Role for CXCR6 in recruitment of activated CD8+ lymphocytes to inflamed liver

Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6(-/-), H-2D(b) lymphocytes into BDF(1), H-2D(bxd) recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6(-/-) or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6(-/-) CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6(-/-) and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.     

Lamellar organization of pigments in chlorosomes, the light harvesting complexes of green photosynthetic bacteria

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations approximately 20 A apart. X-ray scattering from chlorosomes exhibited a feature with the same approximately 20 A spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.     

Murine CD8+ recent thymic emigrants are alphaE integrin-positive and CC chemokine ligand 25 responsive

Recent thymic emigrants (RTE) are an important subpopulation of naive CD8+ T cells because of their ability to reconstitute a diverse immune system after periods of T cell depletion. In neonatal mice, the majority of peripheral T lymphocytes are RTE, cells that have recently left the thymus to populate the periphery. Postulating that these cells could have unique trafficking mechanisms, we compared adhesion molecule and chemokine receptor expression of neonatal RTE with mature adult lymphocytes. Neonatal CD8+ splenocytes uniformly express alpha(E) integrin and exhibit a high responsiveness to CC chemokine ligand (CCL25) (as compared with adult CD8+ splenocytes). Mature CD8+ thymocytes have a similar alpha(E) integrin(+) CCL25 responsive phenotype, as do adult CD8+ RTE identified by intrathymic FITC injection. With increasing age, the frequency of CD8+ alpha(E) integrin(+) splenocytes decreases, roughly correlating with thymic involution. Moreover, halting thymic output by thymectomy accelerates the age-dependent decline in peripheral CD8+ alpha(E) integrin(+) RTE phenotype cells. Low expression of CD44 distinguishes these CD8+ RTE from a population of memory phenotype alpha(E) integrin(+) CD8+ cells that are CD44(high). We conclude that CD8+ RTE have unique adhesive and chemotactic properties that distinguish them from naive CD8+ T cells. These properties may enable specialized microenvironmental and cell-cell interactions contributing to the fate of RTE in the periphery during the early post-thymic period. This phenotype will also facilitate the identification and isolation of RTE for further studies.     

Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children

We have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD(-) B cells are predominantly large, CD38(high), CD27(high), CD138(+/-), CCR6(-), alpha4beta7(+), CCR9(+), CCR10(+), cutaneous lymphocyte antigen-negative (CLA(-)), L-selectin(int/-), and sIgM(+), sIgG(-), sIgA(+/-) lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38(int/-), CD27(int/-), CCR6(+), alpha4beta7(+/-), CCR9(+/-) and CCR10(-), most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.     

Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes

Staphylococcus aureus is the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting, S. aureus is a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and the S. aureus population structure, we compared 61 community-acquired invasive isolates of S. aureus and 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the seven S. aureus sequencing projects. The core genes common to all strains were identified, and 10 dominant lineages of S. aureus were clearly discriminated. Each lineage carried a unique combination of hundreds of "core variable" (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that the S. aureus gene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors.     

Quantification of embryonic atrioventricular valve biomechanics during morphogenesis

Tissue assembly in the developing embryo is a rapid and complex process. While much research has focused on genetic regulatory machinery, understanding tissue level changes such as biomechanical remodeling remains a challenging experimental enigma. In the particular case of embryonic atrioventricular valves, micro-scale, amorphous cushions rapidly remodel into fibrous leaflets while simultaneously interacting with a demanding mechanical environment. In this study we employ two microscale mechanical measurement systems in conjunction with finite element analysis to quantify valve stiffening during valvulogenesis. The pipette aspiration technique is compared to a uniaxial load deformation, and the analytic expression for a uniaxially loaded bar is used to estimate the nonlinear material parameters of the experimental data. Effective modulus and strain energy density are analyzed as potential metrics for comparing mechanical stiffness. Avian atrioventricular valves from globular Hamburger-Hamilton stages HH25-HH34 were tested via the pipette method, while the planar HH36 leaflets were tested using the deformable post technique. Strain energy density between HH25 and HH34 septal leaflets increased 4.6±1.8 fold (±SD). The strain energy density of the HH36 septal leaflet was four orders of magnitude greater than the HH34 pipette result. Our results establish morphological thresholds for employing the micropipette aspiration and deformable post techniques for measuring uniaxial mechanical properties of embryonic tissues. Quantitative biomechanical analysis is an important and underserved complement to molecular and genetic experimentation of embryonic morphogenesis.     

Resilience of inshore, juvenile snapper Pagrus auratus to angling and release

This study assessed the mortality of 157 snapper Pagrus auratus (9-29 cm, total length, L(T) ) after being conventionally angled and then released into cages (along with 48 controls) for 4 days off south-eastern Australia. Fatalities were restricted to 12 angled fish (7·6%) and mostly attributed to the ingestion of hooks and especially their subsequent removal, which caused substantial blood loss and immediate death. Hook ingestion was significantly biased towards smaller fish (<21 cm L(T)) and attributed to a lower chance of anglers initially detecting these individuals on the line (allowing them to consume more of the baits). While mortalities might be reduced in future via (1) choosing terminal rigs that promote mouth hooking and (2) cutting the line on any-hook ingested fish, the results nevertheless validate releasing unwanted angled inshore juvenile P. auratus as a means for managing their exploitation.     

Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii

IL-12-mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2(-/-)) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2(-/-) mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2(-/-) mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2(-/-)CD8(+) DC compared with WT CD8(+) DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2(-/-) animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.     

Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating

Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins'' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.