In this review, we summarize our investigations concerning the differential importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form lipopolysaccharide (LPS) chemotypes and include the results obtained in studies with murine and human TLR4-transgenic mice. Furthermore, we present more recent data on the mechanisms involved in the induction of LPS hypersensitivity by bacterial and viral infections and on the reactivity of the hypersensitive host to non-LPS microbial ligands and endogenous mediators. Finally, the effects of pre-existing hypersensitivity on the course and outcome of a super-infection with Salmonella typhimurium or Listeria monocytogenes are summarized.
Cloned PstI fragments of human adenovirus 35 (AV35) genome were compared with the DNA of representatives of human adenovirus subgroups A (type 12), B (type 7), C (types 1, and 5), D (type 8), and E (type 4), using blot hybridization techniques. The E1b region of AV35 was found to be more distantly related to those of other subgroups than E1a regions sequences and examined by others. DNA hybridization was observed only between E1b of AV35 and the DNA of AV4, thus the recombinant constructed might be applied as B-subgroup-specific diagnostic probe. Common nucleotide sequences were detected within the E3 regions of serotypes 1, 4, 5, 7, 8, and 35. On the basis of inter-subgroup homology, and PstI-fragments it may be concluded, that the structure of E3 sequences of AV7 and AV35 DNA are closely related to those of AV3 DNA sequenced by Signäs et al. [18]. E4 regions were compared only of serotypes representing subgroups B, C, and D. These sequences were subgroup specific, similarly to E1b regions.
The transcription factor junB belongs to the jun family of protooncogenes. The appearence of junB mRNA in hepatic cells is an extremely early and sensitive marker of the action of proinflammatory cytokines including interleukin-6. In this study, a competitive reverse transcription (RT)-PCR assay has been developed that is suitable for the quantitative determination of junB mRNA expression. This nonisotopic assay compared to other methods (e.g., Northern blot) is a fast and convenient way to determine the expression of the junB gene and thus the immediate concentration- and time-dependent action of interleukin-6. Because interleukin-6 and interleukin-6-type cytokines play a highly important regulatory role in various pathophysiologically important processes, such as hepatic acute-phase reaction, the quantitative assay of junB mRNA completes the scale of laboratory approaches in inflammation and among other pathological conditions.
Mouse adenovirus type 1 (MAV-1) produces a lethal disease in newborn or suckling mice characterized by infectious virus and viral lesions in multiple organs. Previous reports of MAV-1 infection of adult mice generally described serologic evidence of infection without morbidity or mortality. However, our current results demonstrate that MAV-1 causes a fatal illness in adult C57BL/6(B6) mice (50% lethal dose, [LD50], 10(3.0) PFU) but not in adult BALB/c mice at all of the doses tested (LD50, > or = 10(5.0) PFU). Adult (BALB/c x B6)F1 mice were intermediately susceptible (LD50, 10(4.5) PFU). Clinically, the sensitive B6 mice showed symptoms of acute central nervous system (CNS) disease, including tremors, seizures, ataxia, and paralysis. Light microscopic examination of CNS tissue from the B6 animals revealed petechial hemorrhages, edema, neovascularization, and mild inflammation in the brain and spinal cord. Analysis by electron microscopy showed evidence of inflammation, such as activated microglia, as well as swollen astrocytic endfeet and perivascular lipid deposition indicative of blood-brain barrier dysfunction. Outside of the CNS, the only significant pathological findings were foci of cytolysis in the splenic white pulp. Assessment of viral replication from multiple tissues was performed by using RNase protection assays with an antisense MAV-1 early region 1a probe. The greatest amounts of viral mRNA in MAV-1-infected B6 animals were located in the brain and spinal cord. Less viral message was detected in the spleen, lungs, and heart. No viral mRNA was detected in BALB/c mouse tissue, with the exception of low levels in the heart. Viral titers of organ tissues were also determined and were concordant with RNase protection findings on the brain and spinal cord but failed to demonstrate significant infectious virus in additional organs. Our experiments demonstrate that MAV-1 has a striking tropism for the CNS that is strain dependent, and this provides an informative in vivo model for the study of adenoviral pathogenesis.
Lung alveolar macrophages (AMs) are in the first line of immune defense against respiratory pathogens and play key roles in the pathogenesis of Mycobacterium tuberculosis (Mtb) in humans. Nevertheless, AMs are available only in limited amounts for in vitro studies, which hampers the detailed molecular understanding of host-Mtb interactions in these macrophages. The recent establishment of the self-renewing and primary Max Planck Institute (MPI) cells, functionally very close to lung alveolar macrophages, opens unique opportunities for in vitro studies of host-pathogen interactions in respiratory diseases. Here, we investigated the suitability of MPI cells as a host cell system for Mtb infection. Bacterial, cellular and innate immune features of MPI cells infected with Mtb were characterized. Live bacteria were readily internalized and efficiently replicated in MPI cells, similarly to primary murine macrophages and other cell-lines. MPI cells were also suitable for the determination of anti-tuberculosis drug activity. The primary innate immune response of MPI cells to live Mtb showed significantly higher and earlier induction of the pro-inflammatory cytokines TNFα, IL-6, IL-1α and IL-1β, as compared to stimulation with heat-killed bacteria. MPI cells previously showed a lack of induction of the anti-inflammatory cytokine IL-10 to a wide range of stimuli, including heat-killed Mtb. In contrast, we show here that live Mtb is able to induce significant amounts of IL-10 in MPI cells. Autophagy experiments using LC3B immunostaining, as well as LysoTracker labelling of acidic vacuoles, demonstrated that MPI cells efficiently control killed Mtb by elimination through phagolysosomes. MPI cells were also able to accumulate lipid droplets in their cytoplasm following exposure to lipoproteins. Collectively, this study establishes the MPI cells as a relevant, versatile host cell model for tuberculosis research, allowing a deeper understanding of AMs functions in this pathology.
Methylation of specific residues within the N-terminal histone tails plays a critical role in regulating eukaryotic gene expression. Although great advances have been made toward identifying histone methyltransferases (HMTs) and elucidating the consequences of histone methylation, little is known about the recruitment of HMTs to regulatory regions of chromatin. Here we report that the sequence-specific DNA-binding transcription factor Yin Yang 1 (YY1) binds to and recruits the histone H4 (Arg 3)-specific methyltransferase, PRMT1, to a YY1-activated promoter. Our data confirm that histone methylation does not occur randomly but rather is a targeted event and provides one mechanism by which HMTs can be recruited to chromatin to activate gene expression.
Polyinosinic:polycytidylic acid (poly IC), a double-stranded RNA, is an effective adjuvant in vivo. IFN-λs (also termed IL-28/29) are potent immunomodulatory and antiviral cytokines. We demonstrate that poly IC injection in vivo induces large amounts of IFN-λ, which depended on hematopoietic cells and the presence of TLR3 (Toll-like receptor 3), IRF3 (IFN regulatory factor 3), IRF7, IFN-I receptor, Fms-related tyrosine kinase 3 ligand (FL), and IRF8 but not on MyD88 (myeloid differentiation factor 88), Rig-like helicases, or lymphocytes. Upon poly IC injection in vivo, the IFN-λ production by splenocytes segregated with cells phenotypically resembling CD8α+ conventional dendritic cells (DCs [cDCs]). In vitro experiments revealed that CD8α+ cDCs were the major producers of IFN-λ in response to poly IC, whereas both CD8α+ cDCs and plasmacytoid DCs produced large amounts of IFN-λ in response to HSV-1 or parapoxvirus. The nature of the stimulus and the cytokine milieu determined whether CD8α+ cDCs produced IFN-λ or IL-12p70. Human DCs expressing BDCA3 (CD141), which is considered to be the human counterpart of murine CD8α+ DCs, also produced large amounts of IFN-λ upon poly IC stimulation. Thus, IFN-λ production in response to poly IC is a novel function of mouse CD8α+ cDCs and their human equivalents.
1 Abstract Macrophages are key antigen presenting cells that also secrete cytokines during inflammation. They can be polarised to M1 or M2 phenotypes. Molecules such as CD206 and inducible Nitric Oxide Synthase are considered macrophage phenotype markers because they are highly expressed in either M1 or M2 macrophages. LPCAT2 is a phospholipid modifying enzyme that influences inflammatory responses in macrophages. However, how LPCAT2 influences inflammation is not fully understood. In this study, we have used genetic technology to study the influence of LPCAT2 on macrophage phenotype markers. Our results show for the first time that overexpression of LPCAT2 promotes the expression of M1 macrophage phenotype markers, and attenuates the expression of M2 macrophage markers.
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