Diphtheria Toxin Receptor Transgenic Mice Research Articles

Science, Medicine, and the Anesthesiologist

Key Papers from the Most Recent Literature Relevant to AnesthesiologistsThe deposition of tau neurofibrillary tangles and amyloid-β plaques are classic histopathologic findings in Alzheimer’s disease, but a correlation between brain tau topography and clinical phenotypes of Alzheimer’s is unknown. Preclinical studies suggest that tau pathology has cell-to-cell propagation properties, thus the present study questioned whether tau aggregation is differentially concentrated in distinct brain network hubs in different Alzheimer’s phenotypes. Positron emission tomography (PET) and structural, diffusion-weighted, and functional magnetic resonance imaging analyses were conducted in control and Alzheimer’s patients with amnestic, posterior cortical atrophy, behavioral/dysexecutive or progressive aphasia phenotypes of Alzheimer’s. The tau-PET tracer MK6240 in each clinical variant of Alzheimer’s matched to a different functional network. Each Alzheimer’s phenotype correlated to a known canonical network: amnestic to the default mode, the posterior cortical atrophy to visuospatial network, the behavioral/dysexecutive to salience network, and the progressive aphasia to the language network. Subsequently, white matter tracts were also shown to be differentially associated with different Alzheimer’s phenotypes, suggesting a possible propagation pathway for tau pathology. The results are consistent with a concept that tau pathology aggregates along intrinsic connectivity pathways via cell-to-cell propagation, highlighting the therapeutic potential of therapies that block the uptake or transmission of tau.Take home message: The functional and structural networks of the human brain provide the framework for tau aggregation propagation which are distinct among clinical phenotypes of Alzheimer’s disease.Parkinson’s disease is the fastest growing neurologic disorder worldwide. Treatment relies on the Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS), which is subjective. Parkinson’s affects respiratory drive and muscle function, which can be detected in nocturnal sleep patterns. Using sleep data from several large U.S. tertiary care and research databases (11,964 nights, more than 120,000 h of nocturnal breathing signals from 757 Parkinson’s disease subjects [mean ± SD age 69 ± 10 yr] and 6,914 control subjects [66 ± 18 yr]) obtained from either a wearable belt or an in-room radio signal (reflected off the patient’s body while they sleep at home), an artificial intelligence model was developed that processes breathing data using a neural network to infer the presence of Parkinson’s disease and assesses severity according to the MDS-UPDRS standard. The artificial intelligence models for diagnosis demonstrated an area under the curve of 0.889 (wearable belt) and 0.906 (wireless radio signal). The artificial intelligence model estimated severity and progression in accordance with the Movement Disorder Society’s Rating Scale (R = 0.94).Take home message: An artificial intelligence model using at-home minimally invasive or noninvasive sleep-derived data may facilitate objective early diagnosis and progression of Parkinson’s disease.Button batteries are now ubiquitous in consumer electronic devices. Data from the National Electronic Injury Surveillance System report an estimated 70,322 battery-related emergency department visits in children younger than 18 y from 2010 to 2019, yielding an annual average of 9.5 visits/100,000 children. This rate was highest in children 5 y and younger, and 12% of these patients were hospitalized with button battery ingestions (85% of cases). These estimates were 2.1 times higher compared with an earlier study period (1990 to 2009). Previous reports have noted an almost sevenfold increase in major complications and deaths after button battery ingestions in children since introduction of the 20 mm CR2032 lithium batteries in 2006. Updated ingestion guidelines from the National Capital Poison Center now include the administration of honey to neutralize the strong alkali reaction before taking the child to the hospital. Sucralfate may also be administered in the emergency department.Take home message: Button battery ingestion is on the rise and accounts for most battery-related hospitalizations. Dissemination of preventative options and early treatment recommendations may help in curbing this public health problem.High-flow nasal cannula and continuous positive airway pressure are popular modes of noninvasive support in the pediatric critical care setting. This was a pragmatic, multicenter, randomized noninferiority clinical trial in 24 U.K. pediatric critical care units of children (0 to 15 yr; median age, 9 months; 39% female) requiring noninvasive respiratory support. Patients were randomly assigned 1:1 to either high-flow nasal cannula at a flow rate based on body weight (n = 301) or continuous positive airway pressure (7 to 8 cm H2O; n = 299). The primary outcome was the time from randomization to liberation from respiratory support (the start of a 48-h period free from all forms of respiratory support). Seven secondary outcomes were assessed, including mortality at critical care unit discharge, intubation within 48 h, and use of sedation. The median time to liberation in the high-flow nasal cannula group was 53 h (95% CI, 46 to 61 h) versus 48 h (95% CI, 41 to 56 h) in the continuous positive airway pressure group (absolute difference, 5 h [95% CI, –10 to 17 h]). This met the criterion for noninferiority. Of the seven prespecified secondary outcomes, use of sedation, mean duration of critical care stay, and mean duration of acute hospital stay were lower in the high-flow nasal cannula group. The most common adverse event was nasal trauma (high-flow nasal cannula: 6 of 295 [2%]; continuous positive airway pressure: 18 of 278 [7%]).Take home message: In this randomized trial of critically ill children requiring noninvasive respiratory support, high-flow nasal cannula was found to be noninferior to continuous positive airway pressure.Although the use aspirin-based thromboprophylaxis has increased over the past decade, limited evidence exists regarding the safety and efficacy of aspirin when used as a sole prophylactic agent. The CRISTAL trial was a cluster-randomized, crossover trial across 31 hospitals (performing greater than 250 knee or hip arthroplasty procedures per year) in Australia. Adult patients undergoing hip or knee arthroplasty procedures were enrolled at each hospital. Hospitals were randomized to administer aspirin (100 mg/d) or enoxaparin (40 mg/d) for 35 days after total hip and for 14 days after total knee arthroplasty. Crossover occurred after the patient enrollment target had been met for the first group. The primary outcome was symptomatic venous thromboembolism within 90 days, including pulmonary embolism and deep venous thrombosis. Enrollment was stopped after an interim analysis (9,711 patients out of 15,562 planned). Within 90 days of surgery, symptomatic venous thromboembolism occurred in 256 patients (79 pulmonary embolism and 18 above-knee and 174 below-knee deep venous thrombosis). The symptomatic venous thromboembolism rate in the aspirin group was 3.45% versus 1.82% in the enoxaparin group (estimated difference, 1.97%; 95% CI, 0.54 to 3.41%). This failed to meet the criterion for noninferiority for aspirin and was significantly superior for enoxaparin (P = 0.007).Take home message: Enoxaparin resulted in a significantly lower 90-day rate of symptomatic venous thromboembolism when compared with aspirin in patients undergoing hip or knee arthroplasty.First-line oral treatments provide only modest benefit in diabetic neuropathic pain. Although patients often receive multiple adjuvants, there are few data on comparative-effectiveness of combination therapy. This double-blind, non–placebo-controlled crossover study randomly assigned 140 patients at 13 U.K. centers with diabetic neuropathy to amitriptyline supplemented by pregabalin, pregabalin supplemented by amitriptyline, and duloxetine supplemented by pregabalin (84 completed at least two pathways). Participants were titrated up to 75 mg amitriptyline, 600 mg pregabalin, and 120 mg duloxetine. At 6 weeks, those reporting pain scores less than 3 out of 10 continued on monotherapy while those with higher scores received supplemental pharmacotherapy. At the 16-week endpoint, the 7-day pain score average (primary outcome) decreased from (mean ± SD) 6.6 ± 1.5 to 3.3 ± 1.8 in all pathways, with the largest difference being –0.1 (98% CI, –0.5 to 0.3) for duloxetine-pregabalin versus amitriptyline-pregabalin and for pregabalin-amitriptyline versus amitriptyline-pregabalin. At 6 weeks, 61% of participants experienced at least 30% pain reduction with no significant differences between groups; at 16 weeks, 66% reported at least 30% pain reduction. Mean pain score reduction in patients on combination therapy was 1.0 ± 1.3 [1.3] versus 0.2 ± 1.5 in those remaining on monotherapy; P = 0.085). Most discontinuations from side effects occurred during monotherapy, with fewer occurring with pregabalin-amitriptyline (5%) than with other combinations (P = 0.031).Take home message: Combination oral treatment for diabetic neuropathic pain was well tolerated and may provide benefit over monotherapy, with all three combination pathways having similar analgesic effectiveness.The appropriate oxygenation target for mechanical ventilation in comatose survivors of out-of-hospital cardiac arrest is not known. This randomized trial with a two-by-two factorial design randomly assigned 789 intubated and mechanically ventilated comatose adults with out-of-hospital cardiac arrest (1:1 ratio) in Denmark of either a restrictive (partial pressure of oxygen [Pao2] 68 to 75 mmHg) or a liberal oxygen target (Pao2 98 to 105 mmHg). Patients were assigned to one of two blood-pressure targets that were reported separately. The primary outcome was a composite of death from any cause or hospital discharge with severe disability or coma (Cerebral Performance Category 3 or 4; range 1 to 5, higher values indicating more severe disability) within 90 days after randomization. Secondary outcomes were neuron-specific enolase levels at 48 h, death from any cause, the score on the Montreal Cognitive Assessment (range 0 to 30, higher scores indicating better cognitive ability), the score on the modified Rankin scale (range 0 to 6, higher scores indicating greater disability), and the Cerebral Performance Category at 90 days. There were no significant differences between groups in the primary outcome (32% vs. 34%, respectively; (hazard ratio, 0.95; 95% CI, 0.75 to 1.21; P = 0.69) nor in any of the secondary outcomes (death 28% vs. 31%), all scores and enolase levels were nonsignificant.Take home message: Targeting a higher or lower partial pressure of oxygen during mechanical ventilation of survivors of out of hospital cardiac arrest had no influence on survival or disability.The appropriate mean blood pressure targets in comatose survivors of out-of-hospital cardiac arrest are not known. The authors report a two-by-two factorial design randomized trial of 789 intubated and mechanically ventilated, comatose adults with out-of-hospital cardiac arrest (1:1 ratio) in Denmark of a higher (77 mmHg) or lower target (63 mmHg) (oxygen targets discussed in preceding summary above). Blood pressure targets were assessed during the period of invasive arterial monitoring; the high-target protocol standardized volume resuscitation, norepinephrine, or supplemental dopamine infusion use. The primary and secondary outcomes are presented in the oxygen target summary above. A total of 789 patients were analyzed (393 high-target, 396 low-target groups). There were no differences in either the primary or secondary outcomes between groups (primary outcome composite 34% high vs. 32% low, hazard ratio, 1.08; 95% CI, 0.84 to 1.37; P = 0.56; mortality 31% vs. 29% hazard ratio, 1.13; 95% CI, 0.88 to 1.46); median Cerebral Performance Category 1 (interquartile range, 1 to 5) in both groups; corresponding median modified Rankin scale scores were 1 (interquartile range, 0 to 6) in both groups, median Montreal Cognitive Assessment scores 27 (interquartile range, 24 to 29) high versus 26 (interquartile range, 24 to 29) low. Median neuron-specific enolase levels were also similar between groups.Take home message: In this randomized trial of survivors of out-of-hospital cardiac arrest requiring mechanical ventilation, there were no significant differences in major physiologic or disability related outcomes in targeting a higher mean arterial blood pressure of 77 mmHg or lower mean pressure of 63 mmHg.Forty percent of heart transplant recipients will suffer from a rejection within the first year after surgery. The strong systemic immunosuppression currently needed after transplantation is responsible for the increased incidence of infections and malignancies. Hence, an alternative approach to achieve allograft immunotolerance is required. This murine heart transplantation model study aimed to investigate how the donor heart’s intrinsic immunity influences allograft acceptance. This study deciphers the specific immunologic roles of tissue resident macrophages and of monocyte-derived macrophages that originate from the donor and recipient, respectively. Using single-cell RNA sequencing, the transcriptional signatures of monocytes, macrophages, and dendritic cells demonstrated that donor and recipient macrophage expression profiles were nonoverlapping. Donor macrophages distinguished themselves based on the expression of the C-C chemokine receptor 2 (CCR2+ or CCR2–) and persisted after heart transplantation (from days 1 to 3), but both populations disappeared due to ongoing rejection (after day 7). Using CCR2-linked diphtheria toxin receptor transgenic mice, selective depletion of donor CCR2+ macrophages resulted in extended allograft survival, while selective depletion of donor CCR2– fostered rapid rejection. CCR2+ macrophages signaled via myeloid differentiation primary response protein 88 (MyD88) and the conditional deletion of MyD88 was sufficient to prevent CCR2+ macrophage activation and extend allograft survival.Take home message: These results from a murine transplant model suggest that donor CCR2+ macrophages are promising targets to prevent heart transplant rejection.The 2022 European Society of Cardiology guidelines recommend a stepwise evaluation of patients integrating clinical risk factors and test results with the estimated stress of the planned surgical procedure and the risks of discontinuing medications such as anticoagulants. Medical therapies, coronary interventions, timing of surgery, and specific surgical and anesthetic techniques, or withholding treatment are recommended to optimize patients’ perioperative outcomes. The recommendations are intended to prevent perioperative cardiovascular morbidity and mortality, specifically perioperative myocardial infarction and injury, stent thrombosis, acute heart failure, arrhythmias, pulmonary embolism, bleeding complications, stroke, and death. Patients younger than 65 yr of age without signs, symptoms, or history of cardiovascular disease or cardiovascular risk factors are considered low risk and can proceed with low- and intermediate-risk surgeries without additional preoperative risk assessment. For patients 65 yr and older, the presence of cardiovascular disease, risk factors for cardiovascular disease, frailty, and functional capacity along with the risk of surgery guide preoperative risk assessment, testing, interventions, and optimization. Clinical examination, patient-reported functional capacity, and noninvasive tests are fundamental for preoperative cardiac assessment. The guidelines acknowledge that evidence is lacking on the value of cardiac biomarkers, stress testing, or routine myocardial revascularization to reduce perioperative cardiovascular complications.Take home message: The European Society of Cardiology has extensively updated their 2014 guidelines for the cardiac evaluation of patients planning noncardiac surgery.Animal and in vitro evidence suggests that stress-inflammatory responses may stimulate tumor growth, increasing metastatic potential. Conversely, inhibition of the catecholamine-induced responses via β-adrenergic receptor blockade may improve survival. Small observational studies have reported mixed results. This is a population-based observational cohort study (two provinces in Australia accounting for 65% of the total population) of 3,844 females age 50 yr or older with a history of cardiovascular conditions undergoing surgery for epithelial ovarian cancer (2002 to 2014) evaluating treatment effects of selective β-blocker or nonselective β-blocker supply perioperatively on survival (through 2016) using covariate-balanced inverse probability of treatment weights with flexible parametric survival models allowing for time-varying survival effects. Of 10,590 with ovarian cancer, 3,844 were eligible for analysis. Administrative databases were used to determine timing of surgery, covariates, and medication prescriptions. Perioperatively, 560 (14.5%) women had active prescriptions for a selective β-blocker and 67 (1.7%) were a nonselective agent (91% propranolol). At 2 yr after surgery, a survival advantage was noted for nonselective use (80%, 95% CI, 68 to 88) versus 69% (67 to 70) for no use and this extended for on average 8 yr after surgery, while no association was noted for those with selective β-blocker prescriptions.Take home message: This population-based cohort analysis of β-blocker prescription availability around the time of surgery for epithelial ovarian cancer suggests a survival benefit associated with the use of nonselective β-blockers.Before the advent of antibiotics, more than half of all deaths were from infections. However, we are at present in the middle of an antibiotic crisis caused by the spread of multidrug-resistant organisms and stagnation in the development of new antibiotics. Many large pharmaceutical companies have abandoned antibiotic innovation because of the high cost of bringing a drug to market and the relatively poor return on investment. There is a need to change investment policies to prioritize societal value over commercial profit. A successful antibiotic needs to be very nontoxic, be able to penetrate and kill senescent-phase organisms (biofilms), and have minimal impact on the patient’s microbiome. Fortunately, there are several emerging drug developments that meet these requirements and have shown some promise. These include narrow-spectrum small molecules, bacteriophages, monoclonal antibodies, and vaccines. The effective use of these narrow-spectrum compounds is also predicated on the simultaneous development of fast, accurate point-of-care molecular diagnostic methods to identify the pathogenic organism, and its virulence and sensitivity.Take home message: With suitable structural coordination of the antibiotic development pipeline, clinicians should be able to look forward to having a diverse battery of tools to treat and prevent bacterial infection.

Lymphotoxin β receptor signaling and CD11c-positive dendritic cells form high endothelial venule-like vessels in the skin in murine atopic dermatitis model

Abstract High endothelial venules (HEVs), which are characterized by the expression of peripheral node addressin (PNAd), are essential routes for the trafficking of naïve T cells and central memory T cells (Tcms) into secondary lymphoid organs. In lymph nodes (LNs), lymphotoxin (LT) α1β2-LTβ receptor (LTβR) signaling from dendritic cells (DCs) is essential for the formation of HEVs. Although it has been reported that PNAd+ HEV-like vessels appear in the skin under some inflammatory condition, such as atopic dermatitis (AD), the formation mechanism of HEV-like vessels and their functions in the skin have remained unclear. To clarify these issues, we established a system to induce PNAd+ HEV-like vessels in the skin, using a mouse AD model induced by calcipotriol. Flow cytometric (FCM) analysis revealed that around 20–30% of vessels were PNAd+ in the AD-like skin lesions. Electron microscopic analysis showed prominent formation of rough endoplasmic reticulum, Golgi apparatus and mitochondria in the HEV-like vessels similar to HEVs in LNs. These vessels were mainly detected in post-capillary venules, around which dermal DCs formed clusters. Administration of LTβR-Fc fusion protein or inhibition of the DC clusters by either pertussis toxin or depletion of DCs using CD11c diphtheria toxin receptor transgenic mice abolished the formation of HEV-like vessels in the skin. Naïve T cells and Tcms infiltration were detected in both AD patients and murine AD model skin by immunohistochemistry and FCM. Collectively, our results suggest that LTβR signaling and CD11c+ DCs mediate the formation of HEV-like vessels in the skin, which may possess functions of HEV and regulate the transmigration of naïve T cells and Tcms.

Natural Killer Cells Dampen the Pathogenic Features of Recall Responses to Influenza Infection.

Despite evidence of augmented Natural Killer (NK) cell responses after influenza vaccination, the role of these cells in vaccine-induced immunity remains unclear. Here, we hypothesized that NK cells might increase viral clearance but possibly at the expense of increased severity of pathology. On the contrary, we found that NK cells serve a homeostatic role during influenza virus infection of vaccinated mice, allowing viral clearance with minimal pathology. Using a diphtheria toxin receptor transgenic mouse model, we were able to specifically deplete NKp46+ NK cells through the administration of diphtheria toxin. Using this model, we assessed the effect of NK cell depletion prior to influenza challenge in vaccinated and unvaccinated mice. NK-depleted, vaccinated animals lost significantly more weight after viral challenge than vaccinated NK intact animals, indicating that NK cells ameliorate disease in vaccinated animals. However, there was also a significant reduction in viral load in NK-depleted, unvaccinated animals indicating that NK cells also constrain viral clearance. Depletion of NK cells after vaccination, but 21 days before infection, did not affect viral clearance or weight loss—indicating that it is the presence of NK cells during the infection itself that promotes homeostasis. Further work is needed to identify the mechanism(s) by which NK cells regulate adaptive immunity in influenza-vaccinated animals to allow efficient and effective virus control whilst simultaneously minimizing inflammation and pathology.

Megakaryocyte Ablation Ameliorates Bone Marrow Fibrosis By Reducing Myofibroblast Differentiation in a Mouse Model of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a chronic myeloproliferative disorder characterized by excessive bone marrow (BM) fibrosis that can lead to ineffective hematopoiesis and reduced survival. PMF patients develop several megakaryocyte (MK) abnormalities including increased proliferation, abnormal morphology and upregulated expression of pro-fibrotic genes. Although several studies support the role of MK in BM fibrosis, the significance of MK in the development of BM fibrosis has not been demonstrated in vivo. Here, we investigated the in vivo role of MK in the pathogenesis of BM fibrosis. First, we reproduced an established bone marrow transplantation (BMT) mouse model of PMF. To achieve this, lethally irradiated wild-type mice were transplanted with wild-type hematopoietic stem cells (HSC) transduced with either MPLW515L gene construct (MPLW515L BMT) or MIGR1 empty vector (MIGR1 BMT). Consistent with previous studies, starting at day 14 post-BMT, MPLW515L BMT mice gradually developed increased number of leukocytes and platelets, increased proliferation of abnormal megakaryocytes in the BM and spleen, hepatosplenomegaly with extra-medullary hematopoiesis, and reticulin fibrosis in the BM. Notably, robust fibrosis was also seen in the spleen and liver. Next, to study the significance of MK in the development of BM fibrosis, we developed a MPLW515L BMT mouse model in which MK lineage could be selectively ablated. We accomplished this using PF4-Cre inducible diphtheria toxin receptor transgenic mice (iDTR+/-/PF4-Cre) as BMT donors. HSCs from iDTR+/-/PF4-Cre mice were transduced with MPLW515L or MIGR1 empty vector and transplanted as described above. Beginning day 14 post-BMT, recipient mice were injected with diphtheria toxin (DT) or water every 48 hours. At day 20 and day 29, DT injection significantly depleted MK in both MPLW515L BMT and MIGR1 BMT groups compared to water injection. Importantly, DT-injected MPLW515L BMT mice displayed attenuated fibrosis in the BM, spleen, and liver compared to water-injected MPLW515L BMT mice. In addition, DT treatment decreased the level of alpha smooth muscle actin in both BM and spleen of MPLW515L BMT mice, which suggests that MKs are critical for myofibroblast differentiation. MK ablation did not leukocytosis, thrombocytosis or hepatosplenomegaly. Together, our study show that successful MK ablation in vivo reduces fibrosis development in the BM, spleen, and liver of the MPLW515L mouse model of PMF. In summary, these results support the essential role of MK in the pathogenesis of BM fibrosis in PMF. Further studies are underway to elucidate the mechanisms by which MK contribute to fibrosis in PMF. Disclosures No relevant conflicts of interest to declare.

Tumor Lymphatic Function Regulates Tumor Inflammatory and Immunosuppressive Microenvironments.

Proliferation of aberrant, dysfunctional lymphatic vessels around solid tumors is a common histologic finding. Studies have shown that abnormalities in lymphatic function result in accumulation of inflammatory cells with an immunosuppressive profile. We tested the hypothesis that dysfunctional lymphatic vessels surrounding solid tumors regulate changes in the tumor microenvironment and tumor-specific immune responses. Using subcutaneously implanted mouse melanoma and breast cancer tumors in a lymphatic endothelial cell-specific diphtheria toxin receptor transgenic mouse, we found that local ablation of lymphatic vessels increased peritumoral edema, as compared with controls. Comparative analysis of the peritumoral fluid demonstrated increases in the number of macrophages, CD4+ inflammatory cells, F4/80+/Gr-1+ (myeloid-derived suppressor cells), CD4+/Foxp3+ (Tregs) immunosuppressive cells, and expression of inflammatory cytokines such as TNFα, IFNγ, and IL1β following lymphatic ablation. Tumors grown in lymphatic ablated mice exhibited reduced intratumoral accumulation of cytotoxic T cells and increased tumor PD-L1 expression, causing rapid tumor growth, compared with tumors grown in nonlymphatic-ablated mice. Our study suggests that lymphatic dysfunction plays a role in regulating tumor microenvironments and may be therapeutically targeted in combination with immunotherapy to prevent tumor growth and progression.

CD11c+ M1-like macrophages (M\u03a6s) but not CD206+ M2-like M\u03a6 are involved in folliculogenesis in mice ovary

Macrophages (MΦs) are involved in folliculogenesis and ovulation. However, it is unknown which type of MΦ, M1 or M2, plays a more essential role in the ovary. CD206 or CD11c diphtheria toxin receptor transgenic (DTR) mice, which enable depletion of CD206+ M2 MΦs and CD11c+ MΦ or CD11c+ Dendritic cells (DCs), respectively, were used. Oocytes were used for in vitro fertilization and embryo transfer. In vitro fertilized embryos derived from M2 MΦ depleted oocytes were transferred to pseudo pregnant wild type mice. CD11c DTR mice were also used to investigate the role of CD11c cells, M1 MΦ and DCs in folliculogenesis. In WT mice, the proportion of CD206+ M2-like MΦs was not increased in follicular induction, while that of CD11c+ M1-like MΦs was increased. In CD206 DTR mice, folliculogenesis was normal and the ovulation number, fertilization rate, and implantation rate were similar to those in WT mice. In CD11c DTR mice, folliculogenesis was impaired with ovarian hemorrhage and the staining of platelet derived growth factor-receptor β (PDGF-Rβ), a marker of pericytes, and CD34, a marker of endothelial cells, was reduced. CD11c+ cells, M1 MΦs or DCs, may be involved in folliculogenesis, while M2 MΦs are not involved in folliculogenesis.

108 - Helminth Exposure Protects Intestinal Epithelium from Acute Injury in IEC Diphtheria Toxin Receptor Transgenic Mice

108 - Helminth Exposure Protects Intestinal Epithelium from Acute Injury in IEC Diphtheria Toxin Receptor Transgenic Mice

Dendritic cells modulate burn wound healing by enhancing early proliferation.

Adequate wound healing is vital for burn patients to reduce the risk of infections and prolonged hospitalization. Dendritic cells (DCs) are antigen presenting cells that release cytokines and are central for the activation of innate and acquired immune responses. Studies have showed their presence in human burn wounds; however, their role in burn wound healing remains to be determined. This study investigated the role of DCs in modulating healing responses within the burn wound. A murine model of full-thickness contact burns was used to study wound healing in the absence of DCs (CD11c promoter-driven diphtheria toxin receptor transgenic mice) and in a DC-rich environment (using fms-like tyrosine kinase-3 ligand, FL- a DC growth factor). Wound closure was significantly delayed in DC-deficient mice and was associated with significant suppression of early cellular proliferation, granulation tissue formation, wound levels of TGFβ1 and formation of CD31+ vessels in healing wounds. In contrast, DC enhancement significantly accelerated early wound closure, associated with increased and accelerated cellular proliferation, granulation tissue formation, and increased TGFβ1 levels and CD31+ vessels in healing wounds. We conclude that DCs play an important role in the acceleration of early wound healing events, likely by secreting factors that trigger the proliferation of cells that mediate wound healing. Therefore, pharmacological enhancement of DCs may provide a therapeutic intervention to facilitate healing of burn wounds.

Investigation of bipotent differentiation of hepatoblasts using inducible diphtheria toxin receptor-transgenic mice.

Hepatic progenitor cells, called hepatoblasts, are highly proliferative and exhibit bipotential differentiation into hepatocytes and cholangiocytes in the fetal liver. Thus, they are the ideal source for transplantation therapy. Although several studies have been performed in vitro, the molecular mechanisms regulating hepatoblast differentiation in vivo following transplantation remain poorly understood. The aim of this study was to investigate an in vivo model to analyze hepatoblast bipotency and proliferative ability. Hepatic transplantation model using Cre-inducible diphtheria toxin receptor-transgenic mice (iDTR), and albafpCre mice expressing Cre under the control of albumin and α-fetoprotein (AFP) regulatory elements were established. Fresh hepatoblasts were transplanted into diphtheria toxin (DT)-injected iDTRalbafpCre mice and we analyzed their differentiation and proliferation abilities by immunostaining and gene expression profiles. Fresh hepatoblasts transplanted into DT-injected iDTRalbafpCre mice engrafted and differentiated into both hepatocytes and cholangiocytes. Additionally, the number of engrafted hepatoblast-derived hepatocytes increased following partial hepatectomy and serial DT injections. Expression levels of hepatic functional genes in transplanted hepatoblast-derived hepatocytes were similar to that of normal hepatocytes. In our iDTRalbafpCre transplantation model, fresh hepatoblasts could differentiate into hepatocytes and cholangiocytes. In addition, these donor cells were induced to proliferate by the following liver injury stimulation. This result suggests that this model is valuable for investigating hepatoblast differentiation pathways in vivo.

Functionally relevant neutrophilia in CD11c diphtheria toxin receptor transgenic mice

Transgenic mice expressing the diphtheria toxin receptor (DTR) in specific cell types are key tools for functional studies in several biological systems. B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J (CD11c.DTR) and B6.Cg-Tg(Itgax-DTR/OVA/EGFP)1Gjh/Crl (CD11c.DOG) mice express the DTR in CD11c(+) cells, allowing conditional depletion of dendritic cells. We report that dendritic-cell depletion in these models caused polymorphonuclear neutrophil (PMN) release from the bone marrow, which caused chemokine-dependent neutrophilia after 6-24 h and increased bacterial clearance in a mouse pyelonephritis model. We present a transgenic mouse line, B6.Cg-Tg(Itgax-EGFP-CRE-DTR-LUC)2Gjh/Crl (CD11c.LuciDTR), which is unaffected by early neutrophilia. However, CD11c.LuciDTR and CD11c.DTR mice showed late neutrophilia 72 h after dendritic cell depletion, which was independent of PMN release and possibly resulted from increased granulopoiesis. Thus, the time point of dendritic cell depletion and the choice of DTR transgenic mouse line must be considered in experimental settings where neutrophils may be involved.

Immunosuppressive Effects of Streptozotocin-Induced Diabetes Result in Absolute Lymphopenia and a Relative Increase of T Regulatory Cells

OBJECTIVEStreptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood.RESEARCH DESIGN AND METHODSThis study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice.RESULTSEarly lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8+ cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice.CONCLUSIONSThese data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

Plasmacytoid Dendritic Cell Ablation Impacts Early Interferon Responses and Antiviral NK and CD8+ T Cell Accrual

Plasmacytoid dendritic cells (pDCs) mediate type I interferon (IFN-I) responses to viruses that are recognized through the Toll-like receptor 7 (TLR7) or TLR9 signaling pathway. However, it is unclear how pDCs regulate the antiviral responses via innate and adaptive immune cells. We generated diphtheria toxin receptor transgenic mice to selectively deplete pDCs by administration of diphtheria toxin. pDC-depleted mice were challenged with viruses known to activate pDCs. In murine cytomegalovirus (MCMV) infection, pDC depletion reduced early IFN-I production and augmented viral burden facilitating the expansion of natural killer (NK) cells expressing the MCMV-specific receptor Ly49H. During vesicular stomatitis virus (VSV) infection, pDC depletion enhanced early viral replication and impaired the survival and accumulation of virus-specific cytotoxic T lymphocytes. We conclude that pDCs mediate early antiviral IFN-I responses and influence the accrual of virus-specific NK or CD8(+) Tcells ina virus-dependent manner.

Identification of the Cellular Sensor That Stimulates the Inflammatory Response to Sterile Cell Death

Cell death provokes a robust inflammatory response. We have previously shown that this response is dependent on IL-1alpha. In this study, we investigate the cellular mechanism used by a host to sense cell death, produce IL-1alpha and also the role of IL-1beta in this response. In almost all cases examined, the IL-1 that stimulated the death-induced inflammatory response came from the host rather than the cell that was dying. In these situations, host bone marrow-derived cells were the key source of the IL-1alpha that was required for the inflammatory response. Conditional cellular depletion and reconstitution in CD11b promoter-driven diphtheria toxin receptor transgenic mice revealed that host macrophages played an essential role in the generation of the inflammatory response and were the source of the required IL-1alpha. In addition, we found a role for IL-1beta in the death-induced inflammatory response and that this cytokine was generated by both bone marrow-derived and radioresistant host cells. The one exception to these findings was that when dendritic cells were injected into mice, they provided a portion of the IL-1 that stimulated inflammation, and this was observed whether the dendritic cells were live or necrotic. Together, these findings demonstrate that macrophages play a key role as the primary sentinels that are required to sense and report cell death in ways that initiate the inflammatory response. One key way they accomplish this important task is by producing IL-1alpha that is needed to initiate the inflammatory response.

Anergy in Memory CD4+ T Cells Is Induced by B Cells

Induction of tolerance in memory T cells has profound implications in the treatment of autoimmune diseases and transplant rejection. Previously, we reported that the presentation of low densities of agonist peptide/MHC class II complexes induced anergy in memory CD4(+) T cells. In the present study, we address the specific interaction of different types of APCs with memory CD4(+) T cells. A novel ex vivo anergy assay first suggested that B cells induce anergy in memory T cells, and an in vivo cell transfer assay further confirmed those observations. We demonstrated that B cells pulsed with defined doses of Ag anergize memory CD4 cells in vivo. We established that CD11c(+) dendritic cells do not contribute to anergy induction to CD4 memory T cells, because diphtheria toxin receptor-transgenic mice that were conditionally depleted of dendritic cells optimally induced anergy in memory CD4(+) T cells. Moreover, B cell-deficient muMT mice did not induce anergy in memory T cells. We showed that B2 follicular B cells are the specific subpopulation of B cells that render memory T cells anergic. Furthermore, we present data showing that anergy in this system is mediated by CTLA-4 up-regulation on T cells. This is the first study to demonstrate formally that B cells are the APCs that induce anergy in memory CD4(+) T cells.

Monocyte/Macrophage Suppression in CD11b Diphtheria Toxin Receptor Transgenic Mice Differentially Affects Atherogenesis and Established Plaques

Although monocytes/macrophages are considered important in atherogenesis, their role in established plaques is unclear. For example, macrophage content is associated with plaque instability, but their loss through cell death is observed at sites of plaque rupture. To examine the role of monocytes/macrophages in atherosclerosis, we developed CD11b-diphtheria toxin (DT) receptor (DTR) transgenic mice, whereby administration of DT selectively kills monocytes/macrophages. DT treatment reduced peripheral blood monocytes and tissue macrophages and inhibited macrophage function in CD11b-DTR mice and apolipoprotein E-null (apoE(-/-)) mice transplanted with CD11b-DTR bone marrow. In atherogenesis experiments, DT markedly reduced plaque development and altered plaque composition, reducing collagen content and necrotic core formation. In mice with established plaques, acute DT treatment induced macrophage apoptosis and reduced macrophage content but did not induce plaque inflammation, thrombosis, or rupture. Furthermore, despite a 50% reduction in monocytes, chronic DT treatment of these mice did not alter plaque extent or composition, most likely because of ongoing recruitment/proliferation of monocytes with recovery of macrophage content. We conclude that monocytes/macrophages are critical to atherogenesis, but established plaques are more resistant to reductions in monocytes.

A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration

A new system for lineage ablation is based on transgenic expression of a diphtheria toxin receptor (DTR) in mouse cells and application of diphtheria toxin (DT). To streamline this approach, we generated Cre-inducible DTR transgenic mice (iDTR) in which Cre-mediated excision of a STOP cassette renders cells sensitive to DT. We tested the iDTR strain by crossing to the T cell- and B cell-specific CD4-Cre and CD19-Cre strains, respectively, and observed efficient ablation of T and B cells after exposure to DT. In MOGi-Cre/iDTR double transgenic mice expressing Cre recombinase in oligodendrocytes, we observed myelin loss after intraperitoneal DT injections. Thus, DT crosses the blood-brain barrier and promotes cell ablation in the central nervous system. Notably, we show that the developing DT-specific antibody response is weak and not neutralizing, and thus does not impede the efficacy of DT. Our results validate the use of iDTR mice as a tool for cell ablation in vivo.