Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection.

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.

Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing.

Essential role of the Ly49A stalk region for immunological synapse formation and signaling.

NK cells use surface NK receptors to discriminate self from non-self. The NK receptor ligand-binding domain (NKD) has been considered the sole regulator of ligand binding. Using a prototypic murine NK receptor, Ly49A, we show that the membrane proximal nonligand binding ecto-domain (the stalk region) is critical to ligand binding and signaling. The stalk region is required for receptor binding to ligand on target cells (trans interaction), but is dispensable for receptor binding to ligand on the same cell (cis interaction). Also, signaling in a trans manner depends on the stalk region mediating the formation of the immunological synapse. Thus, our data modeling receptor function at the cellular level reveal an essential role for the stalk region as a specific mediator of receptor signal integration, by which NKD-ligand interactions at the interface initiate and deliver information to the spatially separated cytoplasmic domain.

Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses.

Advanced age is associated with a low-grade, systemic inflammatory response characterized by increased inflammatory cytokine production both in vitro and in vivo, termed inflamm-aging. It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Here we show that peripheral blood mononuclear cells (PBMC) from old horses compared to young horses have increased inflammatory cytokine production; moreover, fat old horses compared to thin old horses have even greater frequencies of lymphocytes and monocytes producing inflammatory cytokines. Therefore, we proposed that decreasing adiposity in old horses would reduce age-associated increases of inflammatory cytokines both in vitro and in vivo, and increasing adiposity in old horses would increase these measurements. To test this hypothesis further, eight old obese horses (20-28 year) were assigned to two consecutive treatments, dietary restriction (DR) during weeks 1-12 and increased dietary intake (DI) during weeks 13-30. Body weight, body condition score (BCS) and percent body fat were measured weekly. PBMC were stimulated in vitro and interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) production was measured by intracellular staining. Levels of nascent IFNgamma and TNFalpha mRNA expression were examined by RT-PCR. Serum concentrations of TNFalpha protein were also measured weekly. Reducing body weight and fat in old horses significantly reduced the percent of IFNgamma and TNFalpha positive lymphocytes and monocytes, and serum levels of TNFalpha protein. Further, when weight and fat increased in these old horses there was a significant increase in inflammatory cytokine production. Regression analysis also revealed significant relationships. These findings demonstrate that age-related obesity potentially plays a role in the dysregulation of inflammatory cytokine production by the immune system with age or inflamm-aging in the horse.

The effect of age and telomere length on immune function in the horse.

Telomeres, specialized structures present at the ends of linear eukaryotic chromosomes, function to maintain chromosome stability and integrity. Telomeres shorten with each cell division eventually leading to replicative senescence, a process thought to be associated with age-related decline in immune function. We hypothesized that shortened PBMC telomere length is a factor contributing to immunosenescence of the aged horse. Telomere length was assessed in 19 horses ranging in age from 1 to 25 years. Mitogen-induced 3H-thymidine incorporation, total serum IgG, and pro-inflammatory cytokine expression was also determined for each horse. Relative telomere length (RTL) was highly correlated with overall age. RTL was positively correlated with 3H-thymidine incorporation and total IgG. Expression of pro-inflammatory cytokines was negatively correlated with RTL. These measures were also correlated with age, as expected. However, RTL was not correlated with immunosenescence and inflammaging in the oldest horse.