LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus

Cellular attachment factors like the C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can augment viral infection and might promote viral dissemination in and between hosts. The lectin LSECtin is encoded in the same chromosomal locus as DC-SIGN/R and is coexpressed with DC-SIGNR on sinusoidal endothelial cells in liver and lymphnodes. Here, we show that LSECtin enhances infection driven by filovirus glycoproteins (GP) and the S protein of SARS coronavirus, but does not interact with human immunodeficiency virus type-1 and hepatitis C virus envelope proteins. Ligand binding to LSECtin was inhibited by EGTA but not by mannan, suggesting that LSECtin unlike DC-SIGN/R does not recognize high-mannose glycans on viral GPs. Finally, we demonstrate that LSECtin is N-linked glycosylated and that glycosylation is required for cell surface expression. In summary, we identified LSECtin as an attachment factor that in conjunction with DC-SIGNR might concentrate viral pathogens in liver and lymph nodes.     

Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.     

Mice with neonatally induced inactivation of the vascular cell adhesion molecule-1 fail to control the parasite in Toxoplasma encephalitis

Under various inflammatory conditions, cell adhesion molecules are up-regulated in the central nervous system (CNS) and may contribute to the recruitment of leukocytes to the brain. In the present study, the functional role of vascular cell adhesion molecule (VCAM)-1 in Toxoplasma encephalitis (TE) was addressed using VCAM(flox/flox MxCre) mice. Neonatal inactivation of the VCAM-1 gene resulted in a lack of induction of VCAM-1 on cerebral blood vessel endothelial cells, whereas the constitutive expression of VCAM-1 on choroid plexus epithelial cells and the ependyma was unaffected; in these animals, resistance to T. gondii was abolished, and VCAM(flox/flox MxCre) mice died of chronic TE caused by a failure to control parasites in the CNS. Although leukocyte recruitment to the CNS was unimpaired, the B cell response was significantly reduced as evidenced by reduced serum levels of anti-T. gondii-specific IgM and IgG antibodies. Furthermore, the frequency and activation state of intracerebral T. gondii-specific T cells were decreased, and microglial activation was markedly reduced. Taken together, these data demonstrate the crucial requirement of VCAM-1-mediated immune reactions for the control of an intracerebral infectious pathogen, whereas other cell adhesion molecules can efficiently compensate for VCAM-1-mediated homing across cerebral blood vessels.     

High frequency recombination betweenloxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase

An adenovirus vector (AdCre1) expressing Cre recombinase has been used to induce recombination betweenloxP sites in human chromosomes. G418 resistant cells with oneloxP site, generated by transfection with a plasmid containingloxP between the SV40 promoter and the G418 resistance (neo) gene, were infected with AdCre1 and transfected with a plasmid containingloxP adjacent to a promoterless hisD gene. This resulted in integration of hisD downstream of the SV40 promoter with gain of histidinol and loss of G418 resistance. Since AdCre1 is non-replicating and Cre expression transient, histidinol resistant cells containing the hisD gene flanked byloxP sites were stable. Reinfection of these cells with AdCre1 induced excision of hisD in over 90% of infected cells. This high efficiency of site-specific recombination suggests that AdCre1 may be exploited for temporal and tissue-specific regulation of gene expression and for chromosome engineering in vitro and in animals.     

Practical Steps in the Diagnosis and Management of Gout

One of the earliest described conditions, gout continues to plague humanity. It is characterised by the deposition of monosodium urate crystals in the joints and soft tissue. The main clinical features of gout are hyperuricaemia, acute monoarticular arthritis, tophi and chronic arthritis, along with nephrolithiasis. Gout typically occurs in middle age and more commonly in men. Asymptomatic hyperuricaemia does not require treatment. The initial attack of acute gout usually affects a single joint, often the first metatarsal phalangeal joint. Definitive diagnosis requires demonstration of urate crystals in the joint fluid. Treatment of acute gout includes nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine and corticosteroids. The most important factor in success of treatment is how quickly therapy is begun after onset of symptoms. Drug treatment of hyperuricaemia includes allopurinol, sulfinpyrazone, probenecid and benzbromarone and should be used in patients with frequent gout attacks, tophi or urate nephropathy.     

Phosphatidylcholine-Specific Phospholipase C from Listeria monocytogenes Is an Important Virulence Factor in Murine Cerebral Listeriosis

Meningoencephalitis is a serious and often fatal complication of Listeria monocytogenes infection. The aim of the present study was to analyze the role of internalin A (InlA) and B, which are involved in the invasion of L. monocytogenes into cultivated host tissue cells, and that of phosphatidylcholine-specific phospholipase C (PlcB), which mainly promotes the direct cell-to-cell spread of L. monocytogenes, in murine cerebral listeriosis by use of an InlA/B (ΔinlAB2)- and a PlcB (ΔplcB2)-deficient isogenic deletion mutant strain and the wild-type (WT) L. monocytogenes EGD. Listeria strains were directly applied to the brain, a technique which has been employed previously to study the pathogenesis of cerebral listeriosis (D. Schlüter, S. B. Oprisiu, S. Chahoud, D. Weiner, O. D. Wiestler, H. Hof, and M. Deckert-Schlüter, Eur. J. Immunol. 25:2384–2391, 1995). We demonstrated that PlcB, but not InlA or InlB, is an important virulence factor in cerebral listeriosis. Nonimmunized mice infected intracerebrally with the ΔplcB2 strain survived significantly longer and had a reduced intracerebral bacterial load compared to mice infected with the ΔinlAB2 strain or WT bacteria. In addition, immunization with the WT prior to intracerebral infection significantly increased the survival rate of mice challenged intracerebrally with the ΔplcB2 strain compared to that of mice infected with the WT or ΔinlAB2 strain. Histopathology revealed that the major difference between the various experimental groups was a significantly delayed intracerebral spread of the ΔplcB2 mutant strain, indicating that cell-to-cell spread is an important pathogenic feature of cerebral listeriosis. Interestingly, irrespective of the Listeria mutant used, the apoptosis of hippocampal and cerebellar neurons and an internal hydrocephalus developed in surviving mice, indicating that these complications are not dependent on the virulence factors InlA/B and PlcB. In conclusion, this study points to PlcB as a virulence factor important for the intracerebral pathogenesis of murine L. monocytogenes meningoencephalitis.     

Interleukin-6 gene alleles affect the risk of Alzheimer's disease and levels of the cytokine in blood and brain

Two different polymorphic regions of the interleukin-6 (IL-6) gene were investigated in patients with Alzheimer's disease (AD) and non-demented controls. The -174 C allele in the promoter region of IL-6 gene was over-represented in AD patients compared to controls and significantly increased the risk of AD. Moreover, the -174 CC genotype was associated with a high risk of the disease in women. The D allele of a variable number of tandem repeat (VNTR) was in strong linkage disequilibrium with the -174 C allele and slightly increased AD risk. On the other hand, the frequency of the VNTR C allele was decreased in patients with AD and was negatively associated with the risk of developing AD. Both the -174 CC and VNTR DD genotypes were also associated with increased IL-6 levels in the blood and brain from AD. These findings suggest that IL-6 may play a multifaceted role in AD by affecting the turnover of the cytokine.     

Multidimensional Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers, 1

Summary: The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D‐chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC‐CC, MALDI‐TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

2D separation of poly(MPEG‐MM 2), 1^st dimension: LC‐CC, 2^nd dimension: SEC.