Recurrent cardiovascular and limb events in 294,428 patients with coronary or peripheral artery disease or ischemic stroke on antiplatelet monotherapy: The RESRISK cohort study.

BACKGROUND AND AIMS: Utilising real-world data, we quantified the burden of cardiovascular risk factors and long-term residual risk of atherothrombotic events among routine care cohorts with coronary (CAD) or peripheral (PAD) artery disease or ischemic stroke (IS) on guideline-recommended antiplatelet monotherapy (APMT). METHODS: Retrospective cohort study using data (2010-2020) from the United Kingdom Clinical Practice Research Datalink (CPRD) and Hospital Episode Statistics, including adults with CAD, PAD or IS who were first prescribed APMT (CAD/IS: aspirin; PAD: clopidogrel). Primary outcomes (recurrent events): major adverse cardiovascular events (MACE) for CAD/PAD/IS cohorts, major adverse limb events (MALE) for PAD. RESULTS: 266,478 CAD, 13,162 PAD, and 14,788 IS patients were included (mean age: 71 years; women 37.7%-47.5 %). Risk factor burden was high and attainment of recommended goals was low. There were 73,691, 3,121 and 7,137 MACE among CAD, PAD and IS patients, respectively (median follow-up: 89.9, 42.4 and 75.9 months, respectively), and 4,767 MALE among PAD patients. MACE incidence rate per 1000 person-years was higher in IS (268.7; 95%CI 265.3-272.0) than CAD (92.9; 95%CI 92.5-93.4) or PAD cohorts (97.2; 95%CI 94.6-99.8). MALE incidence rate was 195.9 (95%CI 192.2-199.6) per 1000 person-years. IS patients presented a lower rate of hospitalisations and longer time-to-first hospitalisation, but once hospitalised, they had a longer length-of-stay. PAD patients had the highest hospitalisation rate. CONCLUSIONS: Among a contemporary cohort with cardiovascular disease on APMT, long-term residual atherothrombotic risk remains high despite being on APMT. Greater attention to risk factor control and use of appropriate evidence-based therapy is required to reduce residual risk among this very high-risk population.

Mycoplasma contamination and viral immunomodulatory activity: dendritic cells open Pandora's box.

During in vitro investigations on the interaction of classical swine fever virus (CSFV)--an immunosuppressive viral pathogen--with monocyte-derived dendritic cells (MoDC) a soluble factor with a strong anti-proliferative activity for T lymphocytes was found. This activity, with an inhibitory dilution 50% (ID(50)) of 10(3)-10(7), was induced after virus infection of monocytes differentiating into DC. UV--inactivation of the supernatants and blocking experiments with a monoclonal antibody against the E2 envelope protein of CSFV initially indicated a virus-dependency. However, further investigations including filtration and centrifugation experiments as well as antibiotic treatment demonstrated the involvement of mycoplasma. This was confirmed by a Hoechst 33258 staining, PCR and mycoplasma cultures--Mycoplasma hyorhinis was identified as the contaminant. Elucidation of a mycoplasma presence occurred under conditions in which the original virus stocks prepared in SK6 cells were negative for mycoplasma using the above tests. Moreover, conventional passage of the virus on the SK6 cells used for this purpose did not reveal any mycoplasma. It was the passage of virus in MoDC rather than SK6 cells that was required to expose the contamination. Three passages of the anti-proliferative supernatants on MoDC cultures increased the ID(50) 10(3)-fold; only when these MoDC-derived supernatants were employed was the mycoplasma contaminant also detectable on SK6 cells. In conclusion, these data demonstrate that regular testing of cell lines and virus stocks for mycoplasma does not necessarily identify their presence, and that application of passage in MoDC cultures could prove an aid for identifying initially undetectable levels of mycoplasma contamination.

Subset-dependent modulation of dendritic cell activity by circovirus type 2.

Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single-stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon-producing cells (NIPCs)], as well as DC precursors. This PCV2-DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon-alpha/tumour necrosis factor-alpha (IFN-alpha/TNF-alpha), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non-macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG-oligonucleotide (CpG-ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2-induced inhibition of the IFN-alpha and TNF-alpha normally produced following interaction with CpG-ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.

Porcine peripheral blood dendritic cells and natural interferon-producing cells.

Peripheral blood contains two major particular infrequent dendritic cells (DC) subsets linking the innate and specific immune system, the myeloid DC and plasmacytoid DC equivalent to the natural interferon-producing cells (NIPC). The functional characterization of these cells demands large volumes of blood, making a large animal model more appropriate and beneficial for certain studies. Here, two subsets of porcine blood mononuclear cells expressing swine workshop cluster 3 (SWC3, a SIRP family member), are described and compared to monocytes. The blood DC specialized in T-cell stimulation were major histocompatibility complex (MHC) class II+, CD80/86+, CD1+/-, CD4-, and in contrast to monocytes CD14-. A CD16- and a CD16+ subset could be discriminated. Granulocyte-macrophage colony-stimulating factor and interleukin-3 were survival factors for this DC subset, and culture induced an up-regulation of MHC class II and CD80/86. The second subset described, are porcine NIPC, typically CD4++, MHC class IIlow, CD80/86low, CD1-, CD8-/low, CD16-/low and CD45RA-/low. Porcine NIPC had high interleukin-3 binding capacity, and survived in response to this cytokine. Their unique function was strong interferon type I secretion after virus stimulation. Both subsets were endocytically active when freshly isolated, and down-regulated this activity after in vitro maturation. Taken together, the present report has delineated porcine blood DC and NIPC, permitting a more detailed understanding of innate immune defences, particularly in response to infections.

C-kit positive porcine bone marrow progenitor cells identified and enriched using recombinant stem cell factor.

Porcine haematological studies have been hampered by the lack of monoclonal antibodies against porcine CD34 or CD117 expressed on haematological progenitors. The present report describes the enumeration, phenotyping and isolation of porcine haematopoietic progenitor cells expressing stem cell factor (SCF, c-kit ligand) receptor (c-kit, CD117). Recombinant porcine (rp) SCF and granulocyte-macrophage colony-stimulating factor (GM-CSF) were expressed in the mammalian HEK293 cell-based expression system. Both were biologically active and induced the proliferation of the human erythroleukemic cell line TF-1, as well as of porcine bone marrow haematopoietic cells (BMHC), in a concentration-dependent manner. The effect of rpSCF on BMHC proliferation was synergistic with rpGM-CSF. Furthermore, rpSCF had a synergistic effect on the generation of BMHC-derived dendritic cells (DC) induced by GM-CSF and TNF-alpha. RpSCF was expressed with a 6-histidine epitope, permitting both its purification and immunological detection. Binding studies with BMHC demonstrated ligation of SCF to 4-11% of BMHC. These cells represented the SWC3(low/-)SWC8- BMHC subset, with characteristics of immature proliferative progenitor BMHC. In contrast, no expression was noted on the SWC3+SWC8- monocytic, the SWC3+SWC8+ granulocytic or the SWC3-SWC8+ B cell lineage cells. Using magnetic or fluorescence-activated cell sorting, SCF-ligating BMHC were enriched for pluripotent progenitor cells. In this manner, porcine haematological studies can be pursued in a detailed manner not before possible.

Macrophage phagocytosis of foot-and-mouth disease virus may create infectious carriers.

Macrophages play critical roles in innate defences against virus infections, particularly pertinent to the rapid immune response required following emergency vaccination against foot-and-mouth disease virus (FMDV). Consequently, macrophage-FMDV interaction was studied in vitro, in the absence of specific antibodies, to mimic the animal early postvaccination. A gradual loss of infectivity and viral antigen was observed over 48 hr, and no evidence of productive virus replication was found. From the pathological viewpoint, an important observation was that the majority of macrophages carried infectious virus for at least 10 hr. Pronase and mild acid treatments showed the virus to be primarily on the cell surface during the first 4 hr. Thereafter, it became internalized (pronase- and pH resistant), but remained infectious for 10-24 hr. The internalization process was dependent on microfilament activity, while the survival of infectious virus related to live virus-dependent inhibition of macrophage protein synthesis. Infectious centre assays demonstrated that this infectious virus - whether on the cell surface or internalized - was actually being released from the cells. This is interesting considering that FMDV is highly pH labile. Together, these characteristics suggest that the virus had been internalized by a process such as macropinocytosis, and fusion with endosomes was delayed or impaired. This mechanism whereby the virus could 'piggyback' on or in the macrophage, becoming internalized but not degraded for at least 10 hr, are important considerations in FMD pathogenesis. Such 'virus-transporting' macrophages would be in a position to carry infectious FMDV to different sites in the body, where it could be released to infect other cells for replication.