Social enrichment alters the response of brain leukocytes to chemotherapy and tumor development in aged mice.

Aging is a risk factor for the development of breast cancer. Foundational science studies have supported associations among neuroinflammation, breast cancer, and chemotherapy, but to date, these associations are based on studies using young adult rodents. The current study examined the neuroinflammatory effects of chemotherapy in aged, tumor-naïve and tumor-bearing mice with or without social enrichment. Mice received two intravenous injections of doxorubicin (A) and cyclophosphamide (C) at a two-week interval. Brain immune cells were enriched/assessed via flow cytometry, seven days following the second chemotherapy injection. Social enrichment enhanced peripheral immune cell trafficking in aged tumor-naive mice treated with AC. Group housed aged tumor bearing mice receiving AC had reduced percentage of IL-6+ monocytes and granulocytes relative to their singly housed counterparts. Notably, group housing aged experimental mice with young cage partners significantly reduced TNF + monocytes, tumor volume, and tumor mass. These data illustrate the importance of social enrichment in attenuating neuroinflammation and are the first to demonstrate that social support with young housing partners reduces tumor growth in aged mice.

A Survey on Core Flow Cytometry Facilities: Instrument Maintenance, Usage, and Funding.

Flow cytometry is a powerful tool that finds applications in various fields such as immunology, molecular biology, cancer biology, virology, and infectious disease monitoring. A significant portion of the research in these disciplines is supported by flow cytometry shared resource laboratories (SRLs). There are several types of flow cytometers available for use in SRLs, including analyzers, sorters, imaging flow cytometers, and mass cytometers. Each type has different challenges when it comes to maintenance and life expectancy. An independent online survey was conducted to better understand instrument maintenance and turnover in flow cytometry SRLs. Questions regarding instrument uptime (availability), its usage, routine maintenance, and cost associated with it were addressed. The respondents also answered questions pertaining to the frequency of deep cleaning of the instrument and quality control. In addition, the survey queried about the source of funding used to purchase the instruments and possible reasons for a replacement. Presented herein are the results compiled from 146 core facilities that provide a look at the operation within a typical SRL, with the responses reflecting researchers' experiences with handling flow cytometers.

Cell Sorter Cleaning Practices and Their Impact on Instrument Sterility.

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.

The impact of opioid exposure during pregnancy on the human neonatal immune profile.

BACKGROUND: The increasing magnitude of the opioid crisis and rising rates of neonatal abstinence syndrome (NAS) diagnoses highlight the need for increased research into how maternal substance use during pregnancy can impact the neonatal immune profile and its functionality. We hypothesized that neonates with opioid exposure would have reduced proportions of some immune cells, an anti-inflammatory cytokine profile, reduced T cell proliferation, and monocyte bacterial killing activity compared to the control population. METHODS: The present study compares immune cell populations, inflammatory and anti-inflammatory cytokine and chemokine levels in the serum, and monocyte and T cell functional activity using umbilical cord samples from neonates with known opioid exposure during gestation and from control neonates without known exposure. RESULTS: Our findings demonstrated a significant reduction in neutrophils, decreased levels of inflammatory cytokines in the serum, and reduced IL-2 production during in vitro CD4+ T cell proliferation in neonates exposed to opioids compared to controls. The neutrophil findings were supported by retrospective analysis of an extended network of deidentified patient records. CONCLUSIONS: This study is the first of its kind to evaluate differences in neonatal immunity as a result of opioid exposure in the human population that will inform continued mechanistic studies. IMPACT: The opioid epidemic has become a public health crisis in the United States, and the corresponding incidence of neonatal abstinence syndrome (NAS) have risen accordingly. New research is required to understand the short and long-term health impacts of opioid exposure to the neonate. This is the first human study to investigate the immunologic profile and functionality in neonates with known opioid exposure in utero. The abundance of neutrophils and the ratio of neutrophils to lymphocytes is significantly reduced along with inflammatory cytokines and chemokines following opioid exposure during pregnancy. The immune profile in opioid-exposed neonates may promote susceptibility to infection.

Contribution of HCN1 variant to sinus bradycardia: A case report.

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.

Myeloid-Derived Suppressor Cells Gain Suppressive Function during Neonatal Bacterial Sepsis.

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.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Opening large-conductance potassium channels selectively induced cell death of triple-negative breast cancer.

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.

Long-term Immunotoxic Effects of Oral Prenatal and Neonatal Atrazine Exposure.

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.

Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

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.

Effects of Meditation and Music-Listening on Blood Biomarkers of Cellular Aging and Alzheimer's Disease in Adults with Subjective Cognitive Decline: An Exploratory Randomized Clinical Trial.

BACKGROUND: Telomere length (TL), telomerase activity (TA), and plasma amyloid-ß (Aß) 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 Aß 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 Aß(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 Aß40 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 <0.006). Both groups improved in cognitive and psychosocial status (ps ≤0.05), with improvements in stress, mood, and QOL greater in the KK group. Rising Aß levels were correlated with gains in cognitive function, mood, sleep, and QOL at both 3 and 6 months, associations that were particularly pronounced in the KK group. Increases in TL and TA were also correlated with improvements in certain cognitive and psychosocial measures. CONCLUSION: Practice of simple mind-body therapies may alter plasma Aß levels, TL, and TA. Biomarker increases were associated with improvements in cognitive function, sleep, mood, and QOL, suggesting potential functional relationships.

Altering calcium influx for selective destruction of breast tumor.

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.

SOX9 inhibits ß-TrCP-mediated protein degradation to promote nuclear GLI1 expression and cancer stem cell properties.

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, ß-TrCP (also known as F-box/WD repeat-containing protein 1A), and suppressed its association with SKP1 and GLI1, a substrate of SCF-ß-TrCP. SOX9 also tethered ß-TrCP within the nucleus and promoted its degradation. SOX9 bound to ß-TrCP through the SOX9 C-terminal PQA/S domain that mediates transcriptional activation. Suppression of ß-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 ß-TrCP as a latent SOX9-bound tumor suppressor with the potential to degrade oncogenic proteins in tumor cells.

MicroRNAs 206 and 21 cooperate to promote RAS-extracellular signal-regulated kinase signaling by suppressing the translation of RASA1 and SPRED1.

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.

Sibling donor and recipient immune modulation with atorvastatin for the prophylaxis of acute graft-versus-host disease.

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.

Prenatal cadmium exposure alters postnatal immune cell development and function.

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.

Early Postnatal Ozone Exposure Alters Rat Nodose and Jugular Sensory Neuron Development.

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.

Subcellular localization of the amide class herbicide 3,4-dichloropropionanilide (DCPA) in T cells and hepatocytes.

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.

Evaluating macrophages in immunotoxicity testing.

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.