Preclinical evaluation of ELP-004 in mice.

This study provides a detailed understanding of the preclinical pharmacokinetics and metabolism of ELP-004, an osteoclast inhibitor in development for the treatment of bone erosion. Current treatments for arthritis, including biological disease-modifying antirheumatic drugs, are not well-tolerated in a substantial subset of arthritis patients and are expensive; therefore, new treatments are needed. Pharmacokinetic parameters of ELP-004 were tested with intravenous, oral, and subcutaneous administration and found to be rapidly absorbed and distributed. We found that ELP-004 was non-mutagenic, did not induce chromosome aberrations, non-cardiotoxic, and had minimal off-target effects. Using in vitro hepatic systems, we found that ELP-004 is primarily metabolized by CYP1A2 and CYP2B6 and predicted metabolic pathways were identified. Finally, we show that ELP-004 inhibits osteoclast differentiation without suppressing overall T-cell function. These preclinical data will inform future development of an oral compound as well as in vivo efficacy studies in mice.

Functionality, complications, and survivorship of total shoulder arthroplasty in patients under 60 years old.

Background: As total shoulder arthroplasty (TSA) expands to younger patients, it is crucial to weigh the benefits of early intervention against potential complications and implant longevity in patients under 60 years of age. This study examines mid-term outcomes in this patient subset. Methods: Between 2009 and 2019, a retrospective analysis was conducted on 50 patients (25 male, 25 female) who underwent anatomic TSA (TSA) under the age of 60 with minimum 5 years follow-up. Demographic and baseline variables were extracted from medical records. Pre-operative and post-operative outcomes of range of motion (ROM) and strength were recorded. Patient-reported outcomes (PROs) were obtained. Results: Fifty patients were followed for an average of 8.7 ± 2.4 years, having a mean age of 54.1 ± 8.4 years. Comparison of pre-operative and post-operative measurements revealed significant improvements in active ROM, including external rotation (ER) (p < 0.0001), forward elevation (FE) (p < 0.0001), and internal rotation (IR) (p = 0.0001). There were significant improvements in functional strength scores, including ER (p = 0.0005) and FE (p = 0.0002). PROs included visual analog scale (VAS) (2.2 ± 2.6), Single Assessment Numeric Evaluation (SANE) (80.3 ± 17.6), American Shoulder and Elbow Surgeons (ASES) score (76.4 ± 22.8), and Simple Shoulder Test (SST) (8.9 ± 3.2). The 5-year and 10-year implant survival rates were found to be 98.0 % and 83.3 %, respectively. There were 7 postoperative complications in 5 patients (14.0 %), including glenoid loosening (n = 2), infection (n = 1), atraumatic instability (n = 1), lesser tuberosity avulsion (n = 1), painful arthroplasty (n = 1) and traumatic rotator cuff insufficiency (n = 1). Subsequently, all 5 patients underwent revision shoulder arthroplasty at an average of 6.5 years after the initial procedure. Conclusion: Positive mid to long-term outcomes, including significant improvements in ROM and strength, along with high 5-year and 10-year implant survival rates support TSA as an effective treatment option for patients under the age of 60.

The role of body mass index in survivorship and clinical outcomes in total shoulder arthroplasty.

BACKGROUND: Increased body mass index (BMI) is a potential risk factor for poorer outcomes and complications. However, the influence of BMI on the long-term outcomes of anatomic total shoulder arthroplasty (aTSA) and reverse total shoulder arthroplasty (rTSA) remains to be fully elucidated. METHODS: Institutional records were queried to identify patients who underwent primary total shoulder arthroplasty (TSA) between 2009 and 2020 with a minimum of 2 years of clinical follow-up. Retrospective review was performed to collect demographic characteristics; comorbidity status; and range-of-motion and strength measurements in forward elevation, external rotation, and internal rotation. Patients were contacted by telephone to provide patient-reported outcomes (PROs). Patients were stratified into 3 cohorts by BMI: underweight or normal weight (U/NW, BMI ≤25 kg/m2), overweight (OW, BMI >25 to ≤30 kg/m2), and obese (BMI >30 kg/m2). RESULTS: Among 466 TSA patients, 245 underwent aTSA whereas 221 underwent rTSA. In the aTSA cohort, 40 patients were classified as U/NW; 72, as OW; and 133, as obese. Comparatively, the rTSA cohort was composed of 33 U/NW, 79 OW, and 209 obese patients. Patients in the aTSA and rTSA cohorts had an average follow-up period of 5.8 ± 3.2 years and 4.5 ± 2.3 years, respectively. No differences in age at surgery were found in the aTSA group (U/NW vs. obese, 65.2 ± 7.9 years vs. 61.9 ± 8.9 years; P = .133); however, in the rTSA cohort, BMI was found to be inversely related to age at surgery (U/NW vs. obese, 72.4 ± 8.8 years vs. 65.7 ± 8.3 years; P < .001). Across all BMI cohorts, patients saw great improvements in range of motion and strength. Postoperative PROs after TSA did not vary by BMI in terms of Single Assessment Numeric Evaluation, Simple Shoulder Test, visual analog scale pain, and American Shoulder and Elbow Surgeons scores. There was no significant difference in survival rates at 10-year follow-up in the aTSA cohort (U/NW vs. obese, 95.8% vs. 93.2%; P = .753) or rTSA cohort (U/NW vs. obese, 94.7% vs. 94.5%; P = .791). CONCLUSION: With dramatic improvements in range of motion, minimal differences in PROs, and high rates of implant survival, TSA is a safe and effective treatment option for all patients, including overweight and obese patients.

Prenatal cadmium exposure does not induce greater incidence or earlier onset of autoimmunity in the offspring.

We previously demonstrated that exposure of adult mice to environmental levels of cadmium (Cd) alters immune cell development and function with increases in anti-streptococcal antibody levels, as well as decreases in splenic natural regulatory T cells (nTreg) in the adult female offspring. Based on these data, we hypothesized that prenatal Cd exposure could predispose an individual to developing autoimmunity as adults. To test this hypothesis, the effects of prenatal Cd on the development of autoimmune diabetes and arthritis were investigated. Non-obese diabetic (NOD) mice were exposed to Cd in a manner identical to our previous studies, and the onset of diabetes was assessed in the offspring. Our results showed a similar time-to-onset and severity of disease to historical data, and there were no statistical differences between Cd-exposed and control offspring. Numerous other immune parameters were measured and none of these parameters showed biologically-relevant differences between Cd-exposed and control animals. To test whether prenatal Cd-exposure affected development of autoimmune arthritis, we used SKG mice. While the levels of arthritis were similar between Cd-exposed and control offspring of both sexes, the pathology of arthritis determined by micro-computed tomography (µCT) between Cd-exposed and control animals, showed some statistically different values, especially in the female offspring. However, the differences were small and thus, the biological significance of these changes is open to speculation. Overall, based on the results from two autoimmune models, we conclude that prenatal exposure to Cd did not lead to a measurable propensity to develop autoimmune disease later in life.

The function of the calcium channel Orai1 in osteoclast development.

To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1fl/fl -LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.

Prenatal Cadmium Exposure Alters Proliferation in Mouse CD4+ T Cells via LncRNA Snhg7.

Objective: Prenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA host gene 7 (lncSnhg7) in T cell proliferation. Methods: RNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Finally, lncSnhg7 was knocked down in splenic CD4+ T cells with lentivirus to assess its effect on proliferation. Results: We identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Knockdown on lncSnhg7 inhibits proliferation of CD4+ T cells. Conclusion: Prenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression and knockdown of lncSnhg7 inhibited proliferation suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis.

BACKGROUND: Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. METHODS: We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1 oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. RESULTS: We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. CONCLUSION: Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.

The roles of Orai and Stim in bone health and disease.

Orai and Stim proteins are the mediators of calcium release-activated calcium signaling and are important in the regulation of bone homeostasis and disease. This includes separate regulatory systems controlling mesenchymal stem cell differentiation to form osteoblasts, which make bone, and differentiation and regulation of osteoclasts, which resorb bone. These systems will be described separately, and their integration and relation to other systems, including Orai and Stim in teeth, will be briefly discussed at the end of this review.

Environmental perfluoroalkyl acid exposures are associated with liver disease characterized by apoptosis and altered serum adipocytokines.

Exposures to perfluoroalkyl substances (PFAS) including perfluoroalkyl acids (PFAAs) are associated with increased liver enzymes in cohort studies including the C8 Health Study. In animal models, PFAAs disrupt hepatic lipid metabolism and induce apoptosis to cause nonalcoholic fatty liver disease (NAFLD). PFAAs are immunotoxic and inhibit pro-inflammatory cytokine release from stimulated leukocytes in vitro. This cross-sectional study tests the hypothesis that environmental PFAAs are associated with increased hepatocyte apoptosis and decreased pro-inflammatory cytokines in serum. Biomarkers previously associated with PFAS exposures and/or NAFLD were evaluated as secondary endpoints. Two hundred adult C8 Health Study participants were included. Measured serum biomarkers included: perfluorohexane sulfonate (PFHxS); perfluorooctanoic acid (PFOA); perfluorooctane sulfonate (PFOS); perfluorononanoic acid (PFNA); cytokeratin 18 M30 (CK18 M30, hepatocyte apoptosis); adipocytokines; insulin; and cleaved complement 3 (C3a). Confounder-adjusted linear regression models determined associations between PFAS and disease biomarkers with cut-offs determined by classification and regression tree analysis. CK18 M30 was positively associated with PFHxS (ß = 0.889, p = 0.042); PFOA (ß = 2.1, p = 0.005); and PFNA (ß = 0.567, p = 0.03). Tumor necrosis factor α (TNFα) was inversely associated with PFHxS (ß = -0.799, p = 0.001); PFOA (ß = - 1.242, p = 0.001); and PFOS (ß = -0.704, p < 0.001). Interleukin 8 was inversely associated with PFOS and PFNA. PFAAs were also associated with sexually dimorphic adipocytokine and C3a responses. Overall, PFAA exposures were associated with the novel combination of increased biomarkers of hepatocyte apoptosis and decreased serum TNFα. These data support previous findings from cohorts and experimental systems that PFAAs may cause liver injury while downregulated some aspects of the immune response. Further studies of PFAAs in NAFLD are warranted and should evaluate sex differences.

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.

Comparative Pharmacokinetics of High and Low Doses of the Herbicide Propanil in Mice.

We have documented that the herbicide propanil is immunotoxic in mice, and our in vitro tissue culture experiments largely recapitulate the in vivo studies. Laboratory studies on environmental contaminants are the most meaningful when these studies are conducted using concentrations that approximate levels in the environment. Many techniques to measure the distribution and pharmacokinetics (PK) on compounds rely on techniques, such as liquid scintillation counting (LSC) of radio-labeled starting compound, that require concentrations higher than environmental levels. The aim of this study was to compare tissue PK after exposure to propanil concentrations more relevant to levels of exposure to agricultural workers and the general population to concentrations previously reported for laboratory studies. To this end, we conducted a study to measure propanil distribution in three immune organs, using ultrasensitive accelerator mass spectrometry (AMS). We used two doses: the lower dose modeled levels expected in the environment or long-term occupational exposure to low doses, while the higher dose was to model the effects of an accidental exposure. Our results showed that the distribution and PK profiles from these two different concentrations was markedly different. The profile of the high dose (concentration) exposure was indicative of saturation of the detoxifying capability of the animal. In contrast, at the lower environmentally relevant concentration, in vivo concentrations of propanil in spleen, liver, and blood dropped to a very low level by 720 min. In conclusion, these studies highlight the differences in PK of propanil at these two doses, which suggests that the toxicity of this chemical should be re-investigated to obtain better data on toxic effects at doses relevant for humans.

Evaluating Macrophages in Immunotoxicity Testing.

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.

Suppression of arthritis-induced bone erosion by a CRAC channel antagonist.

OBJECTIVE: We have shown in vitro and in vivo that osteoclast maturation requires calcium-release activated calcium (CRAC) channels. In inflammatory arthritis, osteoclasts mediate severe and debilitating bone erosion. In the current study, we assess the value of CRAC channels as a therapeutic target to suppress bone erosion in acute inflammatory arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in mice. The CRAC channel inhibitor 3,4-dichloropropionaniline (DCPA) and a placebo was administered 1 day prior to collagen II booster to induce arthritis. Effects on swelling, inflammatory cell invasion in joints, serum cytokines and bone erosion were measured. RESULTS: Assays, by blinded observers, of arthritis severity showed that DCPA, 21 mg/kg/day, suppressed arthritis development over 3 weeks. Bone and cartilage damage in sections of animal feet was reduced approximately 50%; overall swelling of joints was reduced by a similar amount. Effects on bone density by µCT showed clear separation in DCPA-treated CIA animals from CIA without treatment, while differences between controls without CIA and CIA treated with DCPA differed by small amounts and in most cases were not statistically different. Response was not related to anticollagen titres. There were no adverse effects in the treated group on animal weight or activity, consistent with low toxicity. The effect was maximal 12-17 days after collagen booster, during the rapid appearance of arthritis in untreated CIA. At 20 days after treatment (day 40), differences in arthritis score were reduced and tumour necrosis factor α, interleukin (IL)-1, or IL-6 in the serum of the animals were similar in treated and untreated animals. CONCLUSIONS: DCPA, a novel inhibitor of CRAC channels, suppresses bone erosion associated with acute arthritis in mice and might represent a new treatment modality for acute arthrits.

Mixed methods student evaluation of an online systemic human anatomy course with laboratory.

A fully online section of an existing face-to-face (F2F) systemic human anatomy course with a prosection laboratory was offered for the first time in 2012-2013. Lectures for F2F students (N = 365) were broadcast in both live and archived format to online students (N = 40) using virtual classroom software. Laboratories were delivered online by a teaching assistant who manipulated 3D computer models in the virtual classroom environment. An exploratory sequential mixed methods approach was undertaken to determine the most important deciding factors that drive students' preferences for a given format and then to generate theory on the strengths and weaknesses of the online format. Students (20 online; 310 F2F) volunteered to participate in a crossover period of one week to expose them to the course section in which they were not originally registered. Open ended interviews (20 online; 20 F2F) and quantitative surveys (270 F2F) were conducted following a crossover. Students valued pace control, schedule, and location flexibility of learning from archived materials and being assessed online. In the online laboratory they had difficulty using the 3D models and preferred the unique and hands-on experiences of cadaveric specimens. The F2F environment was conducive to learning in both lecture and laboratory because students felt more engaged by instructors in person and were less distracted by their surroundings. These results suggest the need to improve the online experience by increasing the quality of student-instructor communication and in turn student-content interaction with the 3D models. Anat Sci Educ 9: 272-285. © 2015 American Association of Anatomists.

Improving the risk assessment of lipophilic persistent environmental chemicals in breast milk.

Lipophilic persistent environmental chemicals (LPECs) have the potential to accumulate within a woman's body lipids over the course of many years prior to pregnancy, to partition into human milk, and to transfer to infants upon breastfeeding. As a result of this accumulation and partitioning, a breastfeeding infant's intake of these LPECs may be much greater than his/her mother's average daily exposure. Because the developmental period sets the stage for lifelong health, it is important to be able to accurately assess chemical exposures in early life. In many cases, current human health risk assessment methods do not account for differences between maternal and infant exposures to LPECs or for lifestage-specific effects of exposure to these chemicals. Because of their persistence and accumulation in body lipids and partitioning into breast milk, LPECs present unique challenges for each component of the human health risk assessment process, including hazard identification, dose-response assessment, and exposure assessment. Specific biological modeling approaches are available to support both dose-response and exposure assessment for lactational exposures to LPECs. Yet, lack of data limits the application of these approaches. The goal of this review is to outline the available approaches and to identify key issues that, if addressed, could improve efforts to apply these approaches to risk assessment of lactational exposure to these chemicals.

Evaluation of wearable simulation interface for military training.

OBJECTIVE: This research evaluated the training effectiveness of a novel simulation interface, a wearable computer integrated into a soldier's load-bearing equipment. BACKGROUND: Military teams often use game-based simulators on desktop computers to train squad-level procedures. A wearable computer interface that mimics the soldier's equipment was expected to provide better training through increased realism and immersion. METHOD: A heuristic usability evaluation and two experiments were conducted. Eight evaluators interacted with both wearable and desktop interfaces and completed a usability survey. The first experiment compared the training retention of the wearable interface with a desktop simulator and interactive training video. The second experiment compared the training transfer of the wearable and desktop simulators with a live training environment. RESULTS: Results indicated the wearable interface was more difficult to use and elicited stronger symptoms of simulator sickness. There was no significant difference in training retention between the wearable, desktop, or interactive video training methods. The live training used in the second experiment provided superior training transfer than the simulator conditions, with no difference between the desktop and wearable. CONCLUSION: The wearable simulator interface did not provide better training than the desktop computer interface. It also had poorer usability and caused worse simulator sickness. Therefore, it was a less effective training tool. APPLICATION: This research illustrates the importance of conducting empirical evaluations of novel training technologies. New and innovative technologies are always coveted by users, but new does not always guarantee improvement.

The toxicity of the N-hydroxy and 6-hydroxy metabolites of 3,4-dichloropropionanilide does not depend on calcium release-activated calcium channel inhibition.

Each year ~1 billion kg of herbicides are used worldwide to control the unwanted growth of plants. In the United States, over a quarter of a billion kg of herbicides are used, representing 28% of worldwide use. (Kiely, T., Donaldson, D., and Grube, A. [2004]. Pesticide Industry Sales and Usage. 2000 and 2001 Market Estimates. Available at: http://www.epa.gov/pesticides/pestsales/01pestsales/market_estimates2001.pdf. Accessed October 25, 2012.) Propanil (3,4-dichloropropionanilide [DCPA]) is a commonly used herbicide in the United States, with 2-4 million kg applied annually to 2 million acres of crop land. The immunomodulatory effects of DCPA have been well documented, but limited data are available on the effects of its metabolites. (Salazar, K. D., Ustyugova, I. V., Brundage, K. M., Barnett, J. B., and Schafer, R. [2008]. A review of the immunotoxicity of the pesticide 3,4-dichloropropionanalide. J. Toxicol. Environ. Health B Crit. Rev. 11, 630-645.) In mammals, hepatic enzymes metabolize DCPA, resulting in the production of 3,4-dichloroaniline (DCA). Further biotransformation of DCA leads to the production of 6-hydroxy-3,4-dichloroaniline (6OH-DCA) and N-hydroxy-3,4-dichloroaniline (NOH-DCA). We report, for the first time, the immunotoxic effects of DCPA metabolites on T-cell function. Human Jurkat T cells were exposed to varying concentrations of DCPA or its metabolites and assayed for effects on T-cell function. In addition, fluorine analogs of DCPA and DCA were investigated to determine the relative role of chlorine substituents on T-cell immunotoxicity. Here we report that exposure of Jurkat T cells to DCPA and DCA alters IL-2 secretion, nuclear factor of activated T cells (NFAT) activity, and calcium influx. However, exposure to 6OH-DCA and NOH-DCA reduces IL-2 secretion and NFAT activity but has no effect on calcium flux. When both chlorines in DCPA and DCA were substituted with fluorines all effects were abrogated. Our data indicate that metabolites of DCPA have differential effects on T-cell function and the presence of chlorines plays an important role in eliciting these effects.

Prenatal cadmium exposure produces persistent changes to thymus and spleen cell phenotypic repertoire as well as the acquired immune response.

Cadmium (Cd) is a common environmental contaminant. Adult exposure to Cd alters the immune system, however, there are limited studies on the effects of prenatal exposure to Cd. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10 ppm) and the effects on the immune system of the offspring were assessed at 20 weeks of age. Prenatal Cd exposure caused an increase in the percent of CD4(-)CD8(-)CD44(+)CD25(-) (DN1) thymocytes in both sexes and a decrease in the percent of CD4(-)CD8(-)CD44(-)CD25(+) (DN3) thymocytes in females. Females had an increase in the percent of splenic CD4(+) T cells, CD8(+) T cells, and CD45R/B220(+) B cells and a decrease in the percent of NK cells and granulocytes (Gr-1(+)). Males had an increase in the percent of splenic CD4(+) T cells and CD45R/B220(+) B cells and a decrease in the percent of CD8(+) T cells, NK cells, and granulocytes. The percentage of neutrophils and myeloid-derived suppressor cells were reduced in both sexes. The percent of splenic nTreg cells was decreased in all Cd-exposed offspring. Cd-exposed offspring were immunized with a streptococcal vaccine and the antibody response was determined. PC-specific serum antibody titers were decreased in Cd exposed female offspring but increased in the males. PspA-specific serum IgG titers were increased in both females and males compared to control animals. Females had a decrease in PspA-specific serum IgM antibody titers. Females and males had a decrease in the number of splenic anti-PspA antibody-secreting cells when standardized to the number of B cells. These findings demonstrate that very low levels of Cd exposure during gestation can result in long term sex-specific alterations on the immune system of the offspring.

Multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of Cr(VI)-transformed lung cells.

B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI).