Location rather than CD62L phenotype is critical in the early establishment of influenza-specific T cell memory

The rapid recall of influenza virus-specific CD8+ T cell effector function is protective, although our understanding of T cell memory remains incomplete. Recent debate has focused particularly on the CD62L lymph node homing receptor. The present analysis shows that although functional memory can be established from both CD62Lhi and CD62Llo CD8+ T cell subsets soon after initial encounter between naive precursors and antigen, the optimal precursors are CD8+CD44hiCD25lo immune lymphocytes isolated from draining lymph nodes on day 3.5 after influenza virus infection. Analysis of primed T cells at different times after challenge indicates that the capacity to transfer memory is diminished at the peak of the primary cytotoxic T lymphocyte response, challenging speculations that the transition to memory first requires full differentiation to effector status. It seems that location rather than CD62Lhi/lo phenotype may be the more profitable focus for further dissection of the early establishment of T cell memory.

Mutation of the LUNATIC FRINGE gene in humans Causes spondylocostal dysostosis with a severe vertebral phenotype

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

The fur seal — a model lactation phenotype to explore molecular factors involved in the initiation of apoptosis at involution

Mammary gland involution requires co-ordination of milk production, immune responses, apoptosis and remodeling. Initiation and progression of each of these components involves integral control by the mammary gland. Although cell-based culture models and genetically manipulated animals have shed light on these processes, the factors controlling each step in the involution cascade are still poorly understood. The fur seal displays a unique lactation phenotype. During the lactation cycle the mammary gland downregulates milk production and initiates an immune response but fails to initiate the apoptotic phase of involution, allowing the female fur seal to undertake long foraging trips of up to 28 days between suckling bouts. Upon return to shore the female continues feeding her pup following resumption of lactation and milk production. Expression profiling of genes involved in this lactation cycle provides valuable tools for investigation of the factors responsible for the initiation of apoptosis at involution.

Cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine

Defective efferocytosis may perpetuate inflammation in smokers with or without chronic obstructive pulmonary disease (COPD). Macrophages may phenotypically polarize to classically activated M1 (proinflammatory; regulation of antigen presentation) or alternatively activated M2 (poor antigen presentation; improved efferocytosis) markers. In bronchoalveolar lavage (BAL)–derived macrophages from control subjects and smoker/ex-smoker COPD subjects, we investigated M1 markers (antigen-presenting major histocompatibility complex [MHC] Classes I and II), complement receptors (CRs), the high-affinity Fc receptor involved with immunoglobulin binding for phagocytosis (Fc-gamma receptor, FcγR1), M2 markers (dendritic cell–specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN] and arginase), and macrophage function (efferocytosis and proinflammatory cytokine production in response to LPS). The availability of glutathione (GSH) in BAL was assessed, because GSH is essential for both M1 function and efferocytosis. We used a murine model to investigate macrophage phenotype/function further in response to cigarette smoke. In lung tissue (disaggregated) and BAL, we investigated CRs, the available GSH, arginase, and efferocytosis. We further investigated the therapeutic effects of an oral administration of a GSH precursor, cysteine l-2-oxothiazolidine-4-carboxylic acid (procysteine). Significantly decreased efferocytosis, available GSH, and M1 antigen–presenting molecules were evident in both COPD groups, with increased DC-SIGN and production of proinflammatory cytokines. Increased CR-3 was evident in the current-smoker COPD group. In smoke-exposed mice, we found decreased efferocytosis (BAL and tissue) and available GSH, and increased arginase, CR-3, and CR-4. Treatment with procysteine significantly increased GSH, efferocytosis (BAL: control group, 26.2%; smoke-exposed group, 17.66%; procysteine + smoke-exposed group, 27.8%; tissue: control group, 35.9%; smoke-exposed group, 21.6%; procysteine + smoke-exposed group, 34.5%), and decreased CR-4 in lung tissue. Macrophages in COPD are of a mixed phenotype and function. The increased efferocytosis and availability of GSH in response to procysteine indicates that this treatment may be useful as adjunct therapy for improving macrophage function in COPD and in susceptible smokers.

Incubation environment affects phenotype of naturally incubated green turtle hatchlings

A comparison of body size and flipper size was carried out on green turtle Chelonia mydas) hatchlings produced from natural nests at two beaches on Ascension Island, South Atlantic and one beach in northern Cyprus in the Mediterranean (N=18 nests; N=180 hatchlings). Hatchlings from Ascension Island were significantly larger and heavier than hatchlings in Cyprus, a likely consequence of maternal size effects. Incubation temperature appeared to influence body size of hatchlings on Ascension Island with higher temperatures producing smaller hatchlings. Both hind and fore-flipper area scaled positively with body size. In proportion to body size, hind-flipper area appears relatively consistent among the Atlantic populations but is smaller than hatchlings measured in Hawaii.

Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1.

Swine influenza virus PA and neuraminidase gene reassortment into human H1N1iInfluenza Virus is associated with an altered pathogenic phenotype linked to increased MIP-2 expression

Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. IMPORTANCE: Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Characterizing the molecular phenotype of an Atp7a(T985I) conditional knock in mouse model for X-linked distal hereditary motor neuropathy (dHMNX)

ATP7A is a P-type ATPase essential for cellular copper (Cu) transport and homeostasis. Loss-of-function ATP7A mutations causing systemic Cu deficiency are associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome. We previously identified two rare ATP7A missense mutations (P1386S and T994I) leading to a non-fatal form of motor neuron disorder, X-linked distal hereditary motor neuropathy (dHMNX), without overt signs of systemic Cu deficiency. Recent investigations using a tissue specific Atp7a knock out model have demonstrated that Cu plays an essential role in motor neuron maintenance and function, however the underlying pathogenic mechanisms of ATP7A mutations causing axonal degeneration remain unknown. We have generated an Atp7a conditional knock in mouse model of dHMNX expressing Atp7a(T985I), the orthologue of the human ATP7A(T994I) identified in dHMNX patients. Although a degenerative motor phenotype is not observed, the knock in Atp7a(T985I/Y) mice show altered Cu levels within the peripheral and central nervous systems, an increased diameter of the muscle fibres and altered myogenin and myostatin gene expression. Atp7a(T985I/Y) mice have reduced Atp7a protein levels and recapitulate the defective trafficking and altered post-translational regulatory mechanisms observed in the human ATP7A(T994I) patient fibroblasts. Our model provides a unique opportunity to characterise the molecular phenotype of dHMNX and the time course of cellular events leading to the process of axonal degeneration in this disease.

Mapping the genotype-phenotype relationship in complex disease.

Computational methods to identify harmful variations in humans perform well for rare diseases such as Huntington's but not for common diseases like hypertension or diabetes. A modelling approach that takes protein context into account was illustrated to identify harmful variants involved in complex diseases.