Differences in estimated persistent inward currents between ankle flexors and extensors in humans.

Persistent inward currents (PICs) are responsible for amplifying motoneuronal synaptic inputs and contribute to generating normal motoneuron activation. Delta-F (ΔF) is a well-established method that estimates PICs in humans indirectly from firing patterns of individual motor units. Traditionally, motor unit firing patterns are obtained by manually decomposing electromyography (EMG) signals recorded through intramuscular electrodes (iEMG). A previous iEMG study has shown that in humans the elbow extensors have higher ΔF than the elbow flexors. In this study, EMG signals were collected from the ankle extensors and flexors using high-density surface array electrodes during isometric sitting and standing at 10-30% maximum voluntary contraction. The signals were then decomposed into individual motor unit firings. We hypothesized that comparable to the upper limb, the lower limb extensor muscles (soleus) would have higher ΔF than the lower limb flexor muscles [tibialis anterior (TA)]. Contrary to our expectations, ΔF was higher in the TA than the soleus during sitting and standing despite the difference in cohort of participants and body positions. The TA also had significantly higher maximum discharge rate than the soleus while there was no difference in rate increase. When only the unit pairs with similar maximum discharge rates were compared, ∆F was still higher in the TA than the soleus. Future studies will focus on investigating the functional significance of the findings.NEW & NOTEWORTHY With the use of high-density surface array electrodes and convolutive blind source separation algorithm, thousands of motor units were decomposed from the soleus and tibialis anterior muscles. Persistent inward currents were estimated under seated and standing conditions via delta-F (∆F) calculation, and the results showed that unlike the upper limb, the flexor has higher ∆F than the extensor in the lower limb. Future studies will focus on functional significance of the findings.

Carbohydrate Replacement of Rice or Potato with Lentils Reduces the Postprandial Glycemic Response in Healthy Adults in an Acute, Randomized, Crossover Trial.

Background: The postprandial blood glucose response (PBGR) following carbohydrate replacement of high-glycemic index (GI) foods with pulses, in a mixed meal, has not been accurately defined. Objective: We aimed to determine the extent to which PBGR and relative glycemic response (RGR) are lowered when half of the available carbohydrate (AC) from rice or potato is replaced with cooked lentils. Methods: Using a crossover design, 2 groups of 24 healthy adults randomly consumed 50 g AC from control white rice alone [mean ± SD body mass index (BMI, in kg/m2): 24.3 ± 0.5; mean ± SD age: 27.7 ± 1.2 y], instant potato alone (BMI: 24.0 ± 0.5; age: 27.4 ± 1.2 y), or the same starch source in a 50:50 AC combination with each of 3 types of commercially available lentils (large green, small green, split red). Fasting and postprandial blood samples were analyzed for glucose and insulin, and used to derive incremental area under the curve (iAUC), RGR, and maximum concentration (Cmax). Treatment effects were assessed with the use of repeated-measures ANOVA within the rice and potato treatments. Results: In comparison to rice alone, blood glucose iAUC and Cmax (P < 0.001) were lowered after consumption of rice with large green (P = 0.057), small green (P = 0.002), and split red (P = 0.006) lentils. Blood glucose iAUC and Cmax were also significantly lowered (P < 0.0001) after consumption of potato combined with each lentil, compared to potato alone. Plasma insulin iAUC and Cmax were significantly (P < 0.001) decreased when lentils were combined with potato, but not with rice. The RGRs of rice and potato were lowered by ∼20% and 35%, respectively, when half of their AC was replaced with lentils. Conclusions: Replacing half of the AC from high-GI foods with lentils significantly attenuates PBGR in healthy adults; this can contribute to defining a health claim for pulses and blood glucose lowering. This trial was registered at clinicaltrials.gov as NCT02426606.

Intrinsic excitability of human motoneurons in biceps brachii versus triceps brachii.

The intrinsic excitability of spinal motoneurons is mediated in part by the presence of persistent inward currents (PICs), which amplify synaptic input and promote self-sustained firing. Studies using animal models have shown that PICs are greater in extensor motoneurons over flexor motoneurons, but this difference has not yet been demonstrated in humans. The primary objective of this study was to determine whether a similar difference exists in humans by recording from motor units in biceps and triceps brachii during isometric contractions. We compared firing rate profiles of pairs of motor units, in which the firing rate of the lower-threshold "control" unit was used as an indicator of common drive to the higher-threshold "test" unit. The estimated contribution of the PIC was calculated as the difference in firing rate of the control unit at recruitment versus derecruitment of the test unit, a value known as the delta-F (ΔF). We found that ΔF values were significantly higher in triceps brachii (5.4 ± 0.9 imp/s) compared with biceps brachii (3.0 ± 1.4 imp/s; P < 0.001). This difference was still present even after controlling for saturation in firing rate of the control unit, rate modulation of the control unit, and differences in recruitment time between test and control units, which are known to contribute to ΔF variability. We conclude that human elbow flexor and extensor motor units exhibit differences in intrinsic excitability, contributing to different neural motor control strategies between muscle groups.

Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells.

Aggressive dissemination and metastasis of pancreatic ductal adenocarcinoma (PDAC) results in poor prognosis and marked lethality. Rho monomeric G protein levels are increased in pancreatic cancer tissue. As the mechanisms underlying PDAC malignancy are little understood, we investigated the role for cAMP in regulating monomeric G protein regulated invasion and migration of pancreatic cancer cells. Treatment of PDAC cells with cAMP elevating agents that activate adenylyl cyclases, forskolin, protein kinase A (PKA), 6-Bnz-cAMP, or the cyclic nucleotide phosphodiesterase inhibitor cilostamide significantly decreased migration and Matrigel invasion of PDAC cell lines. Inhibition was dose-dependent and not significantly different between forskolin or cilostamide treatment. cAMP elevating drugs not only blocked basal migration, but similarly abrogated transforming-growth factor-ß-directed PDAC cell migration and invasion. The inhibitory effects of cAMP were prevented by the pharmacological blockade of PKA. Drugs that increase cellular cAMP levels decreased levels of active RhoA or RhoC, with a concomitant increase in phosphorylated RhoA. Diminished Rho signaling was correlated with the appearance of thickened cortical actin bands along the perimeter of non-motile forskolin or cilostamide-treated cells. Decreased migration did not reflect alterations in cell growth or programmed cell death. Collectively these data support the notion that increased levels of cAMP specifically hinder PDAC cell motility through F-actin remodeling.

The effect of sampling strategies on assessment of water quality criteria attainment.

Sample locations for large river studies affect the representativeness of data, and thus can alter decisions made regarding river conditions and the need for interventions to improve water quality. The present study evaluated three water-quality sampling programs for Total Dissolved Solid (TDS) assessment in the Monongahela River from 2008 to 2012. The sampling plans cover the same 145 km of river but differ in frequency, sample location and type (e.g., river water sample vs drinking water plant intake sample). Differences resulting from temporal and spatial variability in sampling lead to different conclusions regarding water quality in the river (including regulatory listing decisions), especially when low flow leads to concentrations at or near the water quality criteria (500mg/L TDS). Drinking water samples exceeded the criteria 82 out of 650 samples (12.6%), while river water samples exceeded the criteria 47 out of 464 samples (10.1%). Different water sample types could provide different pictures of water quality in the river and lead to different regulatory listing decisions.

Source water changes and energy extraction activities in the Monongahela River, 2009-2012.

Fossil fuel wastewaters disposed of to surface waters used as sources for potable water supply have the potential to affect finished drinking water quality since these produced waters contain high concentrations of constituents that are of concern to drinking water providers (including dissolved solids, sulfate, chloride, and bromide). A 3-year field study in the Monongahela River was conducted to determine how constituent concentrations and associated loads changed in the river basin, and whether these changes were caused by surface discharges associated with shale gas extraction. Low flow during the dry summer of 2010 contributed to increases in bromide concentrations at drinking water intakes; however, similar low flow conditions in summer of 2012 did not, indicating bromide loads discharged to the river decreased from 2010 to 2012. Analysis of bromide to chloride ratios as an indicator of fossil fuel associated wastewaters showed significant increases in Br/Cl at drinking water intakes over the first year of the study (2009-2010). This indicator ratio declined in the final year of the study (2011-2012), likely associated with a voluntary decrease in the use of surface-discharging treatment plants for shale gas wastewater disposal. Predictions of future concentrations based on historical flow data are also presented.

Participant satisfaction with Wellness Recovery Action Plan (WRAP).

Outpatient programs are often promoted as vehicles for mental health recovery. Yet, few programs include patients' perspectives about their satisfaction with these programs. This descriptive, cross-sectional survey investigated patients' satisfaction with Wellness Recovery Action Plan (WRAP). Participants completed the Mental Health Statistics Improvement Program instrument (n = 26) and qualitative interviews (n = 18). Data were analyzed using multivariate statistics (α = .05) and content analysis. Three composite variables explained 48% of the variance (p = .00) in patient satisfaction. Four themes emerged: Retrospective Desire for Early WRAP Introduction, Pay It Forward, Unconditional Relational Support, and It Takes Time. Future research is warranted to promote WRAP's use in broader settings.

Demographic and clinical factors associated with variations in opioid administration using conscious sedation during HDR brachytherapy for cervical cancer.

PURPOSE/OBJECTIVES To examine patient characteristics that predispose to higher opioid administration during tandem and ovoid (T&O) high-dose rate (HDR) brachytherapy. METHODS A single-institution retrospective review was performed on patients who underwent brachytherapy for cervical cancer. Patients were included if they received at least one fraction of HDR T&O brachytherapy with analgesia administration recorded in the Medication Administration Record. Fentanyl dose was dichotomized as "low" (mean <125 µg per fraction), or "high" (mean ≥ 125 µg per fraction). Descriptive statistics and multiple logistic regression analysis were performed comparing mean opioid dose per fraction with demographic and clinical information. RESULTS From July 2014 through May 2020, 113 patients underwent 531 T&O HDR brachytherapy fractions with oral benzodiazepine and intravenous opioid fentanyl for conscious sedation. The median opioid dose per fraction was 100 µg fentanyl (range 0-250 µg). Using multiple logistic regression analysis, younger age (OR 1.071, p = 0.002) and higher BMI (OR 1.091, p = 0.019) were associated with increased opioid administration during brachytherapy. Black women received less opioid during brachytherapy when compared to White women (OR 0.296, p = 0.047). FIGO stage, ECOG score, smoking status, prior narcotic use, prior illicit drug use, parity, prior cervical procedure, Smit sleeve placement, and distance to treatment center were not associated with high opioid dose. CONCLUSION Cervical cancer patients who are younger or have higher BMI receive more narcotic analgesia during HDR brachytherapy whereas Black women received less narcotic analgesia, irrespective of age and BMI. This underscores the immediate need to address how pain is assessed and managed during brachytherapy.

Clinical Outcomes of Patients With pT1-T2N0 Oral Tongue Squamous Cell Carcinoma.

OBJECTIVE: The objective of this study was to evaluate the clinical outcomes in a cohort of patients with early-stage oral tongue squamous cell carcinoma (OTSCC). MATERIALS AND METHODS: We conducted a retrospective analysis of patients with pT1-T2N0 (American Joint Committee on Cancer [AJCC] seventh edition) OTSCC treated from 2000 to 2018. Two-year actuarial rates of local regional control, cancer-specific survival, and overall survival were calculated for the entire cohort and patients with/without adjuvant radiation. RESULTS: Ninety-six patients met the criteria with a median follow-up of 4 years; 14 had adjuvant radiation, while 82 had surgery alone. Two-year local regional control was 82.7% (75.4% to 90.8%) for the entire cohort, 84.9% (77.8% to 93.2%) for surgery only, and 70.7% (50.2% to 99.6%) for patients with adjuvant radiation. Two-year progression-free survival was 82.7% (75.3% to 90.8%). Of the 20 patients with recurrence, 11 (55%) were successfully salvaged. CONCLUSION: Local regional recurrence remains modest in early-stage OTSCC, but salvage is possible with high survival rates. LEVEL OF EVIDENCE: Level III-retrospective cohort study.

Motor Unit Discharge Variability Is Increased in Mild-To-Moderate Parkinson's Disease.

Individuals with Parkinson's disease (PD) demonstrate deficits in muscle activation such as decreased amplitude and inappropriate bursting. There is evidence that some of these disturbances are more pronounced in extensor vs. flexor muscles. Surface EMG has been used widely to quantify muscle activation deficits in PD, but analysis of discharge of the underlying motor units may provide greater insight and be more sensitive to changes early in the disease. Of the few studies that have examined motor unit discharge in PD, the majority were conducted in the first dorsal interosseous, and no studies have measured motor units from extensor and flexor muscles within the same cohort. The objective of this study was to characterize the firing behavior of single motor units in the elbow flexor and extensor muscles during isometric contractions in people with mild-to-moderate PD. Ten individuals with PD (off-medication) and nine healthy controls were tested. Motor unit spike times were recorded via intramuscular EMG from the biceps and triceps brachii muscles during 30-s isometric contractions at 10% maximum voluntary elbow flexion and elbow extension torque, respectively. We selected variables of mean motor unit discharge rate, discharge variability, and torque variability to evaluate motor abnormalities in the PD group. The effects of group, muscle, and group-by-muscle on each variable were determined using separate linear mixed models. Discharge rate and torque variability were not different between groups, but discharge variability was significantly higher in the PD group for both muscles combined (p < 0.0001). We also evaluated the asymmetry in these motor variables between the triceps and biceps for each individual participant with PD to evaluate whether there was an association with disease severity. The difference in torque variability between elbow flexion and extension was significantly correlated with both the Hoehn and Yahr scale (rho = 0.71) and UPDRS (rho = 0.62). Our findings demonstrate that variability in motor output, rather than decreased discharge rates, may contribute to motor dysfunction in people with mild-to-moderate PD. Our findings provide insight into altered neural control of movement in PD and demonstrate the importance of measuring from multiple muscles within the same cohort.

Resistance training with instability is more effective than resistance training in improving spinal inhibitory mechanisms in Parkinson's disease.

This study assessed 1) the effects of 12 wk of resistance training (RT) and resistance training with instability (RTI) on presynaptic inhibition (PSI) and disynaptic reciprocal inhibition (DRI) of patients with Parkinson's disease (PD); 2) the effectiveness of RT and RTI in moving PSI and DRI values of patients toward values of age-matched healthy controls (HC; Z-score analysis); and 3) associations between PSI and DRI changes and clinical outcomes changes previously published. Thirteen patients in RT group, 13 in RTI group, and 11 in a nonexercising control group completed the trial. While RT and RTI groups performed resistance exercises twice a week for 12 wk, only the RTI group used unstable devices. The soleus H reflex was used to evaluate resting PSI and DRI before and after the experimental protocol. The HC (n = 31) was assessed at pretest only. There were significant group × time interactions for PSI (P < 0.0001) and DRI (P < 0.0001). RTI was more effective than RT in increasing the levels of PSI (P = 0.0154) and DRI (P < 0.0001) at posttraining and in moving PSI [confidence interval (CI) 0.1-0.5] and DRI (CI 0.6-1.1) levels to those observed in HC. There was association between DRI and quality of life changes (r = -0.69, P = 0.008) and a strong trend toward association between PSI and postural instability changes (r = 0.60, P = 0.051) after RTI. RTI increased PSI and DRI levels more than RT, reaching the average values of the HC. Thus RTI may cause plastic changes in PSI and DRI pathways that are associated with some PD clinical outcomes. NEW & NOTEWORTHY: Patients with Parkinson's disease (PD) have motor dysfunction. Spinal inhibitory mechanisms are important for modulating both supraspinal motor commands and sensory feedback at the spinal level. Resistance training with instability was more effective than resistance training in increasing the levels of presynaptic inhibition and disynaptic reciprocal inhibition of lower limb at rest of the patients with PD, reaching the average values of the healthy controls.

Differences in the Phosphorylation-Dependent Regulation of Prenylation of Rap1A and Rap1B.

Two isoforms of the small GTPase Rap1, Rap1A and Rap1B, participate in cell adhesion; Rap1A promotes steady state adhesion, while Rap1B regulates dynamic changes in cell adhesion. These events depend on the prenylation of Rap1, which promotes its membrane localization. Here, we identify previously unsuspected differences in the regulation of prenylation of Rap1A versus Rap1B, due in part to their different phosphorylation-dependent interactions with the chaperone protein SmgGDS-607. Previous studies indicate that the activation of Gαs protein-coupled receptors (GPCRs) phosphorylates S-179 and S-180 in the polybasic region (PBR) of Rap1B, which inhibits Rap1B binding to SmgGDS-607 and diminishes Rap1B prenylation and membrane localization. In this study, we investigate how phosphorylation in the PBR of multiple small GTPases, including K-Ras4B, RhoA, Rap1A, and Rap1B, affects their binding to SmgGDS, with emphasis on differences between Rap1A and Rap1B. We identify the amino acids in SmgGDS-607 necessary for binding of Rap1A and Rap1B, and present homology models examining the binding between Rap1A or Rap1B and SmgGDS-607. Unlike Rap1B, phosphorylation in the PBR of Rap1A does not detectably inhibit its prenylation or its binding to SmgGDS-607. Activation of GPCRs suppresses Rap1A prenylation, but unlike this effect on Rap1B, the GPCR-mediated suppression of Rap1A prenylation can occur independently of Rap1A phosphorylation and does not detectably diminish Rap1A membrane localization. These data demonstrate unexpected evolutionarily conserved differences in the ability of GPCRs to regulate the prenylation of Rap1B compared to Rap1A.

ß-Adrenergic receptors suppress Rap1B prenylation and promote the metastatic phenotype in breast cancer cells.

A greater understanding of the molecular basis of breast cancer metastasis will lead to identification of novel therapeutic targets and better treatments. Rap1B is a small GTPase that suppresses the metastasis of breast cancer cells by increasing cell-cell adhesion. In breast cancer, a decrease in Rap1B prenylation and subsequent loss of Rap1B at the plasma membrane decreases cell-cell adhesion and increases cell scattering, which promotes the metastatic phenotype. Protein kinase A (PKA) was recently found to phosphorylate Rap1B and inhibit its prenylation. PKA is activated by G protein-coupled receptors (GPCR) that stimulate Gαs. In this study, we investigated whether the general Gαs activator, cholera toxin, and agonists of the ß-adrenergic receptor (ßAR), which is a Gαs-coupled GPCR, promote Rap1B phosphorylation and inhibit its prenylation. We show here that cholera toxin and ßAR activation phosphorylate Rap1B and inhibit its prenylation and membrane localization, reducing cell-cell adhesion and promoting cell scattering. Furthermore, we report that breast cancer cell migration is decreased by the FDA-approved ß-blocker, propranolol. Pharmacological targeting of GPCRs, especially those such as the ßAR that are regulated by FDA-approved drugs, to increase cell adhesion and decrease cell scattering could provide a promising therapeutic approach to reduce breast cancer metastasis.

SmgGDS-558 regulates the cell cycle in pancreatic, non-small cell lung, and breast cancers.

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (NSCLC) xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.

Integrin α(v)ß3 as a PET imaging biomarker for osteoclast number in mouse models of negative and positive osteoclast regulation.

PURPOSE: The goal of this study was to determine the specificity of 64Cu-CB-TE2A-c(RGDyK) (64Cu-RGD) for osteoclast-related diseases, such as Paget's disease or rheumatoid arthritis. PROCEDURES: C57BL/6 mice were treated systemically with osteoprotegerin (OPG) for 15 days or RANKL for 11 days to suppress and stimulate osteoclastogenesis, respectively. The mice were then imaged by positron emission tomography/computed tomography using 64Cu-RGD, followed by determination of serum TRAP5b and bone histology. Standard uptake values were determined to quantify 64Cu-RGD in bones and other tissues. RESULTS: Mice treated with OPG showed decreased bone uptake of 64Cu-RGD at 1, 2, and 24 h post-injection of the tracer (p < 0.01 for all time points) compared to vehicle controls, which correlated with a post-treatment decrease in serum TRAP5b. In contrast, mice treated with RANKL showed significantly increased bone uptake at 2 h post-injection of (64Cu-RGD (p < 0.05) compared to the vehicle control group, corresponding to increased serum TRAP5b and OC numbers as determined by bone histology. CONCLUSIONS: These data demonstrate that 64Cu-RGD localizes to areas in bone with increased osteoclast numbers and support the use of 64Cu-RGD as an imaging biomarker for osteoclast number that could be used to monitor osteoclast-related pathologies and their treatments.

Molecular imaging of very late antigen-4 (α4ß1 integrin) in the premetastatic niche.

UNLABELLED: Despite advances in cancer treatment over the past few decades, metastatic disease remains the primary cause of morbidity and mortality. Recent reports suggest the formation of a "premetastatic niche" before the metastatic cascade, where niche is defined as the microenvironment for tumor cells to be able to engraft and proliferate at secondary sites. Bone marrow-derived (BMD) cells that express vascular endothelial growth factor receptor-1 and very late antigen-4 (VLA-4) have been shown to arrive at sites of metastasis to form a receptive environment for tumor cells. Here we describe experiments toward imaging of VLA-4-positive BMD cells using a high-affinity PET probe, (64)Cu-labeled 11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] hexadecane (CB-TE2A)-LLP2A. METHODS: VLA-4-negative MDA-MB-231/firefly luciferase (fluc) human breast tumor cells were injected intraarterially in the left ventricle in nude mice. Tumor metastasis in mice was monitored for 30 d by bioluminescence imaging and small-animal PET/CT. Small-animal PET images were collected 2 h after mice were injected in the tail vein with (64)Cu-CB-TE2A-LLP2A (5.6-11.1 MBq [150-300 µCi; specific activity, 400 µCi/µg]). Cellular uptake of (64)Cu-CB-TE2A-LLP2A was determined in VLA-4-positive B16F10 mouse melanoma cells and VLA-4-negative MDA-MB-231/fluc human breast cancer tumor cells. Biodistribution experiments in nude mice bearing VLA-4-positive B16F10 subcutaneous tumors in the flank were conducted to validate targeting of VLA-4-positive cells in vivo. RESULTS: Uptake of (64)Cu-CB-TE2A-LLP2A was higher in VLA-4-positive human melanoma B16F10 cells than in VLA-4-negative MDA-MB-231 cells (P < 0.05). In B16F10 tumor-bearing mice, (64)Cu-CB-TE2A-LLP2A had high uptake in the VLA-4-rich organs marrow, spleen, and tumor (11.26% ± 2.59%, 8.36% ± 2.15%, and 3.09% ± 0.58% injected dose/g, respectively). Cumulative standardized uptake value data from 2 independent studies (n = 7 and 8 mice) on nude mice implanted with VLA-4-negative MDA-MB-231/fluc human breast tumor cells suggested an influx of VLA-4-positive BMD cells that corresponded to metastasis (P < 0.05). Immunohistochemical analysis and flow cytometry also showed upregulation of VLA-4-positive cell clusters and BMD cells at the metastatic sites, providing evidence for noninvasive imaging of BMD cells in the premetastatic niche. CONCLUSION: The results of the study demonstrated the potential of PET with VLA-4-targeted (64)Cu-CB-TE2A-LLP2A to visualize BMD cell reorganization and expansion noninvasively in vivo.

Combined effects of calcineurin inhibitors or sirolimus with anti-CD40L mAb on alloengraftment under nonmyeloablative conditions.

The immunosuppressive drugs, cyclosporine A (CsA), tacrolimus, or sirolimus, were analyzed as single agents and in combination with anti-CD40L monoclonal antibody (mAb) for their effects on alloengraftment in mice conditioned with minimal total body irradiation (TBI). Whereas anti-CD40L mAb facilitated chimerism, neither sirolimus nor CsA resulted in substantial alloengraftment. However, sirolimus was synergistic with anti-CD40L mAb for inducing donor chimerism. Contrary to expectations, CsA, a T-cell receptor (TCR) signaling inhibitor, did not abrogate anti-CD40L mAb-facilitated engraftment but rather increased engraftment in anti-CD40L mAb-treated mice. Although tacrolimus alone or with anti-CD40L mAb resulted in similar levels of donor chimerism, donor T-cell reconstitution was very low in tacrolimus-treated mice. At 1 week after transplantation, CsA decreased thymic numbers more profoundly than sirolimus or tacrolimus in anti-CD40L mAb-treated recipients. In contrast, only sirolimus resulted in a decrease in host splenic T-cell numbers in anti-CD40L mAb-treated recipients. Importantly, sirolimus and anti-CD40L mAb induced profound donor tolerance with 100% acceptance of donor skin grafts placed early after bone marrow transplantation (BMT). In contrast, anti-CD40L mAb alone or in combination with CsA resulted in 12% or less donor skin graft acceptance early (1 month) and 60% or less later (3 months) after BMT. These data have clinical relevance and indicate that immunosuppressive pharmacologic agents enhance anti-CD40L mAb-facilitated alloengraftment and tolerance induction under nonmyeloablative conditioning.