NKG2D receptor signaling shapes T cell thymic education.

The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vß chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

Reduced Time to Surgery and Prophylactic Fasciotomy May Result in Improved Outcomes in Popliteal Artery Injuries.

BACKGROUND: Popliteal artery injuries are uncommon and often result in limb loss or long-term limb dysfunction. The aims of this study were (1) to evaluate the association between predictors and outcomes and (2) to validate the rational of systematic early fasciotomy. METHODS: This retrospective cohort study included 122 patients (80% men, n = 100) who underwent surgery for popliteal artery injuries from October 2018 to March 2021 in southern Vietnam. Primary outcomes included primary and secondary amputation. The associations between predictors and primary amputation were analyzed using logistic regression models. RESULTS: Among the 122 patients, 11 (9%) underwent primary amputation, while 2 (1.6%) had secondary amputation. Longer time to surgery was associated with increased odds of amputation (odds ratio = 1.65; 95% confidence interval, 1.2 to 2.2 for every 6 hr). Severe limb ischemia was also associated with a 50-fold increase in the risk of primary amputation (adjusted odds ratio = 49.9; 95% confidence interval, 6 to 418, P = 0.001). Furthermore, 11 patients (9%) without signs of severe limb ischemia and acute compartment syndrome on admission were found to have myonecrosis of at least one muscle compartment during fasciotomy. CONCLUSIONS: The data suggest that among patients with popliteal artery injuries, prolonged time before surgery and severe limb ischemia are associated with increased risk of primary amputation, whereas early fasciotomy may lead to improved outcomes.

Left renal vein as a graft for aortic reconstruction in a patient with primary aortoenteric fistula secondary to a mycotic aneurysm.

Description of the use of the left renal vein for aortic reconstruction in primary aortoenteric fistula secondary to a mycotic aneurysm has not been found in the literature. We report here a case of primary aortoenteric fistula secondary to a mycotic aneurysm with gross retroperitoneal contamination that was successfully treated by using a left renal vein graft for aortic reconstruction.

Eustachian Tube Balloon Dilation: A Systematic Review and Meta-analysis of Treatment Outcomes.

OBJECTIVE: To examine the effectiveness of eustachian tube balloon dilation for the treatment of eustachian tube dysfunction. DATA SOURCES: PubMed, Scopus, and Google Scholar. REVIEW METHODS: A systematic review of eustachian tube balloon dilation for the treatment of eustachian tube dysfunction was conducted following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines to identify randomized control trials and prospective and retrospective studies published prior to January 31, 2019. Meta-analysis of proportions evaluated 7-item Eustachian Tube Dysfunction Questionnaire (ETDQ7) scores, tympanometry, otoscopy findings, and the ability to perform a Valsalva maneuver. RESULTS: The systematic review identified 35 studies. Twelve studies met inclusion for meta-analysis (448 patients). Mean ETDQ7 scores decreased by 2.13 from baseline to 6 weeks (95% CI, -3.02 to -1.24; P < .001). From baseline to 6 weeks, 53.0% of patients had improvement in tympanograms (P < .001). At the long-term point (3-12 months), 50.5% of patients had improved tympanograms from baseline (P < .001). There was no significant difference in the proportion of improved tympanograms at 6 weeks compared to long term (P = .535). Normal otoscopy exams at baseline increased by 30.0% at 6 weeks (P < .001) and 55.4% in the long term (P < .001). There was a 67.8% increase in proportion of patients able to perform a Valsalva maneuver in the long term compared to baseline (P < .001). CONCLUSION: Eustachian tube balloon dilation appears to be associated with improvement in subjective and objective treatment outcome metrics. The improvement appears stable at 3 to 12 months after dilation. Patients with eustachian tube dysfunction are likely to benefit from balloon dilation, particularly those with medication-refractory disease.

Systematic Review and Meta-analysis of SNOT-22 Outcomes after Surgery for Chronic Rhinosinusitis with Nasal Polyposis.

Objective Wide variation exists regarding reported outcomes after endoscopic sinus surgery (ESS) for chronic rhinosinusitis with nasal polyps (CRSwNP). This study seeks to combine data across studies to generate a summary measure and explore factors that might lead to variation. Data Sources OVID Medline, Scopus, EbscoHost, Database of Abstracts and Reviews of Effects, Health Technology Assessment, and National Health Service Economic Evaluation Database. Review Methods A search was performed following the PRISMA guidelines. Two independent researchers conducted a search using the mentioned data sources. Studies published before August 29, 2016, that involved ESS to treat CRSwNP were included. Mean changes in Sinonasal Outcome Test-22 (SNOT-22) scores were determined through metaregression of the following independent variables: publication year, sex, age, allergy status, asthma, tobacco use, prior surgery, follow-up length, and preoperative SNOT-22. Results Fifteen articles with 3048 patients treated with ESS met inclusion criteria. Pooled analyses of SNOT-22 scores revealed a mean change of 23.0 points (95% CI, 20.2-25.8; P < .001). A metaregression of patient factor effects on the mean change of SNOT-22 scores demonstrated that age ( r = 0.71, P = .01), asthma ( r = 0.21, P = .01), prior ESS ( r = 0.29, P = .01), and preoperative SNOT-22 score ( r = 0.4, P < .01) correlated with greater improvement in SNOT-22 scores. Tobacco use ( r = -0.91, P = .01) and longer lengths of follow-up ( r = -0.45, P < .01) were associated with less improvement in SNOT-22 scores. Conclusions Quality-of-life outcomes are significantly improved after ESS among patients with CRSwNP. Patient-specific factors may affect the degree of SNOT-22 change after surgery.

Titanium Ossicular Chain Reconstruction Revision Success and Preoperative Factors Predicting Success.

Objective Determine rates of success after revision titanium ossicular chain reconstruction with either partial or total ossicular replacement prosthesis and assess preoperative factors predicting positive outcomes. Study Design Case series with planned data collection. Setting Tertiary hospital. Subjects and Methods The charts of 76 surgical patients who underwent revision titanium ossicular chain reconstruction from 2003 to 2014 were abstracted from a prospectively maintained database at the Medical University of South Carolina. Postoperative air-bone gap (ABG) after revision surgery at short-term (<6 months) and intermediate to long-term (>1 year) follow-up and preoperative factors associated with postoperative ABG ≤20 dB were recorded. A paired t test or Wilcoxon signed-rank sum test was utilized to compare preoperative, short-term, or intermediate to long-term results. Results Seventy-six patients underwent revision ossiculoplasty and met inclusion criteria. Mean postoperative ABG was 22.5 at short-term follow-up ( P < .0001) and 24.4 at intermediate to long-term follow-up ( P = .003). Postoperative ABG ≤20 dB was achieved in 51.5% of patients. The only preoperative factor associated with postoperative ABG ≤20 dB was location of original primary ossiculoplasty ( P = .01). Conclusions This is one of the larger studies involving revision titanium ossiculoplasty. Revision surgery showed a significant improvement in postoperative ABG. The location of the original ossiculoplasty correlated with success of revision surgery (defined as postoperative ABG ≤20 dB). Patients who had the primary ossiculoplasty at an outside hospital may have better audiometric outcomes than patients who had it at a tertiary hospital.

IL22 regulates human urothelial cell sensory and innate functions through modulation of the acetylcholine response, immunoregulatory cytokines and antimicrobial peptides: assessment of an in vitro model.

Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs suitable for the study of human urinary tract disorders, including interactions between urothelium and IL22-producing cells.

Enhancing T lineage production in aged mice: a novel function of Foxn1 in the bone marrow niche.

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. Whereas reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with early T cell progenitors (ETP), derived from multipotent progenitors (MPP) in the bone marrow (BM). A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1 transgenic (Tg); whether the effect is initiated in the BM with MPP is not known. In this study, we report that Foxn1 is expressed in wild-type BM and overexpressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of hematopoietic stem cells. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, common lymphoid progenitors and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared with wild type. We identified a novel population of lineage(neg/low), CD45(pos) EpCAM(pos), SCA1(pos), CD117(neg), CD138(neg), MHCII(neg) cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, and the Foxn1 BM niche may function in skewing MPP development toward T lineage progenitors.

A 7-year review of the safety of tonsillectomy during short-term medical mission trips.

OBJECTIVE: To evaluate the safety of tonsillectomy in a short-term medical mission setting. STUDY DESIGN: Retrospective chart review. SETTING: Catholic mission hospital in Guatemala. SUBJECTS AND METHODS: During 7 consecutive annual mission trips from 2004 to 2010, patients received tonsillectomy and adenotonsillectomy. Established safety protocol requires candidates for tonsillectomy to agree to stay within 1 hour of the hospital for 10 days following the operation. This study includes all tonsillectomy patients regardless of age or indication for tonsillectomy. The primary outcome measures include posttonsillectomy hemorrhage, nasopharyngeal reflux, readmission for dehydration, and mortality. This is a novel study as the work performed by most short-term medical missions is unregulated and unevaluated. RESULTS: Medical charts were available for 197 (96.6%) of the 204 patients receiving tonsillectomy in the 7-year period; this was the only inclusion criterion. Ninety-nine (50.3%) patients had tonsillectomy concomitantly with adenoidectomy. Patients ranged in age from 3 to 66 years. The mean (SD) age was 17.2 (14.0) years. The study team found documentation of postoperative complications in 3 (1.5%) patients; 2 experienced postoperative hemorrhage, 1 within the first postoperative hour and 1 at 96 hours. The final patient returned to the hospital within 24 hours symptomatic for dehydration. CONCLUSIONS: The authors have evaluated a protocol for tonsillectomy patients in a specific setting and believe their data represent satisfactory outcomes for the reviewed patients. The generalizability of this information is uncertain, but safety protocols should be established on all short-term medical missions to prevent untoward complications.

Mixed lineage kinase 3 modulates ß-catenin signaling in cancer cells.

Expression of ß-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3ß (GSK3ß)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/ß-catenin signaling or mutations of members of the degradation complex) can result in ß-catenin stabilization and activation of ß-catenin/T-cell factor (TCF) signaling. ß-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control ß-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between ß-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce ß-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with ß-catenin and phosphorylate it in vitro. Overexpression of GSK3ß-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3ß pathway. Surprisingly, despite stabilizing ß-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A ß-catenin. These resulted in reduced expression of ß-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease ß-catenin/TCF interaction but promoted interaction between ß-catenin and KLF4, a known repressor of ß-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and ß-catenin resulted in significant G(2)/M arrest. These studies provide a novel insight toward the regulation of ß-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of ß-catenin signaling.

Overexpression of Foxn1 attenuates age-associated thymic involution and prevents the expansion of peripheral CD4 memory T cells.

The forkhead box n1 (Foxn1) transcription factor is essential for thymic organogenesis during embryonic development; however, a functional role of Foxn1 in the postnatal thymus is less well understood. We developed Foxn1 transgenic mice (Foxn1Tg), in which overexpression of Foxn1 is driven by the human keratin-14 promoter. Expression of the Foxn1 transgene increased the endogenous Foxn1 levels. In aged mice, overexpression of Foxn1 in the thymus attenuated the decline in thymocyte numbers, prevented the decline in frequency of early thymic progenitors, and generated a higher number of signal joint TCR excised circle. Histologic studies revealed that structural alterations associated with thymic involution were diminished in aged Foxn1 Tg. Total numbers of EpCAM+ MHC II+ and MHC II(hi) thymic epithelial cells were higher in young and old Foxn1Tg and more EpCAM+ MHC II(hi) TEC expressed Ki-67 in aged Foxn1Tg compared with WT. Furthermore, Foxn1Tg displayed a significant reduction in the expansion of splenic CD4+ memory compartments and attenuated the decline in CD4+ and CD8+ naive compartments. Our data indicate that manipulation of Foxn1 expression in the thymus ameliorates thymopoiesis in aged mice and offer a strategy to combat the age-associated decline in naive T-cell production and CD4 naive/memory ratios in the elderly.

A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

OBJECTIVE: A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. RESULTS: In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. CONCLUSIONS: We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy.

In vitro requirement for periostin in B lymphopoiesis.

B lymphopoiesis arrests in rabbits by 4 months of age. To identify molecules that contribute to this arrest, cDNA-representational difference analysis on BM stromal cells from young and adult rabbits showed that expression of Postn that encodes for the extracellular matrix protein periostin dramatically reduced with age. Postn-small interfering RNA OP9 cells lost their capacity to support B-cell development from rabbit or murine BM cells, and reexpression of periostin restored this potential, indicating an in vitro requirement for periostin in B lymphopoiesis. In our system, we determined that periostin deficiency leads to increased cell death and decreased proliferation of B-lineage progenitors. Further, RGD peptide inhibition of periostin/α(v)ß(3) interaction resulted in a marked decrease in B lymphopoiesis in vitro. Microarray analysis of the Postn-small interfering RNA OP9 cells showed decreased expression of key B-lymphopoietic factors, including IL-7 and CXCL12. In vivo, unidentified molecule(s) probably compensate periostin loss because Postn(-/-) mice had normal numbers of B-cell progenitors in BM. We conclude that the decline in periostin expression in adult rabbit BM does not solely explain the arrest of B lymphopoiesis. However, the interaction of periostin with α(v)ß(3) on lymphoid progenitors probably provides both proliferative and survival signals for cells in the B-cell development pathway.

Migration of immature and mature B cells in the aged microenvironment.

Studies in aged mice show that the architecture of B-cell areas appears disrupted and that newly made B cells fail to incorporate into the spleen. These observations may reflect altered migration of immature and mature B cells. Using adoptive transfer, we tested the effect of the aged microenvironment and the intrinsic ability of donor B cells from aged mice to migrate to spleens of intact hosts. Spleens of aged recipients were deficient in attracting young or old donor immature B cells. In contrast, immature and mature B cells maintained an intrinsic ability to migrate to young recipient spleens, except that as the aged immature B cells matured, fewer appeared to enter the recirculating pool. CXCL13 protein, which is necessary for the organization of B-cell compartments, was elevated with age and differences in CXCL13 distribution were apparent. In aged spleens, CXCL13 appeared less reticular, concentrated in patches throughout the follicles, and notably reduced in the MAdCAM-1(+) marginal reticular cells located at the follicular edge. Despite these differences, the migration of young donor follicular B cells into the spleens of old mice was not impacted; whereas, migration of young donor marginal zone B cells was reduced in aged recipients. Finally, the aged bone marrow microenvironment attracted more donor mature B cells than did the young marrow. Message for CXCL13 was not elevated in the marrow of aged mice. These results suggest that the aged splenic microenvironment affects the migration of immature B cells more than mature follicular B cells.

The effects of microenvironment and internal programming on plasma cell survival.

Two populations of plasma cells (PCs) are formed after immunization. A short-lived population in the spleen and lymph nodes provides rapid protection. A long-lived population, mainly in the bone marrow, provides lasting immunity. The mechanisms responsible for the differences in PCs life span remain largely unknown. The goal of the current study was to compare the intrinsic survival capacity of isolated short-lived (spleen) versus long-lived (bone marrow) PCs. We approached this question by using a previously established in vitro model that measures PC survival in a supportive stromal environment. Regardless of the tissue source or isolation time point after immunization, the two PC populations showed similar intrinsic ability to survive in vitro. To test differences in the stromal microenvironments, stromal cells from marrow, spleen or lymph nodes were evaluated for ability to support PCs survival. Survival of isolated PC was always greater when co-cultured with marrow stromal cells compared with those from spleen (or lymph node) despite the finding that IL-6, necessary for PC survival in culture, was secreted by all three stromal cell sources. Additionally, low expression of B-cell-activating factor belonging to the tumor necrosis factor-family was detected in all three stromal isolates. In contrast, marrow stromal cells were distinguished by cell-surface phenotype and CXC chemokine ligand (CXCL)12, IL-7 and stem cell factor expression. Although CXCL12 has been suggested as a possible survival factor for PC, addition or neutralization of CXCL12 had minimal effect on PC survival. We conclude the mechanisms regulating PC longevity appear extrinsically driven and marrow favored, but the factors that give marrow stromal cells a unique advantage remain unknown.

The serotonin-1A agonist ipsapirone prevents ethanol-associated death of total rhombencephalic neurons and prevents the reduction of fetal serotonin neurons.

Previously, this laboratory showed that in utero and in vitro ethanol exposure significantly reduces developing serotonin (5-HT) neurons and that treatment with a 5-HT1A agonist such as buspirone or ipsapirone prevents the ethanol-associated loss. The present study investigated whether ethanol decreases fetal rhombencephalic neurons, including 5-HT neurons, by causing apoptosis. We also investigated whether ipsapirone prevents the ethanol-associated deficit of fetal rhombencephalic neurons by reducing apoptosis. The results of these studies strongly suggest that the ethanol-associated reduction in fetal rhombencephalic neurons that accompanies both in utero and in vitro exposure to physiological concentrations of ethanol is associated with increased apoptosis in these neurons. A physiological concentration of ethanol (i.e., 50 mM) increases apoptosis in fetal rhombencephalic neurons and decreases the number 5-HT neurons. It also appears that the 5-HT1A agonist ipsapirone provides neuroprotection to these neurons by reducing apoptosis. Another mechanism by which ethanol-associated apoptosis can be blocked is by including serum proteins in the media at a concentration of 1% or higher; this concentration of serum proteins is high in comparison to the protein concentration in cerebrospinal fluid.

Role of stromal cells and their products in protecting young and aged B-lineage precursors from dexamethasone-induced apoptosis.

Bone marrow stromal cells are potent providers of stimuli that induce proliferation of B-cell precursors. We proposed that stromal cells play a role in protecting B-lineage cells from corticosteroid-induced apoptosis. We found that stromal cells protected B-cell precursors from dexamethasone-induced apoptosis, but this did not strictly correlate with interleukin-7 (IL-7) production. To determine if stromal-derived factors were involved in protection of B-cell precursors from apoptosis, we examined the activity of three lymphopoietic growth factors: IL-7, stem cell factor (SCF), and insulin-like growth factor-1 (IGF-1). Either IL-7 or IGF-1 alone protected B-cell precursors from dexamethasone-induced apoptosis. The combined activities of IGF-1 and IL-7 were additive rather than synergistic. SCF did not protect B-cell precursors from apoptosis. Aging altered the ability of B-cell precursors to respond to protective stimuli induced by IL-7 and IGF-1. Precursors from aged animals were deficient in ability to modulate expression of apoptosis regulatory genes Bax, Bcl-2, and Bcl-x in comparison to B-cell precursors from young animals. Taken together, these results suggest that stromal cells can protect B-lineage precursors from a corticosteroid-induced apoptotic signal, protection is mediated by stromal-derived cytokines, and aging decreases the ability of B-cell precursors to respond efficiently to protective stimuli.

Molecular characterization of the mouse involuted thymus: aberrations in expression of transcription regulators in thymocyte and epithelial compartments.

Despite playing a critical role in the development of naive T cells, the thymus is involuted with age. Whether a single age-associated defect or multiple aberrations contribute to thymic involution remains controversial. Here, we determined molecular aberrations in the thymocyte and epithelium compartments of the aging thymus. We demonstrated that total thymocyte numbers declined with a stepwise kinetics; clear demarcations occurred at 1.5, 3, 12 and 22 months of age. By quantitative PCR, a 2.4-fold reduction in the copies of signal joint TCR-excised circle (sjTREC)/10(5) thymocytes was first detected at 3 months; no further reduction observed thereafter. Nevertheless, the combined reductions in thymocyte numbers and sjTREC/10(5) cells caused a 7-fold decrease in sjTREC/thymus by 3 months, 21-fold by 18 months and 72-fold by 22 months as compared to 1 month. We showed aberration in expression of E2A, a transcription regulator critical for TCR beta rearrangement. While E2A expression declined 3-fold by 3 months and 18-fold by 7 months, expression of LMO2, a negative regulator of E2A activities, increased 5-fold by 18 months. Interestingly, expression of pre-T alpha and its transcriptional regulator HEB were not reduced with age. Furthermore, keratin-8 expression, specific for cortical thymic epithelium, declined 3-fold by 7 months and remained stable thereafter. In contrast, Foxn1 expression was reduced 3-fold by 3 months, 16-fold by 12 months and 37-fold by 18 months. IL-7 expression was not reduced until 7 months and reached 15-fold reduction by 22 months. Thus, the data demonstrate that thymic involution results not from a single defect, but culminates from an array of molecular aberrations in both the developing thymocytes and thymic epithelials.

T cell receptor excision circle assessment of thymopoiesis in aging mice.

Signal joint T cell receptor delta (TCRD) excision circles (TRECs) are episomal DNA circles generated by the DNA recombination process that is used by T lymphocytes to produce antigen-specific alpha/beta T cell receptors. Measurement of TRECs in thymocytes and peripheral blood T cells has been used to study thymus output in chickens and humans. We have developed a real-time quantitative-PCR assay for the specific detection and quantification of mouse TCRD episomal DNA circles excised from the TCRA locus during TCRA gene rearrangement (mTRECs). We found that the mouse TCRD TRECs detected with this assay were predominantly in naïve phenotype CD4(+) and CD8(+) T cells. In a series of aged mice (range 6-90-week-old) we determined the absolute number of thymocytes and the number of molecules of mTRECs/100,000 thymocytes. We found that the absolute number of thymocytes dramatically decreased with age (P<0.05) and that molecules of mTREC/100,000 thymocytes also declined with mouse age (P<0.05). Splenocytes were isolated from aging mice and the frequency of naïve phenotype CD4 and CD8 cells determined. There was a significant drop in both CD4 and CD8 naïve peripheral T cells in the aged mice over time. mTREC analysis in purified CD4(+) and CD8(+) splenocytes demonstrated a constant level of mTRECs in the CD4 compartment until age 90 weeks, while the mTRECs in the CD8 compartment fell with age (P<0.05). By combining the mouse TREC assay with T cell phenotypic analysis, we demonstrated that IL-7 administration to young mice induced both increased thymopoiesis and peripheral T cell proliferation. In contrast, IL-7 treatment of aged mice did not augment thymopoiesis, nor induce expansion of splenic T cells. Thus, thymus output continues throughout murine adult life, and the thymic atrophy of aging in mice is not reversed by administration of IL-7.