Rux largely restores lungs in Iraq PM-exposed mice, Up-regulating regulatory T-cells (Tregs).

Background Military personnel post-deployment to Iraq and Afghanistan have noted new-onset respiratory illness. This study's primary objective was to further develop an animal model of Iraq Afghanistan War Lung Injury (IAW-LI) and to test a novel class of anti-injury drug called RuX. Methods Particulate Matter (PM) samples were obtained in Iraq then characterized by spectromicroscopy. C57BL/6 mice underwent orotracheal instillation with PM, followed by drinkable treatment with RuX. Lung histology, inspiratory capacity (FlexiVent), thymic/splenic regulatory T cell (Treg) number, and whole-lung genomics were analyzed. Results Tracheal instillation of Iraq PM led to lung septate thickening and lymphocytic inflammation. PM-exposed mice had suppression of thymic/splenic regulatory T-cells (Tregs). Drinking RuX after PM exposure attenuated the histologic lung injury response, improved lung inspiratory capacity, and increased Tregs. Pooled whole lung genomics suggest differences among gene expression of IL-15 among control, PM, and PM + RuX groups. Conclusions RuX, a ruthenium and alpha-lipoic acid complex, attenuates lung injury by improving histology and inspiratory capacity via upregulation of Tregs in Iraq PM-exposed C57BL/6. Plausible genomic effects may involve IL-15 whole lung gene expression.

Polychlorinated biphenyls, mercury, and antinuclear antibody positivity, NHANES 2003-2004.

Serum antinuclear antibody positivity (ANA) has been associated with elevated serum polychlorinated biphenyls (PCBs) among residents in PCB-polluted areas; however, associations in general populations have not been reported by congener type or with adjustment for mercury. Cross-sectional data on serum PCBs, total blood mercury, ANA, and potential confounders age, race, body mass index, menopausal status, and dietary eicosapentaenoic acid (EPA) were obtained from the 2003-2004 National Health and Nutrition Examination Survey (NHANES) for males and females aged 12-85. PCB congeners were summed separately for dioxin-like and nondioxin-like PCBs; the former were weighted for toxic equivalent factors. Total PCBs by congener type and mercury were analyzed as both continuous log-transformed variables and as categorical quintiles. Logistic regression models were stratified by sex. There were no associations between nondioxin-like PCBs or mercury and ANA among males or females. Among females (n=114 affected and 518 unaffected), adjusting for potential confounders, the prevalence odds for ANA positivity were significantly elevated per incremental increase in log-transformed dioxin-like PCBs (odds ratio {OR}=1.66; 95% confidence interval {CI}=1.24, 2.23); the highest dioxin-like PCB quintile (>0.00425-0.04339ng/g) was significantly associated with 4.04 (95% CI=2.43, 6.70) greater prevalence odds for ANA positivity relative to the lowest quintile (Ptrend<0.001). We present novel findings of an association between low-level dioxin-like PCBs and ANA among women. No associations were observed between mercury and ANA at mercury levels common to the U.S. population.

Total blood mercury and rubella antibody concentrations in US children aged 6-11 years, NHANES 2003-2004.

BACKGROUND: Children are susceptible to mercury toxicity, and mercury has immunomodulatory effects. Lower folate and B-12, and higher homocysteine may represent susceptibility cofactors. A large proportion of variability in rubella immune response is attributable to environmental factors. OBJECTIVE: This study aimed to evaluate the interaction between total blood mercury (Hg) and nutritional and homocysteine status on rubella virus antibody concentrations. DESIGN: Cross-sectional data on rubella IgG antibody concentrations, Hg, homocysteine, methylmalonic acid (MMA, an indicator of B-12 deficiency), and folate were obtained from 2003-2004 NHANES for children aged 6-11 years with rubella seropositivity (n=690). Linear regression was used to evaluate relationships between log-transformed rubella concentrations and Hg, stratified by sex, MMA ≥, folate<, and homocysteine ≥ sample medians, adjusted for demographic and nutritional cofactors. RESULTS: Hg was significantly positively associated with rubella antibody concentrations (ß=0.24; 95% confidence interval (CI)=0.11, 0.38) in children with higher MMA, lower folate and higher homocysteine (n=110), yet inversely associated among all other children (ß=-0.18; 95% CI=-0.34, -0.03) (n=580). Among the former, estimates (ß) were positive across all Hg quartiles relative to the lowest (Q1) (Hg<0.30 µg/L): Q2: ß=0.23 (-.10, 0.56); Q3: ß=0.35 (0.13, 0.57); Q4: ß=0.53 (0.21, 0.84); P(trend)<0.01. CONCLUSION: Findings are consistent with previously reported associations between Hg and measles antibody concentrations, and highlight the importance of considering dynamics between toxicant exposures, pathogens and host susceptibility.

VIP Regulates the Development & Proliferation of Treg in vivo in spleen.

BACKGROUND: Mounting evidence supports a key role for VIP as an anti-inflammatory agent and promoter of immune tolerance. It suppresses TNF-α and other inflammatory cytokines and chemokines, upregulates anti-inflammatory IL-10, and promotes immune tolerant cells called T regulatory (Treg) cells. VIP KO mice have recently been demonstrated to have spontaneous airway and pulmonary perivascular inflammatory responses, as part of asthma-like and pulmonary hypertension phenotypes, respectively. Both inflammatory responses are correctable with VIP. Focusing on this model, we have now investigated the influence of VIP not only on inflammatory cells but also on Treg cells. METHODS: Using flow cytometric analysis, we examined the relative preponderance of CD25+CD4+ cells and anti-inflammatory Treg cells, in extracts of thymus and spleen from VIP KO mice (5 VIP KO; 5 VIP KO+ VIP; 10 wild-type). This method allowed antibody-based flow cytometric identification of Treg cells using surface markers CD25 and CD4, along with the: 1) intracellular activation marker FoxP3; and 2) Helios, which distinguishes cells of thymic versus splenic derivation. CONCLUSIONS: Deletion of the VIP gene results in: 1) CD25+CD4- cell accumulation in the thymus, which is corrected by VIP treatment; 2) more Treg in thymus lacking Foxp3 expression, suggesting VIP is necessary for immune tolerance; and, 3) a tendency towards deficiency of Treg cells in the spleen, which is normalized by VIP treatment. Treg lacking Helios are induced by VIP intrasplenically rather than by migration from the thymus. These results confirm the dual role of VIP as an anti-inflammatory and immune tolerance-promoting agent.

Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients.

Recent research advances show that tumor cell intravasation (entry into the circulation) and metastasis occur very early in breast cancer progression. Clinical studies also illustrate the potential importance of detection of circulating tumor cells (CTCs) in outcomes of patients with metastatic breast cancer. Whether these cells exhibit the invasiveness and express tumor stem or progenitor markers, hallmark of the metastatic phenotype, is less well characterized. To detect CTCs with the invasive phenotype and to explore their molecular features, we applied a functional cell separation method, called collagen adhesion matrix (CAM) assay, as enrichment and identification steps. The CAM-coated device successfully recovered tumor cells spiked in 1 ml of blood with a 54% +/- 9% (n = 18) recovery rate and 0.5-35% purity, and detected invasive tumor cells in 10/10 blood samples (100% yield) from patients with metastatic breast cancer with a range of 18-256 CTCs/ml and average of 126 +/- 25 (mean +/- SD) CTCs/ml. CTCs were detected in blood samples of 28/54 (52%) Stage I-III breast cancer patients with a mean count of 61 CTCs/ml. Furthermore, the relative frequency of these cells correlated to the staging, lymph node-status and survival of patients with early stage breast cancer. CAM-captured cells were capable of propagation in culture. Gene expression and multiplex flow cytometric analyses on CAM-captured cells demonstrated the existence of distinct populations of CTCs including these of epithelial lineage and stem or progenitor cells. Thus, CAM-initiated CTC detection provides advantages for examining invasiveness and tumor progenitor phenotypes.

Cytology and flow cytometry to identify ectopic thymic tissue masquerading as a thyroid nodule in two children.

BACKGROUND: Clinically unapparent thyroid nodules in children pose a significant problem in differential diagnosis and management. Ectopic thymic tissue in the thyroid gland is rare, but may masquerade as a thyroid nodule. This paper demonstrates the utility of flow cytometry as an adjunct to cytology by fine needle aspiration in diagnosing ectopic thymic tissue in the thyroid gland. SUMMARY: By demonstration of T lymphocytes maturing along two cell lineages and the absence of markers for malignant lesions, fine-needle aspiration, cytology, and flow cytometry were used to identify ectopic thymic tissue masquerading as a thyroid nodule in two children. CONCLUSION: Use of this technique prevented surgical intervention that otherwise would have been necessary to obtain an accurate diagnosis of these thyroid nodules.

High dose cyclophosphamide preferentially targets naïve T (CD45/CD4/RA+) cells in CIDP and MS patients.

INTRODUCTION: T cells occupy a central role in MS and CIDP pathogenesis. High dose cyclophosphamide's in-vivo cytotoxic-effect on circulating memory and naïve T cells is unknown. METHOD: Three MS and five CIDP patients received cyclophosphamide (200 mg/kg) for refractory disease. Before and after chemotherapy administration, peripheral blood T-cell subsets were determined. Patients underwent serial neurologic evaluations quarterly. RESULTS: Cyclophosphamide uniformly decreased clinical disease activity. Compared to memory T cells, naïve T cells were preferentially eradicated. DISCUSSION: Cyclophosphamide effectiveness in autoimmune illness may result from Naïve T-cell destruction, as this compartment may be the source of autoreactive lymphocytes.

Dexamethasone therapy interferes with the development of the neutrophil oxidative burst in vitro in very low birthweight infants.

Dexamethasone therapy is used in the treatment of chronic lung disease in very low birthweight (VLBW) neonates. However, several significant side effects have been observed in these patients, including an increased incidence of infection. We initiated a pilot study to examine the in vitro function of neutrophils from dexamethasone-treated VLBW infants. Following a 1-week course of dexamethasone, VLBW infants failed to demonstrate the increased oxidative burst that was observed in the age- and weight-matched untreated infants. These observations support recent evidence raising concern about effects on other organs when using dexamethasone in mechanically ventilated infants. In this example, impaired oxidative burst may be among the cellular factors contributing to the increased risk of infection during corticosteroid use in VLBW neonates in the neonatal intensive care setting.

Analysis of connexin 43 expression on keratinocytes using flow cytometry.

Single-cell suspensions of primary keratinocytes comprise a heterogeneous cell population that consists of basal cells (stem cells, transient amplifying cells, and post-mitotic basal cells) and suprabasal cells at different stages of differentiation. Quantitative data for the differential expression of epitopes on single cells can be obtained using a flow cytometer. Simultaneous analysis of two intracellular epitopes, keratin 14 and connexin 43, using flow cytometry after keratinocyte isolation, fixation, permeabilization, and fluorescent immunolabeling is described. Three subsets of cells could be distinguished: stem cells (basal cells [keratin 14 positive] that lack connexin 43 expression); suprabasal cells (connexin 43-positive, keratin 14-negative cells); and basal cells (keratin 14 positive) that express connexin 43. The last population of keratinocytes includes both transient amplifying cells and postmitotic basal cells. The scatter characteristics of each cell population are also described.

In vivo effect of recombinant human granulocyte colony-stimulating factor on phagocytic function and oxidative burst activity in septic neutropenic neonates.

Neutrophil dysfunction may contribute to an increased risk of sepsis in very-low-birth-weight (VLBW) neonates. The current study was designed to determine whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects absolute neutrophil count (ANC), phagocytic function, and oxidative burst in neutropenic VLBW neonates. Fourteen ventilated VLBW neonates were treated with rhG-CSF (10 microg/kg/day x 3 days i.v.). Phagocytic activity and oxidative burst were assessed before and after treatment with rhG-CSF using flow cytometry and fluorescence labeled opsonized Staphylococcus aureus. Control (nonseptic, nonneutropenic, n = 4), preeclamptic neutropenic (PET; nonseptic, n = 5), and septic neutropenic (n = 5) neonates with a gestational age ranging from 24 to 30 weeks were studied. In both PET and septic neonates, posttreatment phagocytosis more than doubled, but did not achieve matching control levels, whereas rhG-CSF treatment maintained the level of the phagocytic activity in the control group. The oxidative burst increased in all groups, but, again, PET and septic groups did not achieve matching control values. These effects occurred independent of a 2- to 12-fold increase in ANC. These results suggest that other disease-specific factors delay full functional recovery even after rhG-CSF treatment. We speculate that PET and septic neonates may remain susceptible to infection due to deficient neutrophil-killing capacity, even though their ANC returns to normal ranges. Augmenting immune function beyond the immediate period of ANC recovery suggests that prophylaxis with rhG-CSF may be an important risk reduction strategy for susceptible VLBW neonates.