Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (M phi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to M phi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca2+/Mg2+ enhanced DV binding. This argues against involvement of beta (2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains. (C) 2001 Elsevier Science B.V. All rights reserved.

Mast cells and oral inflammation

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-alpha in their granules, and release of this promotes leukocyte infiltration during evolving inflammation in several conditions, including lichen planus, gingivitis, pulpitis, and periapical inflammation, through induction of endothelial-leukocyte adhesion molecules. Mast cell synthesis and release of other mediators exerts potent immunoregulatory effects on other cell types, while several T-lymphocyte-derived cytokines influence mast cell migration and mediator release. Mast cell proteases may contribute to alterations in basement membranes in inflammation in the oral cavity, such as the disruptions that allow cytotoxic lymphocytes to enter the epithelium in oral lichen planus. A close relationship exists among mast cells, neural elements, and laminin, and this explains the preferential distribution of mast cells in tissues. Mast cells are responsive to neuropeptides and, through their interaction with neural elements, form a neural immune network with Langerhans cells in mucosal tissues. This facilitates mast cell degranulation in response to a range of immunological and non-immunological stimuli. Because mast cells play a pivotal role in inflammation, therapies that target mast cell functions could have value in the treatment of chronic inflammatory disorders in the oral cavity.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2(d)), C57BL6 (H-2(b)), DBA/2J (H-2(d)) and CBA/CaH (H-2(k)) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4(+) and CD8(+) T-cell subsets, CD14(+) macrophages and CD19(+) B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8(+) T cells and CD19(+) B cells were found in any of the lesions. The percentages of CD4(+) cells, CD14(+) cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14(+) cells in sham-immunized mice. The percentage of CD14(+) cells was higher than that of CD4(+) cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4(+) and CD14(+) cells predominated in immunized CBA/CaH mice and CD4(+) cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14(+) cells and CD4(+) cells in sham-immunized mice. IgG1(+) plasma cells were more dominant than IgG2a(+) cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a(+) plasma cells were more obvious in sham-immunized mice. IgG2a(+) plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

The VCAM-1 gene that encodes the vascular cell adhesion molecule is a target of the Sry-related high mobility group box gene, Sox18

VCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of crosstalk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosis-lymphedematelangiectasia disorder.

Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: Biological validation of the proposed HLA A24 supertype

Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.

Leukocyte subset responses during exercise under heat stress with carbohydrate or water intake

Purpose: This study investigated leukocyte subset responses to moderate-intensity exercise under heat stress, with water (W) or carbohydrate (CHO) drink ingestion. Methods: In repeated trials, 13 soldiers consumed either a W or CHO drink during 3 h of walking at 4.4 km center dot h(-1) with a 5% gradient (15 min rest per hour) under heat stress (35 C and 55% relative humidity). The soldiers wore combat uniforms and carried water bottles and dummy rifles and ammunition, altogether weighing about 11.5 +/- 1.0 kg. Results: Plasma glucose concentration was significantly higher with CHO than W ingestion during exercise (p < 0.01). There were no significant differences between W and CHO conditions in exercise performance, plasma cortisol concentration, heart rate, or core temperature. CHO ingestion significantly moderated the increases in leukocyte (83% in W, 28% in CHO; p < 0.001), monocyte (60% in W, 34% in CHO; p < 0.05), and granulocyte counts (120% in W, 30% in CHO; p < 0.001), but not in lymphocyte count (41% in W, 25% in CHO). Conclusions: The increases in leukocyte and subset counts during moderate-intensity exercise under heat stress may be comparable to those observed during intense exercise in cool conditions. The response of immune cell counts is blunted by CHO intake during moderate-intensity exercise in the heat, and may not occur through the cortisol pathway.

Investigation of oxidative stress defenses of Neisseria gonorrhoeae by using a human polymorphonuclear leukocyte survival assay

Neisseria gonorrhoeae has well-characterized oxidative stress defense systems that protect against oxidative killing in in vitro assays. In contrast, mutant strains of N. gonorrhoeae lacking oxidative stress defenses are identical to the wild type when tested in an ex vivo survival assay using human polymorphonuclear leukocytes.

Bcl-2 in endothelial cells is increased by vitamin E and alpha-lipoic acid supplementation but not exercise training

Atherosclerotic plaque contains apoptotic endothelial cells with oxidative stress implicated in this process. Vitamin E and a-lipoic acid are a potent antioxidant combination with the potential to prevent endothelial apoptosis. Regular exercise is known to increase myocardial protection, however, little research has investigated the effects of exercise on the endothelium. The purpose of these studies was to investigate the effects of antioxidant supplementation and/or exercise training on proteins that regulate apoptosis in endothelial cells. Male rats received a control or antioxidant-supplemented diet (vitamin E and alpha-lipoic acid) and were assigned to sedentary or exercise-trained groups for 14 weeks. Left ventricular endothelial cells (LVECs) were isolated and levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were measured. Antioxidant supplementation caused a fourfold increase in Bcl-2 (P < 0.05) with no change in Bax (P > 0.05). Bcl-2:Bax was increased sixfold with antioxidant supplementation compared to non-supplemented animals (P < 0.05). Exercise training had no significant effect on Bcl-2, Bax or Bcl-2:Bax either alone or combined with antioxidant supplementation (P > 0.05) compared to non-supplemented animals. However, Bax was significantly lower (P < 0.05) in the supplemented trained group compared to non-supplemented trained animals. Cultured bovine endothelial cells incubated for 24 h with vitamin E and/or a-lipoic acid showed the combination of the two antioxidants increased Bcl-2 to a greater extent than cells incubated with the vehicle alone. In summary, vitamin E and a-lipoic acid increase endothelial cell Bcl-2, which may provide increased protection against apoptosis. (c) 2005 Elsevier Ltd. All rights reserved

Correlation of habitual physical activity levels with flow mediated dilation of the brachial artery in 5-10 year old children

Endothelial dysfunction is an early key event of atherogenesis. Both fitness level and exercise intervention have been shown to positively influence endothelial function. In a cross-sectional study of 47 children, the relationship between habitual physical activity and flow-mediated dilation (FMD) of the brachial artery was explored. Habitual physical activity levels (PALs) were assessed using a validated stable isotope technique, and FMD of the brachial artery was measured via high-resolution ultrasound. The results showed that habitual physical activity significantly correlated with FMD (r=0.39, P=0.007), and remained the most influential variable on dilation in multivariate analysis. Although both fitness level and exercise intervention have previously been shown to positively influence FMD, this is the first time that a relationship with normal PALs has been investigated, especially, at such a young age. These data support the concept that physical activity exerts its protective effect on cardiovascular health via the endothelium and add further emphasis to the importance of physical activity in childhood.

The deep-sea teleost cornea: a comparative study of gadiform fishes

The corneal structure of three deep-sea species of teleosts (Gadiformes, Teleostei) from different depths (250-4000 m) and photic zones are examined at the level of the light and electron microscopes. Each species shows a similar but complex arrangement of layers with a cornea split into dermal and scleral components. The dermal cornea comprises an epithelium overlying a basement membrane and a dermal stroma with sutures and occasional keratocytes. Nezumia aequalis is the only species to possess a Bowman's layer, although it is not well-developed. The scleral cornea is separated from the dermal cornea by a mucoid layer and, in contrast to shallow-water species, is divided into three main layers; an anterior scleral stroma, a middle or iridescent layer and a posterior scleral stroma. The iridescent layer of collagen and intercalated cells or cellular processes is bounded by a layer of cells and the posterior scleral stroma overlies a Descemet's membrane and an endothelium. In the relatively shallow-water Microgadus proximus, the keratocytes of the dermal stroma, the cells of the iridescent layer and the endothelial cells all contain aligned endoplasmic reticulum, which may elicit an iridescent reflex. No alignment of the endoplasmic reticulum was found in N. aequalis or Coryphanoides (Nematonurus) armatus. The relative differences between shallow-water and deep-sea corneas are discussed in relation to the constraints of light, depth and temperature.