Function of herpes simplex virus type 1 gD mutants with different receptor-binding affinities in virus entry and fusion

We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(inverted Delta 34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(inverted Delta 34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(inverted Delta 243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(inverted Delta 243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.     

Growth characteristics of Protoheliolites norvegicus (Tabulata; Upper Ordovician; Estonia)

Protoheliolites is an early heliolitine coral characterized by closely spaced corallites separated in places by sparse coenenchyme. Growth characteristics in the type species, P. norvegicus, are revealed by detailed analysis based on serial peels and thin sections of coralla from the uppermost Katian of north‐western Estonia. Colonies of this species had a strong ability to recover from damage and partial mortality, resulting in various forms of rejuvenation, regeneration, fusion and reorganization of corallites; in some cases, this involved relatively large areas of undifferentiated soft parts. The shells of commensal cornulitids became enclosed in host coralla during colony growth. Coralla of P. norvegicus exhibit distinctive growth cycles due to responses to seasonal changes. The production of new corallites by coenenchymal increase usually occurred in low‐density bands, in which corallites generally display round to subrounded transverse outlines. In high‐density bands, the corallites became crenulated, their wall thickness increased, septal development was more pronounced, and the amount of coenenchyme increased. In addition to these cyclomorphic changes, there were significant astogenetic changes during growth. Compared with the early stage of colony development, distinctive characteristics in the late astogenetic stage include a decrease in the growth rate of the colony, better coordination among corallites, maximum development of corallite crenulations and septa in high‐density bands, more numerous coenenchymal tubules and a greater proportion of corallum area occupied by coenenchyme. In general, the role of polyps in determining morphological characteristics of individual corallites, such as tabularium area, corallite crenulations and wall thickness, was subordinate to the astogeny of the colony. Growth characteristics including colony‐wide coordination of polyp behaviour and subjugation of individuals to restore the colony following damage suggest a strong astogenetic control and high level of colony integration. Protoheliolites probably arose from a heliolitine genus rather than from a nonheliolitine group as some authors have proposed.     

Surfactant Protein A Binds Flagellin Enhancing Phagocytosis and IL-1β Production

Surfactant protein A (SP-A), a pulmonary collectin, plays a role in lung innate immune host defense. In this study the role of SP-A in regulating the inflammatory response to the flagella of Pseudomonas aeruginosa (PA) was examined. Intra-tracheal infection of SP-A deficient (SP-A-/-) C57BL/6 mice with wild type flagellated PA (PAK) resulted in an increase in inflammatory cell recruitment and increase in pro-inflammatory cytokines IL-6 and TNF-α, which was not observed with a mutant pseudomonas lacking flagella (fliC). SP-A directly bound flagellin, via the N-linked carbohydrate moieties and collagen-like domain, in a concentration dependent manner and enhanced macrophage phagocytosis of flagellin and wild type PAK. IL-1β was reduced in the lungs of SP-A-/- mice following PAK infection. MH-s cells, a macrophage cell line, generated greater IL-1β when stimulated with flagellin and SP-A. Historically flagella stimulate IL-1β production through the toll-like receptor 5 (TLR-5) pathway and through a caspase-1 activating inflammasome pathway. IL-1β expression became non-detectable in SP-A and flagellin stimulated MH-s cells in which caspase-1 was silenced, suggesting SP-A induction of IL-1β appears to be occurring through the inflammasome pathway. SP-A plays an important role in the pathogenesis of PA infection in the lung by binding flagellin, enhancing its phagocytosis and modifying the macrophage inflammatory response.     

Leishmania donovani amastigotes mobilize organic and inorganic osmolytes during regulatory volume decrease

The protozoan parasite Leishmania donovani encounters large fluctuations in osmolality as it cycles between its insect vector and human host. The flagellated promastigote exhibits regulatory volume responses involving organic and inorganic osmolytes, but little is known about volume regulation in the clinically relevant amastigote that multiplies within the parasitophorous vacuoles of mammalian host cells. Using a combination of morphological, X-ray microanalytical, and biochemical approaches we determined that non-motile amastigotes respond to hypotonic stress with (1) an amino acid and l-alanine-mediated regulatory volume decrease, and (2) a parallel release of Na+, K+, P (presumably as negatively charged phosphates), and subsequently Cl- from cytoplasm and the cell as a whole. In addition P, Zn2+, and subsequently Ca2+ increase in acidocalcisomes as Cl- content declines in this compartment. This evidence is the first to document subcellular translocation of, and thus a potential role for, zinc in volume regulatory responses. These coordinated changes in organic and inorganic osmolytes demonstrate that amastigote subcellular compartments, particularly acidocalcisomes, function in maintaining ionic homeostasis in the response of Leishmania amastigotes to hypo-osmotic stress.     

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria

FoxP3+ regulatory CD4 T cells (T_regs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that T_regs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing T_reg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of T_regs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ T_regs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of T_regs from peripheral blood was accompanied by reduced in vitro induction of T_regs by parasite antigen and decreased expression of TNFR2 on T_regs among children who had intense prior exposure to malaria. While T_reg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower T_reg frequencies. These data demonstrate that chronic malaria exposure results in altered T_reg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.     

Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth

The thymus is the most rapidly aging tissue in the body, with progressive atrophy beginning as early as birth and not later than adolescence. Latent regenerative potential exists in the atrophic thymus, because certain stimuli can induce quantitative regrowth, but qualitative function of T lymphocytes produced by the regenerated organ has not been fully assessed. Using a genome-wide computational approach, we show that accelerated thymic aging is primarily a function of stromal cells, and that while overall cellularity of the thymus can be restored, many other aspects of thymic function cannot. Medullary islet complexity and tissue-restricted antigen expression decrease with age, representing potential mechanisms for age-related increases in autoimmune disease, but neither of these is restored by induced regrowth, suggesting that new T cells produced by the regrown thymus will probably include more autoreactive cells. Global analysis of stromal gene expression profiles implicates widespread changes in Wnt signaling as the most significant hallmark of degeneration, changes that once again persist even at peak regrowth. Consistent with the permanent nature of age-related molecular changes in stromal cells, induced thymic regrowth is not durable, with the regrown organ returning to an atrophic state within 2 weeks of reaching peak size. Our findings indicate that while quantitative regrowth of the thymus is achievable, the changes associated with aging persist, including potential negative implications for autoimmunity.     

Systems genetic analysis of multivariate response to iron deficiency in mice

The aim of this study was to identify genes that influence iron regulation under varying dietary iron availability. Male and female mice from 20+ BXD recombinant inbred strains were fed iron-poor or iron-adequate diets from weaning until 4 mo of age. At death, the spleen, liver, and blood were harvested for the measurement of hemoglobin, hematocrit, total iron binding capacity, transferrin saturation, and liver, spleen and plasma iron concentration. For each measure and diet, we found large, strain-related variability. A principal-components analysis (PCA) was performed on the strain means for the seven parameters under each dietary condition for each sex, followed by quantitative trait loci (QTL) analysis on the factors. Compared with the iron-adequate diet, iron deficiency altered the factor structure of the principal components. QTL analysis, combined with PosMed (a candidate gene searching system) published gene expression data and literature citations, identified seven candidate genes, Ptprd, Mdm1, Picalm, lip1, Tcerg1, Skp2, and Frzb based on PCA factor, diet, and sex. Expression of each of these is cis-regulated, significantly correlated with the corresponding PCA factor, and previously reported to regulate iron, directly or indirectly. We propose that polymorphisms in multiple genes underlie individual differences in iron regulation, especially in response to dietary iron challenge. This research shows that iron management is a highly complex trait, influenced by multiple genes. Systems genetics analysis of iron homeostasis holds promise for developing new methods for prevention and treatment of iron deficiency anemia and related diseases.