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Neonates are at an increased risk of an infectious disease. This is consistent with an increased abundance of myeloid-derived suppressor cells (MDSCs) compared with older children and adults. Using a murine model of neonatal bacterial sepsis, we demonstrate that MDSCs modulate their activity during an infection to enhance immune suppressive functions. A gene expression analysis shows that MDSCs increased NOS2, Arg-1 and IL-27p28 expression in vitro and in vivo in response to Escherichia coli O1:K1:H7 and this is regulated at the level of the gene expression. Changes in the effector gene expression are consistent with increased enzymatic activity and cytokine secretion. The neonatal MDSCs express toll-like receptor (TLR) 2, 4 and 5 capable of recognizing pathogen-associated molecular patterns (PAMPS) on E. coli. However, a variable level of effector expression was achieved in response to LPS, peptidoglycan or flagellin. Individual bacterial PAMPs did not stimulate the expression of Arg-l and IL-27p28 equivalently to E. coli. However, the upregulation of NOS2 was achieved in response to LPS, peptidoglycan and flagella. The increased immune suppressive profile translated to an enhanced suppression of CD4+ T cell proliferation. Collectively, these findings increase our understanding of the dynamic nature of MDSC activity and suggest that these cells abundant in early life can acquire activity during an infection that suppresses protective immunity.

Atrazine and its metabolites are present at high concentrations in many water supplies in agro-intensive areas. Because residents in these areas drink water from sources fed from these contaminated supplies, we investigated the long-term immunotoxicity of combined prenatal and neonatal (perinatal) exposure to atrazine via drinking water, on the immune system in mice. At 6 months of age, upon immunization with heat-killed Streptococcus pneumoniae, the serum IgG antibody response against the T independent antigen phosphorylcholine was significantly higher in male, but not female, atrazine-exposed mice as compared with that in untreated controls. No alterations were present in all offspring in the serum antibody response against the T-dependent antigen pneumococcal surface protein A (PspA). ELISpot analysis showed only a small, insignificant reduction in PspA-specific IgG producing splenocytes in atrazine-treated male offspring. Interestingly, upon ex vivo stimulation with anti-CD3 and anti-CD28 antibodies, significant decreases in interleukin (IL)-2, tumor necrosis factor-α, interferon-γ, and IL-17A and a decreasing trend in IL-10 were observed in splenocytes from atrazine-exposed male, but not female mice. Analysis of thymic and splenic cell populations showed no effects of atrazine exposure in either sex. This is the first time that long-term changes in the immune response were observed after a perinatal exposure to atrazine and it demonstrates that these early life exposures can result in permanent changes to the immune system as well as a male bias in these effects.

KEY POINTS: Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. Using a diabetic rat model, we identified a >130‐fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. Using combined single microvessel perfusion and systemic cross‐transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP‐mediated pathogenesis of diabetes‐associated multi‐organ microvascular dysfunction. ABSTRACT: In various cardiovascular diseases, microparticles (MPs), the membrane‐derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease‐induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin‐induced diabetic rat model with well‐characterized microvascular functions. Our study revealed a >130‐fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre‐coated with a lipid‐binding protein, annexin V, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi‐organ vascular dysfunction that commonly occurs in diabetic patients.

Background Missense mutations in the hyperpolarization‐activated cyclic nucleotide‐modulated (HCN) channel 4 (HCN4) are one of the genetic causes of cardiac sinus bradycardia. Objective To investigate possible HCN4 channel mutation in a young patient with profound sinus bradycardia. Methods Direct sequencing of HCN4 and whole‐exome sequencing were performed on DNA samples from the indexed patient (P), the patient's son (PS), and a family unrelated healthy long‐distance running volunteer (V). Resting heart rate was 31 bpm for P, 67 bpm for PS, and 50 bpm for V. Immunoblots, flow cytometry, and immunocytofluorescence confocal imaging were used to study cellular distribution of channel variants. Patch‐clamp electrophysiology was used to investigate the properties of mutant HCN1 channels. Results In P no missense mutations were found in the HCN4 gene; instead, we found two heterozygous variants in the HCN1 gene: deletion of an N‐terminal glycine triplet (72GGG74, “N‐del”) and a novel missense variant, P851A, in the C‐terminal region. N‐del variant was found before and shared by PS. These two variations were not found in V. Compared to wild type, N‐del and P851A reduced cell surface expression and negatively shifted voltage‐activation with slower activation kinetics. Conclusion Decreased channel activity HCN1 mutant channel makes it unable to contribute to early depolarization of sinus node action potential, thus likely a main cause of the profound sinus bradycardia in this patient., HCN4 cardiac pacemaker channel mutations are one of the genetic mechanisms for sinus bradycardia. We identified a new variant in a different pacemaker channel, HCN1, in a patient of profound sinus bradycardia.

Background Unlike other breast cancer subtypes that may be treated with a variety of hormonal or targeted therapies, there is a need to identify new, effective targets for triple-negative breast cancer (TNBC). It has recently been recognized that membrane potential is depolarized in breast cancer cells. The primary objective of the study is to explore whether hyperpolarization induced by opening potassium channels may provide a new strategy for treatment of TNBC. Methods Breast cancer datasets in cBioPortal for cancer genomics was used to search for ion channel gene expression. Immunoblots and immunohistochemistry were used for protein expression in culture cells and in the patient tissues. Electrophysiological patch clamp techniques were used to study properties of BK channels in culture cells. Flow cytometry and fluorescence microscope were used for cell viability and cell cycle studies. Ultrasound imaging was used to study xenograft in female NSG mice. Results In large datasets of breast cancer patients, we identified a gene, KCNMA1 (encoding for a voltage- and calcium-dependent large-conductance potassium channel, called BK channel), overexpressed in triple-negative breast cancer patients. Although overexpressed, 99% of channels are closed in TNBC cells. Opening BK channels hyperpolarized membrane potential, which induced cell cycle arrest in G2 phase and apoptosis via caspase-3 activation. In a TNBC cell induced xenograft model, treatment with a BK channel opener significantly slowed tumor growth without cardiac toxicity. Conclusions Our results support the idea that hyperpolarization induced by opening BK channel in TNBC cells can become a new strategy for development of a targeted therapy in TNBC.

Cells isolated using electrostatic cell sorters are subsequently evaluated in a variety of in vitro and in vivo applications. Thus, manipulations to the cells during the pre- and post-sort processing as well as when the cells are being analyzed by and passing through the sorter fluidics has the potential to affect the experimental results. There are many variables to consider when seeking to preserve cellular integrity and function during the cell-sorting process. A previous study by the Association of Biomolecular Resource Facilities Flow Cytometry Research Group (FCRG) investigated downstream effects on different cell types as a function of sorting variables such as pressure, nozzle size, and temperature. This multisite study revealed site-to-site variability based on differential gene expression when the same cell type and sort conditions were used. These results indicated the possibility that environmental factors such as the presence of contaminants in the sorter fluidics could exhibit effects on downstream molecular assays (ie, endotoxins or RNases). In the study described here, the FCRG sought to better understand how sorters are maintained and evaluated for contaminants such as bacteria, endotoxin, and RNases. In addition, the efficacy of an endotoxin decontamination method was evaluated. The results demonstrated that the majority of sorters in shared resource laboratories are free of RNase activity and bacteria; however, many are contaminated with endotoxin. The efficacy of a hydrogen peroxide cleaning procedure was tested and found to exhibit only a short-term effectiveness in eliminating endotoxin contamination.

BACKGROUND: Telomere length (TL), telomerase activity (TA), and plasma amyloid-β(Aβ) levels have emerged as possible predictors of cognitive decline and dementia. OBJECTIVE: To assess the: 1) effects of two 12-week relaxation programs on TL, TA, and Aβ levels in adults with subjective cognitive decline; and 2) relationship of biomarker changes to those in cognitive function, psychosocial status, and quality of life (QOL). METHODS: Participants were randomized to a 12-week Kirtan Kriya meditation (KK) or music listening (ML) program and asked to practice 12 minutes/day. Plasma Aβ(38/40/42) and peripheral blood mononuclear cell TL and TA were measured at baseline and 3 months. Cognition, stress, sleep, mood, and QOL were assessed at baseline, 3 months, and 6 months. RESULTS: Baseline blood samples were available for 53 participants (25 KK, 28 ML). The KK group showed significantly greater increases in Aβ(40) than the ML group. TA rose in both groups, although increases were significant only among those with higher practice adherence and lower baseline TA. Changes in both TL and TA varied by their baseline values, with greater increases among participants with values ≤50th percentile (ps-interaction

Background Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. Methods We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. Results Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. Conclusions We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.

Purpose Graft-versus-host disease (GVHD) is major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Atorvastatin is a potent immunomodulatory agent that holds promise as a novel and safe agent for acute GVHD prophylaxis. Patients and Methods We conducted a phase II trial to evaluate the safety and efficacy of atorvastatin administration for GVHD prophylaxis in both adult donors and recipients of matched sibling allogeneic HCT. Atorvastatin (40 mg per day orally) was administered to sibling donors, starting 14 to 28 days before the anticipated first day of stem-cell collection. In HCT recipients (n = 30), GVHD prophylaxis consisted of tacrolimus, short-course methotrexate, and atorvastatin (40 mg per day orally). Results Atorvastatin administration in healthy donors and recipients was not associated with any grade 3 to 4 adverse events. Cumulative incidence rates of grade 2 to 4 acute GVHD at days +100 and +180 were 3.3% (95% CI, 0.2% to 14.8%) and 11.1% (95% CI, 2.7% to 26.4%), respectively. One-year cumulative incidence of chronic GVHD was 52.3% (95% CI, 27.6% to 72.1%). Viral and fungal infections were infrequent. One-year cumulative incidences of nonrelapse mortality and relapse were 9.8% (95% CI, 1.4% to 28%) and 25.4% (95% CI, 10.9% to 42.9%), respectively. One-year overall survival and progression-free survival were 74% (95% CI, 58% to 96%) and 65% (95% CI, 48% to 87%), respectively. Compared with baseline, atorvastatin administration in sibling donors was associated with a trend toward increased mean plasma interleukin-10 concentrations (5.6 v 7.1 pg/mL; P = .06). Conclusion A novel two-pronged strategy of atorvastatin administration in both donors and recipients of matched sibling allogeneic HCT seems to be a feasible, safe, and potentially effective strategy to prevent acute GVHD.

Sensory neurons originating in nodose and jugular ganglia that innervate airway epithelium (airway neurons) play a role in inflammation observed following exposure to inhaled environmental irritants such as ozone (O(3)). Airway neurons can mediate airway inflammation through release of the neuropeptide substance P (SP). While susceptibility to airway irritants is increased in early life, the developmental dynamics of afferent airway neurons are not well characterized. The hypothesis of this study was that airway neuron number might increase with increasing age, and that an acute, early postnatal O(3) exposure might increase both the number of sensory airway neurons as well as the number SP-containing airway neurons. Studies using Fischer 344 rat pups were conducted to determine if age or acute O(3) exposure might alter airway neuron number. Airway neurons in nodose and jugular ganglia were retrogradely labeled, removed, dissociated, and counted by means of a novel technique employing flow cytometry. In Study 1, neuron counts were conducted on postnatal days (PD) 6, 10, 15, 21, and 28. Numbers of total and airway neurons increased significantly between PD6 and PD10, then generally stabilized. In Study 2, animals were exposed to O(3) (2 ppm) or filtered air (FA) on PD5 and neurons were counted on PD10, 15, 21, and 28. O(3) exposed animals displayed significantly less total neurons on PD21 than FA controls. This study shows that age-related changes in neuron number occur, and that an acute, early postnatal O(3) exposure significantly alters sensory neuron development.

Cadmium (Cd) is both an environmental pollutant and a component of cigarette smoke. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/beta-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. This study was designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/beta-catenin pathways. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose (10 ppm) of Cd throughout pregnancy and effects on the thymus were assessed on the day of birth. Thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways were assessed using real-time RT-PCR and western blot, respectively. Prenatal Cd exposure increased the number of CD4(+) cells and a subpopulation of double-negative cells (DN; CD4(-)CD8(-)), DN4 (CD44(-)CD25(-)). Shh and Wnt/beta-catenin signaling were both decreased in the thymus. Target genes of Shh (Patched1 and Gli1) and Wnt/beta-catenin (c-fos, and c-myc) were affected differentially among thymocyte subpopulations. These findings suggest that prenatal exposure to Cd dysregulates two signaling pathways in the thymus, resulting in altered thymocyte development.

3,4-Dichloropropionanilide (DCPA), or propanil, a post-emergent herbicide used on rice and wheat crops in the United States, is immunotoxic in vivo and in vitro. Although it has been documented that DCPA exerts differential effects on specific immune cell types and is toxic to the liver, the way in which DCPA modulates intracellular functions leading to these effects is less understood. In this study, Jurkat T cells and hepatocytes from C57Bl/6 mice were exposed to 100 microM DCPA for 1.5 h. Following incubation, subcellular fractions of each cell type were isolated. DCPA, when present, was removed from each cell fraction by liquid-liquid extraction. The extraction product was then analyzed for the presence of DCPA using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cellular uptake of DCPA was monitored by detection of the molecular ion and product ion of DCPA. The analyses demonstrate that DCPA, a lipophilic compound, localizes primarily in the cytosol of T cells and hepatocytes. These results indicate that DCPA is able to cross the plasma membrane and is accessible to intracellular immunomodulatory effectors.

Flow cytometry is one of the fundamental research tools available to the life scientist. The ability to observe multidimensional changes in protein expression and activity at single-cell resolution for a large number of cells provides a unique perspective on the behavior of cell populations. However, the analysis of complex multidimensional data is one of the obstacles for wider use of polychromatic flow cytometry. Recent enhancements to an open-source platform-R/Bioconductor-enable the graphical and data analysis of flow cytometry data. Prior examples have focused on high-throughput applications. To facilitate wider use of this platform for flow cytometry, the analysis of a dataset, obtained following isolation of CD4(+)CD62L(+) T cells from Balb/c splenocytes using magnetic microbeads, is presented as a form of tutorial. A common workflow for analyzing flow cytometry data was presented using R/Bioconductor. In addition, density function estimation and principal component analysis are provided as examples of more complex analyses. The compendium presented here is intended to help illuminate a path for inquisitive readers to explore their own data using R/Bioconductor (available as Supporting Information).

The pesticide 3,4-dichloropropionanilide (propanil or, alternatively, DCPA) is a member of the acetanilide chemical family and is predominantly used for the control of weeds on commercial rice crops worldwide. This article was written to provide a brief review of the general toxicity of propanil followed by a detailed summary of the immunotoxicity studies that were performed to date in mammalian in vivo and in vitro models. Propanil affects the immune system at organ, cellular, and molecular levels. Studies demonstrated that it produces thymic atrophy and splenomegaly and decreases developing T- and B-cell populations in the thymus and bone marrow. Natural killer (NK) cells and macrophages are critical components of the innate immune system. NK cell cytotoxicity and the ability of macrophages to phagocytose, kill pathogenic bacteria, and produce inflammatory cytokines are suppressed by propanil. Propanil also affects the respiratory burst of macrophages, inhibiting reactive oxygen and nitrogen species production. Molecular mechanisms responsible for propanil's effects have begun to be elucidated and include alterations in nuclear factor (NF)-kappaB transcription factor activity and intracellular Ca(2+) signaling. Propanil exposure alters a number of functions of mature T lymphocytes and B lymphocytes that impacts the adaptive immune response. T-cell cytotoxic activity and cytokine production are major T-cell functions inhibited by propanil. The humoral antibody response to model antigens and intact bacteria is differentially affected after propanil exposure. How these changes in innate and adaptive immune responses impact the host response to bacterial challenge or vaccination has begun to be examined.

The activation of transcription factor NF-kappaB (nuclear factor-kappaB) plays a central role in the induction of many inflammatory response genes. This process is characterized by either oscillations or stable induction of NF-kappaB nuclear binding. Changes in dynamics of binding result in the expression of distinct subsets of genes leading to different physiological outcomes. We examined NF-kappaB DNA binding activity in lipopolysaccharide (LPS)-stimulated IC-21 cells by electromobility shift assay and nonradioactive transcription factor assay and interpreted the results using a kinetic model of NF-kappaB activation. Both assays detected damped oscillatory behavior of NF-kappaB with differences in sensitivity and reproducibility. 3,4-Dichloropropionaniline (DCPA) was used to modulate the oscillatory behavior of NF-kappaB after LPS stimulation. DCPA is known to inhibit the production of two NF-kappaB-inducible cytokines, IL-6 and tumor necrosis factor alpha, by reducing but not completely abrogating NF-kappaB-induced transcription. DCPA treatment resulted in a potentiation of early LPS-induced NF-kappaB activation. The nonradioactive transcription factor assay, which has a higher signal/noise ratio than the electromobility shift assay, combined with in silico modeling, produced results that revealed changes in NF-kappaB dynamics which, to the best of our knowledge, have never been previously reported. These results highlight the importance of cell type and stimulus specificity in transcription factor activity assessment. In addition, assay selection has important implications for network inference and drug discovery.

Stimulation of T cells through the T-cell receptor results in the activation of a series of signaling pathways that leads to the secretion of interleukin (IL)-2 and cell proliferation. Influx of calcium (Ca(2+)) from the extracellular environment, following internal Ca(2+) store depletion, provides the elevated and sustained intracellular calcium concentration ([Ca(2+)](i)) critical for optimal T-cell activation. Our laboratory has documented that exposure to the herbicide 3,4-dichloropropionanilide (DCPA) inhibits intracellular signaling events that have one or more Ca(2+) dependent steps. Herein we report that DCPA attenuates the normal elevated and sustained [Ca(2+)](i) that follows internal store depletion in the human leukemic Jurkat T cell line and primary mouse T cells. DCPA did not alter the depletion of internal Ca(2+) stores when stimulated by anti-CD3 or thapsigargin demonstrating that early inositol 1,4,5-triphosphate-mediated signaling and depletion of Ca(2+) stores were unaffected. 2-Aminoethyldiphenol borate (2-APB) is known to alter the store-operated Ca(2+) (SOC) influx that follows Ca(2+) store depletion. Exposure of Jurkat cells to either DCPA or 50 microM 2-APB attenuated the increase in [Ca(2+)](i) following thapsigargin or anti-CD3 induced store depletion in a similar manner. At low concentrations, 2-APB enhances SOC influx but this enhancement is abrogated in the presence of DCPA. This alteration in [Ca(2+)](i), when exposed to DCPA, significantly reduces nuclear levels of nuclear factor of activated T cells (NFAT) and IL-2 secretion. The plasma membrane polarization profile is not altered by DCPA exposure. Taken together, these data indicate that DCPA inhibits T-cell activation by altering Ca(2+) homeostasis following store depletion.

There is a substantial literature reporting that the developing immune system is more sensitive to toxic insult and that the measurable phenotype resulting from prenatal/neonatal exposure often differs from that seen in adult exposure models (reviewed in Holladay and Steven, and Smialowicz et al.). Atrazine is a common herbicidal contaminant of groundwater in agricultural areas in the USA. The potential immunotoxicity of atrazine has been extensively studied using adult-exposure models; however, few studies have explored its immunotoxicity in a prenatal and/or lactational exposure model. Prenatal/lactational atrazine exposure affects the function of young adult rodent immune systems in both sex- and age-dependant manners. In our studies, the humoural and cell-mediated immune responses of offspring from atrazine-exposed dams were assessed at two ages, 3 and 6 months of age to test the hypothesis that prenatal/lactational atrazine exposure would cause greater health complications as the mice aged. Male offspring showed a significant immunopotentiation at three moa that was not apparent at 6 months. Three-month-old female offspring showed no significant difference in immune response from controls. However, at 6 months, female litter mates showed a significant depression in their immune function. These results indicate a decreasing trend in immune capacity. Rooney et al. showed a significant depression of the immune function of young male rat exposure prenatally and lactationally to atrazine. These results demonstrate a sex- and age-dependant effect of prenatal exposure to atrazine on the immune system of the adult offspring using two rodent strains.

The high mobility group box protein SOX9 and the GLI1 transcription factor play protumorigenic roles in pancreatic ductal adenocarcinoma (PDA). In Kras transgenic mice, each of these factors are crucial for the development of PDA precursor lesions. SOX9 transcription is directly regulated by GLI1, but how SOX9 functions downstream of GLI1 is unclear. We observed positive feedback, such that SOX9-deficient PDA cells have severely repressed levels of endogenous GLI1, attributed to loss of GLI1 protein stability. SOX9 associated with the F-box domain of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase component, β-TrCP (also known as F-box/WD repeat-containing protein 1A), and suppressed its association with SKP1 and GLI1, a substrate of SCF-β-TrCP. SOX9 also tethered β-TrCP within the nucleus and promoted its degradation. SOX9 bound to β-TrCP through the SOX9 C-terminal PQA/S domain that mediates transcriptional activation. Suppression of β-TrCP in SOX9-deficient PDA cells restored GLI1 levels and promoted SOX9-dependent cancer stem cell properties. These studies identify SOX9–GLI1 positive feedback as a major determinant of GLI1 protein stability and implicate β-TrCP as a latent SOX9-bound tumor suppressor with the potential to degrade oncogenic proteins in tumor cells.

Polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA and IgA-coated immune complexes from the lamina propria across epithelia and into secretions. The effect of reovirus infection on regulation of pIgR expression in the human intestinal epithelial cell line HT-29 was characterized in this report. Both replication-competent and UV-inactivated reovirus at m.o.i. equivalents of 1–100 p.f.u. per cell upregulated pIgR mRNA by 24 h post-infection and intracellular pIgR protein was increased at 48 h following exposure to UV-inactivated virus. Binding of virus to HT-29 cells was required, as pre-incubating virus with specific antiserum, but not non-immune serum, inhibited reovirus-mediated pIgR upregulation. Endosomal acidification leading to uncoating of virus is a required step for pIgR upregulation, as ammonium chloride or bafilomycin A1 pre-treatment inhibited virus-induced pIgR upregulation. Inhibition experiments using the calpain inhibitorN-acetyl-leucyl-leucyl-norleucinal suggested that calpains are involved in reovirus-mediated pIgR upregulation. Upregulation of pIgR following virus infection appears to be an innate immune response against invading pathogens that could help the host clear infection effectively. Signalling induced by microbes and their products may serve to augment pIgR-mediated transcytosis of IgA, linking the innate and acquired immune responses to viruses.

Despite the low prevalence of activating point mutation of RAS or RAF genes, the RAS–extracellular signal-regulated kinase (ERK) pathway is implicated in breast cancer pathogenesis. Indeed, in triple-negative breast cancer (TNBC), there is recurrent genetic alteration of pathway components. Using short hairpin RNA (shRNA) methods, we observed that the zinc finger transcription factor Krüppel-like factor 4 (KLF4) can promote RAS-ERK signaling in TNBC cells. Endogenous KLF4 bound to the promoter regions and promoted the expression of two microRNAs (miRs), miR-206 and miR-21 (i.e., miR-206/21). Antisense-mediated knockdown (anti-miR) revealed that miR-206/21 coordinately promote RAS-ERK signaling and the corresponding cell phenotypes by inhibiting translation of the pathway suppressors RASA1 and SPRED1. In TNBC cells, including cells with mutation of RAS, the suppression of either RASA1 or SPRED1 increased the levels of GTP-bound, wild-type RAS and activated ERK 1/2. Unlike the control cells, treatment of RASA1- or SPRED1-suppressed cells with anti-miR-206/21 had little or no impact on the level of activated ERK 1/2 or on cell proliferation and failed to suppress tumor initiation. These results identify RASA1 and SPRED1 mRNAs as latent RAS-ERK pathway suppressors that can be upregulated in tumor cells by anti-miR treatment. Consequently, KLF4-regulated miRs are important for the maintenance of RAS-ERK pathway activity in TNBC cells.

Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2weeks of age. At 7weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4(+)FoxP3(+)CD25(+) (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8(+)CD223(+) T cells were markedly decreased in the spleens in all offspring at 7weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can result in long term detrimental effects on the immune system of the offspring and these effects are to some extent sex-specific.

Publisher Summary This chapter appraises the reaction of the human immune system when it is exposed to different types of pesticides. Chemicals such as pesticides have the potential to alter the immune system, making an individual more susceptible to infection. There are four potential outcomes to pesticide exposure. The first outcome, and the most common, is no alteration to the immune system as a result of pesticide exposure. The second potential outcome is an increase in activation of the immune system with the potential to develop into an autoimmune disease. A third possible outcome of pesticide exposure is a decrease in the immune system activity resulting in immunosuppression. The final potential outcome is the development of hypersensitivity. Depending on the pesticide, these alterations can include a decrease in neutrophil and macrophage function, a decrease in the number of thymocytes, a decrease/increase in mitogen-induced proliferation, a decrease in antibody-dependent cell cytotoxicity, and a decrease/increase in cytokine secretion. As in other types of pesticide-induced toxicities, the route of exposure, length of exposure, dose, and timing of exposure of a pesticide must be taken into consideration when evaluating the potential of a pesticide to be immunotoxic. In addition, these factors can also influence whether a pesticide is immunosuppressive or potentiates the immune system.

Macrophages are the heterogeneous grouping of cells that are derived from monocytes. They have a multitude of functions depending on their final differentiated state. These functions range from phagocytosis to antigen presentation to bone destruction, to name a few. Their importance in both the innate and acquired immune functions is undeniable. Xenobiotics that degrade their functional status can have grave consequences. In this chapter, we provide an overview of the types of macrophages, their hematopoietic origin and a general discussion of the many different assays that are used to assess their functional status.

Macrophages are a heterogeneous group of cells that have a multitude of functions depending on their differentiation state. While classically known for their phagocytic and antigen presentation abilities, it is now evident that these cells fulfill homeostatic functions beyond the elimination of invading pathogens. In addition, macrophages have also been implicated in the downregulation of inflammatory responses following pathogen removal, tissue remodeling, repair, and angiogenesis. Alterations in macrophage differentiation and/or activity due to xenobiotic exposure can have grave consequences on organismal homeostasis, potentially contributing to disease due to immunosuppression or chronic inflammatory responses, depending upon the pathways affected. In this chapter, we provide an overview of the macrophages subtypes, their origin and a general discussion of several different assays used to assess their functional status.

There is a substantial literature reporting that the developing immune system is more sensitive to toxic insult and that the measurable phenotype resulting from prenatal/neonatal exposure often differs from that seen in adult exposure models (reviewed in Holladay and Steven, and Smialowicz et al.). Atrazine is a common herbicidal contaminant of groundwater in agricultural areas in the USA. The potential immunotoxicity of atrazine has been extensively studied using adult-exposure models; however, few studies have explored its immunotoxicity in a prenatal and/or lactational exposure model. Prenatal/lactational atrazine exposure affects the function of young adult rodent immune systems in both sex- and age-dependant manners. In our studies, the humoural and cell-mediated immune responses of offspring from atrazine-exposed dams were assessed at two ages, 3 and 6 months of age to test the hypothesis that prenatal/lactational atrazine exposure would cause greater health complications as the mice aged. Male offspring showed a significant immunopotentiation at three moa that was not apparent at 6 months. Three-month-old female offspring showed no significant difference in immune response from controls. However, at 6 months, female litter mates showed a significant depression in their immune function. These results indicate a decreasing trend in immune capacity. Rooney et al. showed a significant depression of the immune function of young male rat exposure prenatally and lactationally to atrazine. These results demonstrate a sex- and age-dependant effect of prenatal exposure to atrazine on the immune system of the adult offspring using two rodent strains.

Macrophages are a critical part of the innate immune response and natural surveillance mechanisms. As such, proper macrophage function is crucial for engulfing bacterial pathogens through phagocytosis and destroying them by generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The production of a number of cytokines by macrophages, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, plays an important role in the initiation of the acquired immune response creating an inflammatory environment favorable for fighting a bacterial infection. 3,4-Dichloropropionaniline (DCPA) suppresses several inflammatory parameters, including TNF-alpha production through a mechanism where nuclear factor-kappaB (NF-kappaB)-DNA binding is inhibited but not entirely abrogated. The goal of the present study was to evaluate the effects of DCPA on the inflammatory mediators of macrophages, including ROS and RNS in both murine peritoneal exudate cells and the human monocytic cell line, THP-1. The ability to perform phagocytosis and directly kill Listeria monocytogenes was also assessed. The results indicate that DCPA decreases the ability of both types of macrophages to phagocytize beads and generate both types of reactive species, which was correlated with a decrement in listericidal activity. These results demonstrate that DCPA has profound effects on macrophage function and provide insight into the potential mechanisms of immunosuppression by DCPA.

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

Tumor cell heterogeneity poses a major hurdle in the treatment of cancer. Mammary cancer stem-like cells (MaCSCs), or tumor-initiating cells, are highly tumorigenic sub-populations that have the potential to self-renew and to differentiate. These cells are clinically important, as they display therapeutic resistance and may contribute to treatment failure and recurrence, but the signaling axes relevant to the tumorigenic phenotype are poorly defined. The zinc-finger transcription factor Kruppel-like factor 4 (KLF4) is a pluripotency mediator that is enriched in MaCSCs. KLF4 promotes RAS-extracellular signal-regulated kinase pathway activity and tumor cell survival in triple-negative breast cancer (TNBC) cells. In this study, we found that both KLF4 and a downstream effector, microRNA-206 (miR-206), are selectively enriched in the MaCSC fractions of cultured human TNBC cell lines, as well as in the aldehyde dehydrogenase-high MaCSC sub-population of cells derived from xenografted human mammary carcinomas. The suppression of endogenous KLF4 or miR-206 activities abrogated cell survival and in vivo tumor initiation, despite having only subtle effects on MaCSC abundance. Using a combinatorial approach that included in silico as well as loss- and gain-of-function in vitro assays, we identified miR-206-mediated repression of the pro-apoptotic molecules programmed cell death 4 (PDCD4) and connexin 43 (CX43/GJA1). Depletion of either of these two miR-206-regulated transcripts promoted resistance to anoikis, a prominent feature of CSCs, but did not consistently alter MaCSC abundance. Consistent with increased levels of miR-206 in MaCSCs, the expression of both PDCD4 and CX43 was suppressed in these cells relative to control cells. These results identify miR-206 as an effector of KLF4-mediated prosurvival signaling in MaCSCs through repression of PDCD4 and CX43. Consequently, our study suggests that a pluripotency factor exerts prosurvival signaling in MaCSCs, and that antagonism of KLF4-miR-206 signaling may selectively target the MaCSC niche in TNBC.

Atrazine is a widely used herbicide applied to corn, sugar and other crops as a broad leaf weed inhibitor. Using the Balb/c mouse model, we have determined that prenatal/lactational exposure to atrazine alters adult immune function. Pregnant Balb/c dams were exposed subcutaneously for 21 days via time release pellets to 700 μg per day of atrazine beginning between days 10 and 12 of pregnancy. Prenatal/Lactational exposure caused no overt physical malformations in the offspring and had no effect on the number of litters carried to term or the litter size. Upon reaching early adulthood (approximately 3 months of age), the state of their immune system was evaluated. There were no changes in body weight or in the organ to body weight ratio of the spleen. Additionally, no changes were observed in the number of CD8+ T cell, CD4+ T cell, or B220+ B cell subpopulations in the spleen. T cell function was assessed by measuring proliferation and cytolytic activity after in vitro allogeneic stimulation. Male mice which had been prenatally/lactationally exposed to atrazine had an increase in both T cell proliferation and cytolytic activity. The humoral immune response was assessed after immunization with heat killed Streptococcus pneumoniae (HKSP). There was a significant increase in the number of HKSP-specific IgM secreting B cells in the spleen of prenatal/lactational exposed male mice. Inasmuch as atrazine is a widespread environmental contaminant, this immunopotentiation raises concerns that it may potentiate clinical diseases, such as autoimmune disease and hypersensitivity, and needs to be carefully monitored and studied.

Previous studies in our laboratory have demonstrated that the lipophilic herbicide 3,4-dicholoropropionanilide (DCPA) adversely affects cytokine production by activated macrophages and T lymphocytes. The purpose of this study was to test the hypothesis that DCPA alters the mobility of plasma membrane lipid hydrocarbon chains, which interferes with normal T-lymphocyte activation and macrophage function. Electron spin reasonance (ESR) spectroscopy of stearic acid spin labels incorporated into each cell type was used to test the effects of DCPA on lipid hydrocarbon chain mobility in the absence and presence of specific agents that activate each cell type. The results indicated that DCPA treatment had no significant effect on hydrocarbon chain mobility in either cell type per se. However, for T lymphocytes, but not macrophages, DCPA treatment increased a small population of lipid molecules that exhibited reduced hydrocarbon chain mobility near the bilayer hydrocarbon core following cell stimulation. In contrast, there were no significant effects of DCPA on hydrocarbon chain mobility near the head group region of the bilayer for either cell type. The identity of this subpopulation of lipids and its motional properties could not be elucidated from these studies. Nevertheless, data show that DCPA alters the distribution of lipids in distinct motional environments in the membrane of activated T lymphocytes.

VEGF signaling plays an important role in the development and maintenance of blood vessels and it has received much attention due to its role in tumor development and other pathologies. Recent developments in therapeutics have highlighted the complex and often contradictory features of cell signaling mediated by the VEGF ligand and receptor family. The aim of this study is to constrain the effective parameters of a minimal mathematical model [1] of the first steps in VEGF signaling, namely, the molecular transformations that occur from the presentation of VEGF ligand in the extracellular space to the formation of activated ligand-receptor complexes on the cell membrane. This study addresses unique properties of VEGF due to its bivalent nature. Bivalence complicates signal initiation, which requires the formation of a complex of two receptors cross-linked by a ligand. Unlike the initial capture of ligand from the surrounding volume, the second binding step occurs between proteins bound to the cell membrane, and is therefore modulated by the lateral mobility of the receptors, which in turn reflects the limitations to free movement due to various features of the cell membrane. The kinetics of cross-linking are reflected in (i) the degree of reversibility of VEGF binding to the cell, and (ii) the phenomenon of high dose inhibition. Thus, the empirically derived kinetics that defines the well mixed model [1] help integrate binding data with high resolution imaging and single particle tracking studies of the cell membrane, two classes of experimental information that are hard to compare directly. The binding of VEGF to human umbilical vascular endothelial cells (HUVEC) has been well studied in the literature, and was the basis of the original parameterization of the model [1]. However, those experiments did not pursue the distinction between the two different binding steps, and the kinetics of cross-linking was not well determined. To address this, we use a synthetic compound, single-chain VEGF [2], conjugated to a fluorescent tag (scVEGF-Cy5.5). This ligand has two functional binding sites and its binding affinity and physiological effect on cells expressing VEGFR-2 closely reproduce those of VEGF165. We used flow cytometry to quantify the binding of scVEGF-Cy5.5 to HUVEC in vitro. We obtained dose response curves and time courses for total binding in solution, for incubation times from 30 - 120 minutes and ligand concentrations from 0.1 - 200 nM. The data show possible signs of high dose inhibition in the 100 nM range, and allow us to estimate a range for the characteristic time of cross-linking. Subsequent experiments will directly probe this time range, based on use of this versatile probe for imaging studies of receptor localization and mobility in the cell membrane. References: [1] MacGabhann F and Popel AS, Biophys.Chem. 128:125-139 (2007) [2] Backer MV et al, Nat.Med. 13:504-509 (2007) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3970. doi:1538-7445.AM2012-3970