Phospholipase Cγ2 plays a role in TCR signal transduction and T cell selection.

One of the important signaling events following TCR engagement is activation of phospholipase Cγ (PLCγ). PLCγ has two isoforms, PLCγ1 and PLCγ2. It is known that PLCγ1 is important for TCR signaling and TCR-mediated T cell selection and functions, whereas PLCγ2 is critical for BCR signal transduction and BCR-mediated B cell maturation and functions. In this study, we report that PLCγ2 was expressed in primary T cells, and became associated with linker for activated T cells and Src homology 2-domain containing leukocyte protein of 76 kDa and activated upon TCR stimulation. PLCγ1/PLCγ2 double-deficient T cells displayed further block from CD4 and CD8 double-positive to single-positive transition compared with PLCγ1 single-deficient T cells. TCR-mediated proliferation was further impaired in PLCγ1/PLCγ2 double-deficient T cells compared with PLCγ1 single-deficient T cells. TCR-mediated signal transduction, including Ca²âº mobilization and Erk activation, was further impaired in PLCγ1/PLCγ2 double-deficient relative to PLCγ1 single-deficient T cells. In addition, in HY TCR transgenic mouse model, thymic positive and negative selections were reduced in PLCγ1 heterozygous- and PLCγ2 homozygous-deficient (PLCγ1⁺/⁻PLCγ2⁻/⁻) relative to wild-type, PLCγ2 single-deficient (PLCγ2⁻/⁻), or PLCγ1 heterozygous-deficient (PLCγ1⁺/⁻) mice. Taken together, these data demonstrate that PLCγ2 participates in TCR signal transduction and plays a role in T cell selection.

A critical role of TAK1 in B-cell receptor-mediated nuclear factor kappaB activation.

The kinase TAK1 is essential for T-cell receptor (TCR)-mediated nuclear factor kappaB (NF-kappaB) activation and T-cell development. However, the role of TAK1 in B-cell receptor (BCR)-mediated NF-kappaB activation and B-cell development is not clear. Here we show that B-cell-specific deletion of TAK1 impaired the transition from transitional type 2 to mature follicular (FO) B cells and caused a marked decrease of marginal zone (MZ) B cells. TAK1-deficient B cells exhibited an increase of BCR-induced apoptosis and impaired proliferation in response to BCR ligation. Importantly, TAK1-deficient B cells failed to activate NF-kappaB after BCR stimulation. Thus, TAK1 is critical for B-cell maturation and BCR-induced NF-kappaB activation.

The Role of Permission, Supervision, and Precipitating Events in Childhood Pool/Spa Submersion Incidents, United States, 2000-2017.

Drowning is a leading cause of fatality among children in the United States, and pool/spa aquatic structures represent common locations of submersion incidents. This study employed narrative case review to understand characteristics related to permission, supervision, and precipitating events in childhood submersion incidents. Retroactive analysis of 1537 fatal and non-fatal submersion incidents among children age 13 years old and younger was conducted using the U.S. Consumer Products Safety Commission In-Depth Investigations dataset from 2000-2017. Narrative descriptions were coded according to the themes of permission, supervision, and precipitating events. In most (86%) incidents, the child did not have permitted water access, and 80% of narratives indicated the child was alone at time of incident. These attributes were significantly associated with a fatal outcome (No permission: OR 11.98, 95% CI 7.97-18.06; Alone: OR 34.93, 95% CI 19.69-61.96). The average length of inactive supervision time was 15.6 min; this duration significantly differed by non-fatal (3.2 min) and fatal (16.1 min) outcomes (p < 0.001). More than half of cases occurred under the supervision type of a parent (56%), followed by grandparents (14%) and childcare provider (10%). Submersion incidents with a non-parent supervisor were two times more likely to result in a fatal outcome (OR 1.87, 95% CI 1.07-3.64). The most frequently occurring precipitating events included outdoor play (46%), a social gathering (36%), and previous water play (15%). Narrative excerpts further illustrate how tragic submersion events can unfold quickly and unpredictably. Education campaigns should target all adults that supervise children and reiterate key findings in that many submersion incidents occur (1) without permitted pool use, (2) without active supervision, and (3) when a caregiver is distracted. Multiple strategies should be utilized to add layers of projection against submersion injury.

Distinct roles of phosphoinositide-3 kinase and phospholipase Cgamma2 in B-cell receptor-mediated signal transduction.

During B-cell receptor (BCR) signaling, phosphoinositide-3 kinase (PI3K) is thought to function upstream of phospholipase Cgamma2 (PLCgamma2). PLCgamma2 deficiency specifically impedes transitional type 2 (T2) to follicular (FO) mature B-cell transition. Here, we demonstrate that PI3K deficiency specifically impaired T2-to-FO mature B-cell transition and marginal zone B-cell development. Furthermore, we investigated the functional relationship between PI3K and PLCgamma2 using PI3K-/-, PLCgamma2-/-, and PI3K-/- PLCgamma2-/- B cells. Interestingly, PLCgamma2 deficiency had no effect on BCR-mediated PI3K activation, whereas PI3K deficiency only partially blocked activation of PLCgamma2. Moreover, whereas PI3K-/- PLCgamma2-/- double deficiency did not affect hematopoiesis, it resulted in embryonic lethality. PI3K-/- PLCgamma2-/- fetal liver cells transplanted into B-cell null JAK3-/- mice failed to restore development of peripheral B cells and failed to progress through early B-cell development at the pro-B- to pre-B-cell transition, a more severe phenotype than was observed with either PI3K or PLCgamma2 single-deficiency B cells. Consistent with this finding, BCR signaling was more severely impaired in the absence of both PI3K and PLCgamma2 genes than in the absence of either one alone. Taken together, these results demonstrate that whereas PI3K functions upstream of PLCgamma2, activation of PLCgamma2 can occur independently of PI3K and that PI3K and PLCgamma2 also have distinct functions in BCR signal transduction.

Essential role of phospholipase C gamma 2 in early B-cell development and Myc-mediated lymphomagenesis.

Phospholipase Cgamma2 (PLCgamma2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCgamma2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCgamma2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCgamma2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCgamma2(-/-) Emu-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Emu-Myc transgenics. Furthermore, lymphomas from PLCgamma2(-/-) Emu-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCgamma2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

Activin-A and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis.

The use of human pluripotent cell progeny for cardiac disease modeling, drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success, recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR, which in turn induces the Activin-A and Bmp pathways, is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis, and to ultimately promote myocyte maturation, we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq, induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors, followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm, whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency, even when CHIR alone was ineffective. Moreover, co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis, as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast, co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings, which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression, are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages, while Bmp levels regulate subsequent allocation into mesodermal cell types.

Inhibition of Listeria monocytogenes in pH-adjusted pasteurized liquid whole egg.

Although the transmission of L. monocytogenes to humans via pasteurized egg products has not been documented, L. monocytogenes and other Listeria species have been isolated from commercially broken raw liquid whole egg (LWE) in both the United States and Ireland. Recent Listeria thermal inactivation studies indicate that conventional minimal egg pasteurization processes would effect only a 2.1- to 2.7-order-of-magnitude inactivation of L. monocytogenes in LWE; thus, the margin of safety provided by conventional pasteurization processes is substantially smaller for L. monocytogenes than for Salmonella species (a 9-order-of-magnitude process). The objective of this study was to evaluate the inhibitory effects of nisin on the survival and growth of L. monocytogenes in refrigerated and pH-adjusted (pH 6.6 versus pH 7.5) ultrapasteurized LWE and in a liquid model system. The addition of nisin (1,000 IU/ml) to pH-adjusted ultrapasteurized LWE reduced L. monocytogenes populations by 1.6 to > 3.3 log CFU/ml and delayed (pH 7.5) or prevented (pH 6.6) the growth of the pathogen for 8 to 12 weeks at 4 and 10 degrees C. Bioactive nisin was detected in LWE at both pH values for 12 weeks at 4 degrees C. In subsequent experiments, Listeria reductions of > 3.0 log CFU/ml were achieved within 24 h in both LWE and broth plus nisin (500 IU/ml) at pH 6.6 but not at pH 7.5, and antilisterial activity was enhanced when nisin was added as a solution rather than in dry form.

Phospholipase C{gamma}1 is essential for T cell development, activation, and tolerance.

Phospholipase Cgamma1 (PLCgamma1) is an important signaling effector of T cell receptor (TCR). To investigate the role of PLCgamma1 in T cell biology, we generated and examined mice with T cell-specific deletion of PLCgamma1. We demonstrate that PLCgamma1 deficiency affects positive and negative selection, significantly reduces single-positive thymocytes and peripheral T cells, and impairs TCR-induced proliferation and cytokine production, and the activation of ERK, JNK, AP-1, NFAT, and NF-kappaB. Importantly, PLCgamma1 deficiency impairs the development and function of FoxP3(+) regulatory T cells, causing inflammatory/autoimmune symptoms. Therefore, PLCgamma1 is essential for T cell development, activation, and tolerance.

Thermal Resistance of Salmonella spp. and Listeria monocytogenes in Liquid Egg Yolk and Egg White †.

Decimal reduction times (D values) were determined for Salmonella spp. and Listeria monocytogenes (five pooled strains per pathogen) in raw liquid egg yolk (pH 6.3) and liquid egg white (pH 8.2 versus 9.1) by using a low-volume (0.05 ml per sample) immersed sealed-glass capillary-tube procedure. For Salmonella , D values ranged from 0.087 min (at 62.2°C) to 0.28 min (at 60°C) in yolk. and from 1.00 min (at 58.3°C) to 7.99 min (at 55.1° C) in egg white (pH 8.2). For Listeria , D values ranged from 0.58 min (at 62.2°C) to 1.34 min (at 60°C) in yolk, and from 2.41 min (at 58.3°C) to 7.59 min (at 55. 1°C) in egg white (pH 9.1). Mean ZD values for Salmonella ranged from 3.54 to 4.33°C; for Listeria , ZD values ranged from 6.06 to 9.43°C. In egg white, the heat sensitivity of both pathogens was enhanced at pH 9.1, although this trend was more evident for Salmonella spp. than for L. monocytogenes over the temperature range tested. The results indicate that USDA-prescribed minimal pasteurization requirements for liquid egg yolk (equivalent to 3.9- to 22.1-D processes, on the basis of the present study) would be far more lethal to the Salmonella and L. monocytogenes strains tested than would the corresponding thermal processes for liquid egg white (equivalent to 0.7- to 2.2-D processes).

Thermal Resistance of Aeromonas hydrophila in Liquid Whole Egg †.

Aeromonas hydrophila (AH) is a psychrotrophic spoilage bacterium and potential pathogen which has been isolated from a variety of refrigerated foods of animal origin, including raw milk, red meat, poultry, and commercially broken raw liquid whole egg (LWE). Decimal reduction times (D values) of 4 strains of AH (1 egg isolate, 2 egg processing plant isolates, 1 ATCC type strain) were determined in LWE using an immersed sealed capillary tube (ISCT) procedure. Initial populations (7.0 to 8.3 log CFU/tube in 0.05 ml LWE) were heated at 48, 51, 54, 57, and 60°C, and survivors were plated onto starch ampicillin agar (48 h at 28°C). D values ranged from 3.62 to 9.43 min (at 48°C) to 0.026 to 0.040 min (at 60°C). Both processing plant isolates were more heat resistant than the ATCC strain. Decimal reduction time curves (r2 ≤ 0.98) yielded ZD values of 5.02 to 5.59°C, similar to those for other non-spore-forming bacteria. D values of the most heat resistant AH strain were also determined in LWE at 48, 51, and 54°C using a conventional capped test tube procedure (10 ml/tube). Cells heated in test tubes yielded nonlinear (tailing) survivor curves and larger (P ≤ 0.05) apparent D values at each temperature than those obtained using the ISCT method. This study provides the first thermal resistance data for AH in LWE and the first evidence that straight-line semilogarithmic thermal inactivation kinetics may be demonstrated for Aeromonas using the ISCT procedure.

Identification of Shp-2 as a Stat5A phosphatase.

Stat5A, a member of the signal transducers and activators of transcription (Stat) family, is activated upon a single tyrosine phosphorylation. Although much is known about the activation process, the mechanism by which the tyrosine-phosphorylated Stat5A proteins are inactivated is largely unknown. In this report, we demonstrate that down-regulation of the tyrosine-phosphorylated Stat5A was via dephosphorylation. Using tyrosine-phosphorylated peptides derived from Stat5A, we were able to purify protein-tyrosine phosphatase Shp-2 from cell lysates. Shp-2, but not Shp-1, specifically interacted with Stat5A in vivo, and the interaction was tyrosine phosphorylation-dependent. Moreover, Shp-2 was able to accelerate Stat5A dephosphorylation, and dephosphorylation of Stat5A was dramatically delayed in Shp-2-deficient cells. Therefore, we conclude that Shp-2 is a Stat5A phosphatase, which down-regulates the active Stat5A in vivo.

Phospholipase Cgamma2 provides survival signals via Bcl2 and A1 in different subpopulations of B cells.

PLCgamma2 plays a critical role in B cell receptor (BCR) signaling and its targeted deletion results in defective B cell development and function. Here, we show that PLCgamma2 deficiency specifically blocks B cell maturation at the transitional type 2 (T2) to follicular (FO) B cell transition and the PLCgamma2 pathway regulates survival of B cells. BCR-induced apoptosis is dramatically enhanced in all subsets of splenic PLCgamma2-deficient B cells, especially in T2 and FO B cell subpopulations. We also find that all splenic PLCgamma2-deficient B cell subpopulations express abnormally low levels of Bcl-2 protein. In addition, PLCgamma2 deficiency disrupts BCR-mediated induction of A1 expression. Enforced expression of Bcl-2 prevents BCR-induced apoptosis in all splenic PLCgamma2-deficient B cell subpopulations and partially restores the numbers of PLCgamma2-deficient FO B cells. In contrast to Bcl-2, enforced expression of A1 preferentially prevents BCR-induced apoptosis in PLCgamma2-deficient FO B cells and partially restores the numbers of these B cells. Therefore, the PLCgamma2 pathway provides a survival signal via regulation of Bcl-2 in all splenic B cell subpopulations and via additional induction of A1 in mature FO B cells.

An important role of phospholipase Cgamma1 in pre-B-cell development and allelic exclusion.

Phospholipase Cgamma1 (PLCgamma1) has been reported to be expressed predominantly in T cells and to play an important role in T-cell receptor signaling. Here we show that PLCgamma1 is expressed throughout B-cell development, with high expression in B-cell progenitors, and is involved in pre-B-cell receptor (pre-BCR) signaling. Reduced expression of PLCgamma1, in the absence of PLCgamma2 (PLCgamma1+/-PLCgamma2-/-), impedes early B-cell development at the pro-B- to pre-B-cell transition and impairs immunoglobulin heavy chain allelic exclusion, hallmarks of defective pre-BCR signaling. In contrast, early B-cell development is largely normal, whereas late B-cell maturation is impaired in the absence of PLCgamma2 alone (PLCgamma2-/-) and overexpression of PLCgamma1 in PLCgamma2-/- mice fails to restore BCR-mediated B-cell proliferation and maturation. These studies reveal an essential role of PLCgamma1, distinct from that of PLCgamma2, in B-cell development.

The Relationship Between Botulinal Toxin Production and Spoilage of Fresh Tomatoes Held at 13 and 23°C Under Passively Modified and Controlled Atmospheres and Air.

The formation of botulinal toxin relative to spoilage of fresh whole tomatoes was investigated at 13 and 23°C under passively modified (MA) and controlled atmospheres (CA) and air. Tomatoes were subsurface inoculated with a composite of type A and proteolytic and nonproteolytic type B strains of Clostridium botulinum spores. Some were also inoculated with Alternaria mold spores. MA (1.0-2.9% O2) was passively established by a combination of product respiration and package permeability. CA was established by placing tomatoes in continuously flushed (1% O2, 20% CO2, balance N2) Plexiglass plastic containers. Tomatoes were tested for botulinum toxin by the mouse assay at the time when they first became inedible based on predefined stages of decay rather than specific storage times in order to determine the relationship between spoilage and botulinal toxigenesis. All tomatoes became inedible according to the established criteria within 17 to 46 d depending on the storage temperature and atmosphere. Botulinum toxin was not detected in the 24 composite samples of inedible tomatoes (representing 99 tomatoes) which were tested at the time they first became inedible. Toxin was detected in four of five additional composite samples (representing 10 tomatoes) which were held 2 to 9 d beyond the time they were first determined to be inedible. These data indicate that MA-packaged tomatoes can become toxic but only after becoming severely spoiled beyond the point of being organoleptically acceptable. The risk of botulism from consumption of extended shelf life whole tomatoes appears to be insignificant.