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.

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.

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.

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.

Molecular Regulation of Bone Turnover in Juvenile Idiopathic Arthritis: Animal Models, Cellular Features and TNFα.

We review the abnormal bone turnover that is the basis of idiopathic inflammatory or rheumatoid arthritis and bone loss, with emphasis on Tumor Necrosis Factor-alpha (TNFα)-related mechanisms. We review selected data on idiopathic arthritis in juvenile human disease, and discuss mouse models focusing on induction of bone resorbing cells by TNFα and Receptor Activator of Nuclear Factor kappa B Ligand (RANKL). In both humans and animal models, macrophage-derived cells in the joint, particularly in the synovium and periosteum, degrade bone and cartilage. Mouse models of rheumatoid arthritis share with human disease bone resorbing cells and strong relation to TNFα expression. In humans, differences in therapy and prognosis of arthritis vary with age, and results from early intervention for inflammatory cytokines in juvenile patients are particularly interesting. Mechanisms that contribute to inflammatory arthritis reflect, in large part, inflammatory cytokines that play minor roles in normal bone turnover. Changes in inflammatory cytokines, particularly TNFα, are many times larger, and presented in different locations, than cytokines that regulate normal bone turnover. Recent data from in vitro and mouse models include novel mechanisms described in differentiation of bone resorbing cells in inflammatory arthritis dependent on the Transient Receptor Potential Channel (TRPC) family of calcium channels. Low-molecular weight (MW) inhibitors of TRPC channels add to their potential importance. Associations with inflammatory arthritis unrelated to TNFα are briefly summarized as pointing to alternative mechanisms. We suggest that early detection and monoclonal antibodies targeting cytokines mediating disease progression deserves emphasis.

Functional outcomes and survivorship following aseptic revision shoulder arthroplasty.

Background: Revision shoulder arthroplasty (SA) is a surgical procedure performed to address complications or failures of primary total SA. However, limited evidence exists regarding the functional outcomes and longevity of implants following revision. Methods: A retrospective analysis was conducted on patients who underwent revision SA for failed primary arthroplasty at a single institution between 2009 and 2021 with a minimum of 2-years follow-up. Data was collected from medical records, including type of arthroplasty (anatomic total SA [TSA], reverse total SA [RSA], or hemi-SA [HSA]), demographics and patient-specific information, functional measurements, and implant survival. Patient reported outcomes were obtained during follow-up by phone. Results: The mean age at index and revision surgeries was 60.5 ± 12.1 years and 64.8 ± 11.1 years, respectively, and average total follow-up was 5.5 ± 3.5 years. The average time to revision was 4.5 ± 5.2 years (range 0.01-24.5 years). Among 99 revision shoulder arthroplasty procedures, 28 were TSA/HA to TSA/HA, 51 were TSA/HA to RSA, 18 were RSA to RSA, and 2 were RSA to HA. Revision surgery significantly improved functional outcomes in forward elevation (preoperative: 79.8 ± 41.0 vs postoperative: 118.5 ± 38.3; p < 0.001), external rotation (preoperative: 27.8 ± 19.3 vs postoperative: 34.3 ± 16.2; p = 0.028), internal rotation (preoperative: glute vs postoperative: S1; p = 0.002), and forward elevation strength (preoperative: 4+/5 vs postoperative: 5/5; p = 0.002). Postoperative patient reported outcomes included: VAS pain (2.2 ± 2.9), SANE (72.6 ± 21.5), ASES (73.3 ± 20.4), and SST (7.7 ± 2.8) scores. The overall 2-, 5-, and 10-year post-revision implant survival rate was 85.48%, 83.06%, and 79.84%, respectively. Patients who had an initial RSA and were revised to RSA were at higher risk of implant failure and subsequent re-revision (RSA to RSA: 1.5 ± 2.5 years vs. TSA/HA to RSA: 2.5 ± 2.1 years vs. TSA/HA to TSA/HA: 4.0 ± 3.5 years; p = 0.0046). Conclusion: Revision shoulder arthroplasty improved patient outcomes post-index arthroplasty failure. Revisions were more likely to be successful when revising from TSA/HA to RSA. Level of evidence: Level III - retrospective comparative study.

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.

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.

Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development.

Calcium signaling plays a central role in the regulation of bone cells, although uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared the mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1(-/-) mice lacked multinucleated osteoclasts. Yet, they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1(-/-) mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1(-/-) mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1(-/-) animals compared with controls; bone deposition was markedly decreased in the knockout mice. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1(-/-) and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation.

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.

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.

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells.

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A 'pro-cancer' gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment.

Femoral nerve block with selective tibial nerve block provides effective analgesia without foot drop after total knee arthroplasty: a prospective, randomized, observer-blinded study.

BACKGROUND: Sciatic nerve block when combined with femoral nerve block for total knee arthroplasty may provide superior analgesia but can produce footdrop, which may mask surgically induced peroneal nerve injury. In this prospective, randomized, observer-blinded study, we evaluated whether performing a selective tibial nerve block in the popliteal fossa would avoid complete peroneal motor block. METHODS: Eighty patients scheduled for primary total knee arthroplasty were randomized to receive either a tibial nerve block in the popliteal fossa or a sciatic nerve block proximal to its bifurcation in combination with femoral nerve block as part of a multimodal analgesia regimen. Local anesthetic solution of sufficient volume to encircle the target nerve was administered for the block, up to a maximum of 20 mL. General anesthesia was administered for surgery. After emergence from anesthesia, in the recovery room, the presence or absence of peroneal sensory and motor block was noted. Pain scores and opioid consumption were recorded for 24 hours after surgery. RESULTS: The tibial nerve block and sciatic nerve block were performed 1.7 cm (99% CI, 1.3 to 2.1) and 9.4 cm (99% CI, 8.3 to 10.5) proximal to the popliteal crease, respectively (99% CI for difference between means: 6.4 to 9.0; P < 0.001). A lower volume of ropivacaine 0.5% was used for the tibial nerve block, 8.7 mL (99% CI, 7.9 to 9.4) versus 15.2 mL (99% CI, 14.9 to 15.5), respectively (99% CI for difference between means, 5.6 to 7.3; P < 0.001). No patient receiving a tibial nerve block developed complete peroneal motor block compared to 82.5% of patients with sciatic nerve block (P < 0.001). There were no significant differences in the pain scores and opioid consumption between the groups. CONCLUSIONS: Tibial nerve block performed in the popliteal fossa in close proximity to the popliteal crease avoided complete peroneal motor block and provided similar postoperative analgesia compared to sciatic nerve block when combined with femoral nerve block for patients undergoing total knee arthroplasty.

The role of calcium release activated calcium channels in osteoclast differentiation.

Osteoclasts are specialized macrophage derivatives that secrete acid and proteinases to mobilize bone for mineral homeostasis, growth, and replacement or repair. Osteoclast differentiation generally requires the monocyte growth factor m-CSF and the TNF-family cytokine RANKL, although differentiation is regulated by many other cytokines and by intracellular signals, including Ca(2+). Studies of osteoclast differentiation in vitro were performed using human monocytic precursors stimulated with m-CSF and RANKL, revealing significant loss in both the expression and function of the required components of store-operated Ca(2+) entry over the course of osteoclast differentiation. However, inhibition of CRAC using either the pharmacological agent 3,4-dichloropropioanilide (DCPA) or by knockdown of Orai1 severely inhibited formation of multinucleated osteoclasts. In contrast, no effect of CRAC channel inhibition was observed on expression of the osteoclast protein tartrate resistant acid phosphatase (TRAP). Our findings suggest that despite the fact that they are down-regulated during osteoclast differentiation, CRAC channels are required for cell fusion, a late event in osteoclast differentiation. Since osteoclasts cannot function properly without multinucleation, selective CRAC inhibitors may have utility in management of hyperresorptive states.

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.

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.

Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in altered thymocyte 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.

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.

Interleukin 12 a key immunoregulatory cytokine in infection applications.

Interleukin 12 (termed IL-12p70 and commonly designated IL-12) is an important immunoregulatory cytokine that is produced mainly by antigen-presenting cells. The expression of IL-12 during infection regulates innate responses and determines the type of adaptive immune responses. IL-12 induces interferon-gamma (IFN-gamma) production and triggers CD4(+) T cells to differentiate into type 1 T helper (Th1) cells. Studies have suggested that IL-12 could play a vital role in treating many diseases, such as viral and bacterial infections and cancers. The unique heterodimeric structure, which IL-12 shares with its family members including IL-23, IL-27, and IL-35, has recently brought more attention to understanding the mechanisms that regulate the functions of IL-12. This article describes the structure and biological activities of IL-12 in both the innate and adaptive arms of the immune system, and discusses the applications of IL-12 in treating and preventing infections.

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.