Into the Unknown: Navigating a Path as an Early-stage Physician-scientist in Obstetrics and Gynecology.

This piece is a reflection of one early-stage physician-scientist's professional journey. It highlights a few challenges of navigating this path while calling for continued investment and support for physician-scientists to enhance maternal and child lifelong health.

Neonatal outcomes and rationale for timing of birth in perinatal diabetes: a retrospective cohort study.

BACKGROUND: The American College of Obstetricians and Gynecologists recommends delivery in the 39th week of pregnancy for patients with pregestational and medication-controlled gestational diabetes with consideration for earlier delivery among those with poor glucose control. OBJECTIVE: We sought to evaluate the impact of birth before 39 weeks' gestation exclusively for diabetes-related indications on neonatal outcomes and clinician rationale for these recommendations. STUDY DESIGN: This was a retrospective cohort study of all singleton, nonanomalous pregnancies complicated by diabetes. Patients were identified through an obstetrical database containing information of 90,185 births from 2011 to 2021. Patients who delivered in a given week of gestation exclusively for diabetes-related indications were compared with ongoing pregnancies. Recommended births for other obstetrical indications were excluded from the diabetes-related indications cohorts. The primary outcome was neonatal intensive care unit admission. Secondary outcomes included neonatal intensive care unit length of stay, stillbirth, neonatal death, hypoglycemia, respiratory distress syndrome, and shoulder dystocia. For all births before 39 weeks' gestation, the electronic medical records were reviewed to confirm the rationale for the intervention for a diabetes-indicated condition. RESULTS: From the 90,185 recorded births that occurred in 2011 to 2021, 4750 patients with diabetes were identified. Of those, 30.5% (n=1449) had a recommended birth for a diabetes-related indications with 2.2% of those (n=32) occurring at 36 weeks' gestation, 7.9% (n=114) at 37 weeks' gestation, 9.7% (n=141) at 38 weeks' gestation, and 63.0% (n=913) at 39 weeks' gestation. Births that occurred at 36 and 37 weeks' gestation exclusively for diabetes-related indications had higher rates of neonatal intensive care unit admission than the respective ongoing pregnancies (62.5% vs 8.7%; P<.001 and 25.4% vs 7.2%; P<.001). There was no difference in neonatal intensive care unit admission for births at 38 or 39 weeks' gestation when compared with ongoing pregnancy. For neonates born at 36 and 37 weeks' gestation in comparison with ongoing pregnancies, the median neonatal intensive care unit length of stay was 11.0 vs 2.8 days, (P<.001) and 4.4 vs 2.6 days (P=.026), respectively. There were significantly increased rates of neonatal hypoglycemia and respiratory distress syndrome among births that occurred at 36, 37, and 38 weeks' gestation when compared with ongoing pregnancies. There were no differences in the rate of stillbirth in this cohort. Primary factors cited for early birth were poor glycemic control (71.4%), recommendation by a maternal-fetal medicine specialist (38.7%), and suspected fetal macrosomia (27.9%). Overall, 46.7%, 32.8%, and 20.6% of patients had 1, 2, or ≥3 indications, respectively, listed as rationale for early birth. Overall, few objective measures were used to recommend birth before 39 weeks' gestation owing to diabetes. CONCLUSION: In pregnancies complicated by diabetes, early birth exclusively for diabetes-related indications was associated with increased neonatal intensive care unit admission and length of stay and with neonatal morbidity. Little objective data are documented by clinicians to support their recommendations for early birth associated with diabetes. Additional clinical guidelines are needed to define suboptimal glucose control necessitating birth before 39 weeks' gestation.

Metformin impairs trophoblast metabolism and differentiation in a dose-dependent manner.

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing the risk of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin's impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 µM (therapeutic range) and 2000 µM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 µM metformin-treated cells, 2000 µM metformin impaired oxidative metabolism and increased the abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 µM, but not 200 µM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impair trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

Mitochondrial citrate metabolism and efflux regulate BeWo differentiation.

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established BeWo cell culture model of trophoblast differentiation. Differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

Metformin impairs trophoblast metabolism and differentiation in dose dependent manner.

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing risks of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin's impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 µM (therapeutic range) and 2000 µM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 µM metformin treated cells, 2000 µM metformin impaired oxidative metabolism and increased abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 µM, but not 200 µM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impairs trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

Mitochondrial citrate metabolism and efflux regulates trophoblast differentiation.

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

The Effect of Financial Incentives on Adherence to Glucose Self-Monitoring during Pregnancy among Patients with Insulin-Requiring Diabetes: A Randomized Clinical Trial.

OBJECTIVE: Glucose self-monitoring is critical for the management of diabetes in pregnancy, and increased adherence to testing is associated with improved obstetrical outcomes. Incentives have been shown to improve adherence to diabetes self-management. We hypothesized that use of financial incentives in pregnancies complicated by diabetes would improve adherence to glucose self-monitoring. STUDY DESIGN: We conducted a single center, randomized clinical trial from May 2016 to July 2019. In total, 130 pregnant patients, <29 weeks with insulin requiring diabetes, were recruited. Participants were randomized in a 1:1:1 ratio to one of three payment groups: control, positive incentive, and loss aversion. The control group received $25 upon enrollment. The positive incentive group received 10 cents/test, and the loss aversion group received $100 for >95% adherence and "lost" payment for decreasing adherence. The primary outcome was percent adherence to recommended glucose self-monitoring where adherence was reliably quantified using a cellular-enabled glucometer. Adherence, calculated as the number of tests per day divided by the number of recommended tests per day×100%, was averaged from time of enrollment until admission for delivery. RESULTS: We enrolled 130 participants and the 117 participants included in the final analysis had similar baseline characteristics across the three groups. Average adherence rates in the loss aversion, control and positive incentive groups were 69% (SE=5.12), 57% (SE = 4.60), and 58% (SE=3.75), respectively (p=0.099). The loss aversion group received an average of $50 compared with $38 (positive incentive) and $25 (control). CONCLUSION: In this randomized clinical trial, loss aversion incentives tended toward higher adherence to glucose self-monitoring among patients whose pregnancies were complicated by diabetes, though did not reach statistical significance. Further studies are needed to determine whether use of incentives improve maternal and neonatal outcomes. KEY POINTS: · Self-glucose monitoring is a critical part of diabetes management in pregnancy.. · Loss aversion financial incentives may increase adherence to glucose self-monitoring in pregnancy.. · The impact of testing incentives on maternal and neonatal outcomes requires further investigation..

More than grit: growing and sustaining physician-scientists in obstetrics and gynecology.

Obstetricians know the statistics-1 out of every 10 babies is born premature; preeclampsia affects 1 in 25 pregnant people; the United States has the highest rate of maternal mortality in the developed world. Yet, physicians and scientists still do not fully understand the biology of normal pregnancy, let alone what causes these complications. Obstetrics and gynecology-trained physician-scientists are uniquely positioned to fill critical knowledge gaps by addressing clinically-relevant problems through fundamental research and interpreting insights from basic and translational studies in the clinical context. Within our specialty, however, physician-scientists are relatively uncommon. Inadequate guidance, lack of support and community, and structural barriers deter fellows and early stage faculty from pursuing the physician-scientist track. One approach to help cultivate the next generation of physician-scientists in obstetrics and gynecology is to demystify the process and address the common barriers that contribute to the attrition of early stage investigators. Here, we review major challenges and propose potential pathways forward in the areas of mentorship, obtaining protected research time and resources, and ensuring diversity, equity, and inclusion, from our perspective as early stage investigators in maternal-fetal medicine. We discuss the roles of early stage investigators and leaders at the institutional and national level in the collective effort to retain and grow our physician-scientist workforce. We aim to provide a framework for early stage investigators initiating their research careers and a starting point for discussion with academic stakeholders. We cannot afford to lose the valuable contributions of talented individuals due to modifiable factors or forfeit our voices as advocates for the issues that impact pregnant populations.

Redesigned Care Delivery for Insulin-Requiring Diabetes in Pregnancy Improves Perinatal Glycemic Control While Reducing Neonatal Intensive Care Admissions, Length of Stay, and Costs.

Objective: We sought to improve perinatal glycemic control and downstream neonatal outcomes through redesigned ambulatory management for women with insulin-requiring diabetes in pregnancy. Methods: To address gaps in perinatal glycemic management of women with insulin-requiring diabetes in pregnancy, redesigned care delivery (RCD) utilized integrated practice unit and minimally disruptive medicine concepts with incorporation of cellular-enabled glucose monitoring. Primary outcomes of RCD (N = 129) included hemoglobin A1c ([HbA1c], within RCD cohort), and gestational age (GA) at delivery, neonatal intensive care (NICU) admission, and NICU length of stay (LOS) compared with a preredesign care cohort (Pre-RCD; N = 122). Secondary outcomes included facility, payer reimbursement, and program costs. Generalized linear models assessed continuous variables while logistic regression methods assessed categorical outcomes. Results: Utilizing RCD, 92% of women with an initial HbA1c <6.5% maintained glycemic control until delivery, and 67.2% with an initial HbA1c ≥6.5% achieved delivery levels <6.5%. NICU admissions and GA-adjusted LOS decreased significantly [Pre-RCD vs. RCD: NICU admissions, 41.0% vs. 27.3%, p < 0.024; NICU LOS (95% confidence interval [CI]), 21.9 (17.1-26.6) vs. 14.6 (9.1-20.1), p = 0.045]. Every 10 days of redesigned management decreased mean NICU LOS by 1 day. Mean payer neonatal reimbursements decreased over $18,000 per delivery (p = 0.08) compared with implementation costs of $1,942 per delivery. Conclusion: Redesigned perinatal diabetes care with remote glucose monitoring demonstrated improved outcomes and value through downstream neonatal outcomes and lower payer costs. Therefore, subsequent dissemination and sustainability of similar programs' improved outcomes will likely require payer support.

Cellular-Enabled Glucometers and Maternal Glucose Control: A Quality Improvement Initiative.

BACKGROUND: Management of diabetes in pregnancy is burdensome due to self-glucose monitoring, recording, and reporting demands. Cellular-enabled glucometers provide real-time transmission of glucose values independent of internet access and cell phone data plans. We describe a quality improvement (QI) intervention that introduced cellular-enabled glucometers for use during pregnancies complicated by diabetes. METHODS: Our aim was to improve maternal glucose control in a cohort of insulin-requiring pregnant women enrolled in a telemedicine diabetes program. During initial establishment of a QI program, women were offered cellular-enabled glucometers but could elect to keep their standard meter. The primary outcome evaluated was glycosylated hemoglobin A1c (HbA1c) at delivery. RESULTS: Baseline characteristics including initial HbA1c were similar between women using a standard glucometer (n = 45) and those using a cellular-enabled glucometer (n = 72). Women who used a cellular-enabled glucometer had a lower HbA1c at delivery compared to those using a standard glucometer (5.8% vs 6.3%, P = .03). This improvement was particularly notable for women with poor glucose control (defined as HbA1c >6.5%) at initial obstetric visit. Women with poor glucose control who used a cellular-enabled glucose monitor had significantly lower HbA1c at delivery (6.0% vs 6.8%, P = .03) and greater change from initial visit compared to those using a standard glucometer (-2.6% vs -1.4%, P = .02). No statistically significant differences were detected in tracked neonatal outcomes. CONCLUSION: For pregnancies complicated by insulin-requiring diabetes, use of cellular-enabled glucometers as part of a perinatal diabetes program improves glucose control at delivery with timely transmission of accurate values throughout gestation.

Adherence to self-glucose monitoring recommendations and perinatal outcomes in pregnancies complicated by diabetes mellitus.

BACKGROUND: Self-glucose monitoring is critical for the management of diabetes mellitus in pregnancy; yet, validated reports of adherence to testing recommendations and associated perinatal outcomes are limited. OBJECTIVE: Using cloud-based, self-glucose monitoring technology, we sought to answer the following questions: (1) Are there differences in the rates of testing adherence based on type of diabetes mellitus in pregnancy? (2) Is adherence to glucose monitoring recommendations associated with perinatal outcomes in pregnancies that are complicated by diabetes mellitus? We hypothesized that adherence to glucose testing recommendations varies by type of diabetes mellitus and that increased adherence to testing recommendations would be associated with improved perinatal outcomes. STUDY DESIGN: This single-center, prospective cohort study included women with type 2 diabetes mellitus and gestational diabetes mellitus who were enrolled in a perinatal diabetes program at <29 weeks gestation between December 2015 and June 2018. All women received a cellular-enabled glucometer that uploaded glucose values to a cloud-based, Health Insurance Portability and Accountability Act-compliant platform in real time that ensured transmission of accurate glucose values. The primary outcome was adherence to self-glucose monitoring recommendations. Four glucose checks were advised daily, and percentage of adherence was calculated. Secondary outcomes were preeclampsia, cesarean delivery, large-for-gestational-age neonates, and neonatal hypoglycemia. The study was powered to detect a 10% difference in the primary outcome of adherence to advised self-glucose monitoring by diabetes mellitus type. Adjusted risk ratios and 95% confidence intervals were generated with the use of logistic regression. RESULTS: This study included 103 eligible women. Baseline characteristics differed between groups, with women with type 2 diabetes mellitus having higher initial HgbA1c and body mass index when compared with women with gestational diabetes mellitus. No differences were noted in age or parity. Adherence was calculated over 20±6 weeks for women with type 2 diabetes mellitus compared with 9±4 weeks for women with gestational diabetes mellitus. Overall adherence to glucose monitoring was significantly less for women with type 2 diabetes mellitus compared with those with gestational diabetes mellitus. Mean testing adherence rates were 51%, 66%, and 70% for type 2 diabetes mellitus, and gestational diabetes mellitus, class A1 and A2, respectively (P=.016). We found that, for every 10% increase in adherence to testing recommendations, the odds of cesarean delivery, neonatal hypoglycemia, and large-for-gestational-age fetuses decreases by 15-20%. There was no association between adherence and rates of preeclampsia. CONCLUSION: This study shows that overall adherence to testing recommendations differs by diabetes mellitus type and is associated with neonatal outcomes. Improved outcomes with higher adherence may reflect more timely medication adjustments in response to real-time glucose values. Programs aimed at improving adherence could prove beneficial.

The focal adhesion kinase inhibitor PF-562,271 impairs primary CD4+ T cell activation.

The focal adhesion kinase inhibitor, PF-562,271, is currently in clinical development for cancer, however it is not known how PF-562,271 affects T cell function. Here, we demonstrate inhibitory effects of PF-562,271 on the activation of primary human and mouse T cells. PF-562,271 inhibits T cell receptor signaling-induced T cell adhesion to intercellular adhesion molecule-1 and T cell interactions with antigen-presenting cells. An additional focal adhesion kinase inhibitor, PF-573,228, and genetic depletion of focal adhesion kinase also impair T cell conjugation with antigen-presenting cells. PF-562,271 blocks phosphorylation of the signaling molecules zeta chain associate protein of 70 kDa, linker of activated T cells, and extracellular signal-regulated kinase, and impairs T cell proliferation. The effects observed on T cell proliferation cannot solely be attributed to focal adhesion kinase inhibition, as genetic depletion did not alter proliferation. The effect of PF-562,271 on T cell proliferation is not rescued when proximal T cell receptor signaling is bypassed by stimulation with phorbol-12-myristate-13-acetate and ionomycin. Taken together, our findings demonstrate that focal adhesion kinase regulates integrin-mediated T cell adhesion following T cell receptor activation. Moreover, our findings suggest that PF-562,271 may have immunomodulatory effects that could impact its therapeutic applications.

Acute hypotension associated with intraoperative cell salvage using a leukocyte depletion filter during management of obstetric hemorrhage due to amniotic fluid embolism.

Amniotic fluid embolism (AFE) is a rare but catastrophic obstetric complication that can lead to profound coagulopathy and hemorrhage. The role of cell salvage and recombinant human Factor VIIa (rFVIIa) administration in such cases remains unclear. We present a case of AFE and describe our experience with the use of cell salvage and rFVIIa administration during the resuscitation. Cell salvage and transfusion through a leukocyte depletion filter was attempted after the diagnosis of AFE was made, but the attempted transfusion was immediately followed by hypotension and a worsening of hemodynamics. rFVIIa, on the contrary, was used with clinical improvement in coagulopathy and without apparent adverse thrombotic effect.

Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays.

Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays.

Contact-dependent T cell activation and T cell stopping require talin1.

T cell-APC contact initiates T cell activation and is maintained by the integrin LFA-1. Talin1, an LFA-1 regulator, localizes to the immune synapse (IS) with unknown roles in T cell activation. In this study, we show that talin1-deficient T cells have defects in contact-dependent T cell stopping and proliferation. Although talin1-deficient T cells did not form stable interactions with APCs, transient contacts were sufficient to induce signaling. In contrast to prior models, LFA-1 polarized to T cell-APC contacts in talin1-deficient T cells, but vinculin and F-actin polarization at the IS was impaired. These results indicate that T cell proliferation requires sustained, talin1-mediated T cell-APC interactions and that talin1 is necessary for F-actin polarization and the stability of the IS.

PIPKI gamma 90 negatively regulates LFA-1-mediated adhesion and activation in antigen-induced CD4+ T cells.

T cell activation requires the formation and maintenance of stable interactions between T cells and APCs. The formation of stable T cell-APC contacts depends on the activation of the integrin LFA-1 (CD11aCD18). Several positive regulators of LFA-1 activation downstream of proximal TCR signaling have been identified, including talin; however, negative regulators of LFA-1 activity remain largely unexplored. Extended isoform of phosphatidylinositol phosphate kinase type I γ (PIPKIγ90) is a member of the type I phosphatidylinositol phosphate kinase family that has been shown previously to modulate talin activation of integrins through production of phosphatidylinositol 4,5-bisphosphate and direct binding to talin. In this study, we show that PIPKIγ90 negatively regulates LFA-1-mediated adhesion and activation of T cells. Using CD4(+) T cells from PIPKIγ90-deficient mice, we show that CD4(+) T cells exhibit increased LFA-1-dependent adhesion to ICAM-1 and increased rates of T cell-APC conjugate formation with enhanced LFA-1 polarization at the synapse. In addition to increased adhesiveness, PIPKIγ90-deficient T cells exhibit increased proliferation both in vitro and in vivo and increased production of IFN-γ and IL-2. Together, these results demonstrate that PIPKIγ90 is a negative regulator of Ag-induced T cell adhesion and activation.

Calpain 4 is not necessary for LFA-1-mediated function in CD4+ T cells.

BACKGROUND: T cell activation and immune synapse formation require the appropriate activation and clustering of the integrin, LFA-1. Previous work has reported that the calpain family of calcium-dependent proteases are important regulators of integrin activation and modulate T cell adhesion and migration. However, these studies have been limited by the use of calpain inhibitors, which have known off-target effects. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used a LoxP/CRE system to specifically deplete calpain 4, a small regulatory calpain subunit required for expression and activity of ubiquitously expressed calpains 1 and 2, in CD4+ T cells. CD4+ and CD8+ T cells developed normally in Capn4(F/F):CD4-CRE mice and had severely diminished expression of Calpain 1 and 2, diminished talin proteolysis and impaired casein degradation. Calpain 4-deficient T cells showed no difference in adhesion or migration on the LFA-1 ligand ICAM-1 compared to control T cells. Moreover, there was no impairment in conjugation between Capn4(F/F):CD4-CRE T cells and antigen presenting cells, and the conjugates were still capable of polarizing LFA-1, PKC-theta and actin to the immune synapse. Furthermore, T cells from Capn4(F/F):CD4-CRE mice showed normal proliferation in response to either anti-CD3/CD28 coated beads or cognate antigen-loaded splenocytes. Finally, there were no differences in the rates of apoptosis following extrinsic and intrinsic apoptotic stimuli. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that calpain 4 is not necessary for LFA-1-mediated adhesion, conjugation or migration. These results challenge previous reports that implicate a central role for calpains in the regulation of T cell LFA-1 function.

Calpain inhibition impairs TNF-alpha-mediated neutrophil adhesion, arrest and oxidative burst.

Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in many chronic inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils into areas of inflammation. TNF-alpha induces a stop signal that promotes neutrophil firm adhesion and inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates cytoskeletal reorganization during cell migration. Here, we show that calpain inhibition impairs TNF-alpha-induced neutrophil firm adhesion to fibrinogen-coated surfaces and the formation of vinculin-containing focal complexes. Calpain inhibition induces random migration in TNF-alpha-stimulated cells and prevents the generation of reactive oxygen species, but does not alter TNF-alpha-mediated activation of p38 MAPK and ERK MAPK. These findings suggest that the TNF-alpha-induced neutrophil arrest requires the activity of calpain independent of p38 MAPK and ERK signaling seen after TNF-alpha stimulation. Together, our data suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF-alpha-induced inflammatory responses.