and CXCR4. The findings suggest that loss of DCs in HIV infection may contribute to disease progression.

Human peripheral blood contains two populations of dendritic cells (DC) but their developmental relationship has not been established. Freshly isolated CD11c– DC possessed a lymphoid morphology, lacked myeloid markers but expressed lymphoid markers (CD4+ CD10+) whilst the CD11c+ DC were monocytoid in appearance and expressed myeloid markers. Although both populations were allostimulatory, only the CD11c+ DC were able to take up antigen. Irrespective of the culture conditions the CD11c– cells developed into CD11c– CD13– CD33– CD4+ CD1a– CD83+/– DC. In contrast, cultured CD11c+ cells developed the phenotype CD11c+ CD13+ CD33+/– CD4– CD1a+ CD83+ CD9+. Only the CD11c+ DC expressed macrophage colony‐stimulating factor (M‐CSF) receptor and gave rise to CD14+, esterase+, phagocytic macrophages when cultured in M‐CSF. These data suggest that these two populations of DC represent distinct lineages of antigen‐presenting DC.

Recently it has been shown that the 2 populations of blood dendritic cells (DCs), termed plasmacytoid (pcDCs) and myeloid (myDCs), are reduced in HIV-1 infection. This study aimed to determine whether these 2 populations are targets for HIV-1 infection and whether their ability to stimulate T-lymphocyte proliferation is affected. Highly purified populations of myDCs and pcDCs were isolated from the blood of antiretroviral treatmentnaive patients and assessed for the level of HIV provirus by polymerase chain reaction (PCR). We show that both populations are targets for HIV-1 infection as indicated by the presence of provirus in 12 of 14 pcDC and 13 of 14 myDC samples tested. A proportion of this provirus is integrated in myDCs. The ability of both myDCs and pcDCs from HIV-1-infected patients to stimulate allogeneic T-lymphocyte proliferation in a 6-day mixed leukocyte reaction was severely impaired, IntroductionPrimary adaptive immune responses are initiated by dendritic cells (DCs) through the presentation of antigen to T lymphocytes. DCs are also important stimulators of innate immune responses. 1,2 DCs constitute about 1% of the mononuclear cell population of blood and can be divided into 2 major subpopulations that are phenotypically and functionally distinct. 3,4 Myeloid DCs (myDCs) express CD11c, CD13, CD33, and the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) and secrete interleukin 12 (IL-12). They are precursors of classical antigen-presenting cells, including Langerhans cells and dermal and interstitial DCs. 5 By contrast, plasmacytoid DCs (pcDCs) are CD11c Ϫ ; lack myeloid markers but express CD68, CD36, IL-3 receptor-␣ (IL-3R␣), immunoglobulin-like transcript 3 (ILT3); and produce high amounts of interferon-␣ (IFN-␣), 6,7 a potent inhibitor of HIV-1 replication. 8 Unlike myDCs, which migrate to the tissues and intercept invading pathogens before migrating to the lymph nodes, pcDCs migrate directly from blood to the secondary lymphoid tissue where they differentiate into cells originally termed plasmacytoid T cells on the basis of their extensive endoplasmic reticulum. 9,10 It has long been known that during HIV-1 infection there is a progressive decline in CD4 T-lymphocyte numbers, and in the later stages of disease there is loss of HIV-specific cytotoxic Tlymphocyte activity. 11 Recently, we and others have reported that there is also a progressive loss in the number of blood DCs. [12][13][14][15] However, to fully understand the role of DCs in the pathogenesis of HIV-1, it is important to determine whether the 2 DC populations are targets for HIV-1 infection and whether their ability to stimulate T lymphocytes in infected individuals is affected. Evidence of infection would be important as it would provide a mechanism to explain DC loss and might facilitate infection of T lymphocytes during antigen presentation. Down-regulation of T-lymphocyte stimulatory capacity together with reduced numbers would severely suppress cell-mediated immunity and contribute to the immunosuppr...

SummaryReasons for performing study : We hypothesised that seasonal pasture myopathy (SPM), which closely resembles atypical myopathy (AM), was caused by ingestion of a seed-bearing plant abundant in autumn pastures. Objectives: To identify a common seed-bearing plant among autumn pastures of horses with SPM, and to determine whether the toxic amino acid hypoglycin A was present in the seeds and whether hypoglycin metabolites were present in SPM horse serum or urine. Methods: Twelve SPM cases, 11 SPM pastures and 23 control farms were visited to identify a plant common to all SPM farms in autumn. A common seed was analysed for amino acid composition (n = 7/7) by GC-MS and its toxic metabolite (n = 4/4) identified in conjugated form in serum [tandem mass spectrometry (MS/MS)] and urine [gas chromatography (GC) MS]. Serum acylcarnitines and urine organic acid profiles (n = 7) were determined for SPM horses. Results: Seeds from box elder trees (Acer negundo) were present on all SPM and 61% of control pastures. Hypoglycin A, known to cause acquired multiple acyl-CoA dehydrogenase deficiency (MADD), was found in box elder seeds. Serum acylcarnitines and urine organic acid profiles in SPM horses were typical for MADD. The hypoglycin A metabolite methylenecyclopropylacetic acid (MCPA), known to be toxic in other species, was found in conjugated form in SPM horse serum and urine. Horses with SPM had longer turn-out, more overgrazed pastures, and less supplemental feeding than control horses. Potential relevance: For the first time, SPM has been linked to a toxin in seeds abundant on autumn pastures whose identified metabolite, MCPA, is known to cause acquired MADD, the pathological mechanism behind SPM and AM. Further research is required to determine the lethal dose of hypoglycin A in horses, as well as factors that affect annual seed burden and hypoglycin A content in Acer species in North America and Europe.

Attachment to the plasma membrane by linkage to a glycosylphosphatidylinositol (GPI) anchor is a mode of protein expression highly conserved from protozoa to mammals. As a clinical entity, deficiency of GPI has been recognized as paroxysmal nocturnal hemoglobinuria, an acquired clonal disorder associated with somatic mutations of the X-linked PIGA gene in hematopoietic cells. We have identified a novel disease characterized by a propensity to venous thrombosis and seizures in which deficiency of GPI is inherited in an autosomal recessive manner. In two unrelated kindreds, a point mutation (c --> g) at position -270 from the start codon of PIGM, a mannosyltransferase-encoding gene, disrupts binding of the transcription factor Sp1 to its cognate promoter motif. This mutation substantially reduces transcription of PIGM and blocks mannosylation of GPI, leading to partial but severe deficiency of GPI. These findings indicate that biosynthesis of GPI is essential to maintain homeostasis of blood coagulation and neurological function.

Plasmacytoid dendritic cells (pcDC) and myeloid dendritic cells (myDC) are shown to express CD4 and low levels of CCR5 and CXCR4, but only myDC express DC SIGN, a C-type lectin that binds human immunodeficiency virus but does not mediate virus entry. Both DC types were more susceptible to infection with a macrophage than a lymphotropic strain of human immunodeficiency virus type 1, but pcDC were more readily infected than myDC.

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.

Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription.