Biomarkers from subcutaneous engineered tissues predict acute rejection of organ allografts.

Invasive graft biopsies assess the efficacy of immunosuppression through lagging indicators of transplant rejection. We report on a microporous scaffold implant as a minimally invasive immunological niche to assay rejection before graft injury. Adoptive transfer of T cells into Rag2-/- mice with mismatched allografts induced acute cellular allograft rejection (ACAR), with subsequent validation in wild-type animals. Following murine heart or skin transplantation, scaffold implants accumulate predominantly innate immune cells. The scaffold enables frequent biopsy, and gene expression analyses identified biomarkers of ACAR before clinical signs of graft injury. This gene signature distinguishes ACAR and immunodeficient respiratory infection before injury onset, indicating the specificity of the biomarkers to differentiate ACAR from other inflammatory insult. Overall, this implantable scaffold enables remote evaluation of the early risk of rejection, which could potentially be used to reduce the frequency of routine graft biopsy, reduce toxicities by personalizing immunosuppression, and prolong transplant life.

Rewiring of cortical glucose metabolism fuels human brain cancer growth.

The brain avidly consumes glucose to fuel neurophysiology. Cancers of the brain, such as glioblastoma (GBM), lose aspects of normal biology and gain the ability to proliferate and invade healthy tissue. How brain cancers rewire glucose utilization to fuel these processes is poorly understood. Here we perform infusions of 13 C-labeled glucose into patients and mice with brain cancer to define the metabolic fates of glucose-derived carbon in tumor and cortex. By combining these measurements with quantitative metabolic flux analysis, we find that human cortex funnels glucose-derived carbons towards physiologic processes including TCA cycle oxidation and neurotransmitter synthesis. In contrast, brain cancers downregulate these physiologic processes, scavenge alternative carbon sources from the environment, and instead use glucose-derived carbons to produce molecules needed for proliferation and invasion. Targeting this metabolic rewiring in mice through dietary modulation selectively alters GBM metabolism and slows tumor growth. Significance: This study is the first to directly measure biosynthetic flux in both glioma and cortical tissue in human brain cancer patients. Brain tumors rewire glucose carbon utilization away from oxidation and neurotransmitter production towards biosynthesis to fuel growth. Blocking these metabolic adaptations with dietary interventions slows brain cancer growth with minimal effects on cortical metabolism.

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions. This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands.

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.

Transient blockade of delta-like Notch ligands prevents allograft rejection mediated by cellular and humoral mechanisms in a mouse model of heart transplantation.

Rejection remains a major clinical challenge limiting allograft survival after solid organ transplantation. Both cellular and humoral immunity contribute to this complication, with increased recognition of Ab-mediated damage during acute and chronic rejection. Using a mouse model of MHC-mismatched heart transplantation, we report markedly protective effects of Notch inhibition, dampening both T cell and Ab-driven rejection. T cell-specific pan-Notch blockade prolonged heart allograft survival and decreased IFN-γ and IL-4 production by alloreactive T cells, especially when combined with depletion of recipient CD8(+) T cells. These effects were associated with decreased infiltration by conventional T cells and an increased proportion of regulatory T cells in the graft. Transient administration of neutralizing Abs specific for delta-like (Dll)1/4 Notch ligands in the peritransplant period led to prolonged acceptance of allogeneic hearts, with superior outcome over Notch inhibition only in T cells. Systemic Dll1/4 inhibition decreased T cell cytokines and graft infiltration, germinal center B cell and plasmablast numbers, as well as production of donor-specific alloantibodies and complement deposition in the transplanted hearts. Dll1 or Dll4 inhibition alone provided partial protection. Thus, pathogenic signals delivered by Dll1/4 Notch ligands early after transplantation promote organ rejection through several complementary mechanisms. Transient interruption of these signals represents an attractive new therapeutic strategy to enhance long-term allograft survival.

The high diagnostic accuracy of combined test of thyroid transcription factor 1 and Napsin A to distinguish between lung adenocarcinoma and squamous cell carcinoma: a meta-analysis.

BACKGROUND: Accurate classification of non-small cell lung cancer (NSCLC) using morphological features has several limitations. However, the use of thyroid transcription factor 1 (TTF-1) and Napsin A as markers for the identification of various subtypes of NSCLC has shown promise. This meta-analysis was designed to evaluate the diagnostic value of combined TTF-1 and Napsin A test to distinguish lung adenocarcinoma from squamous cell carcinoma. METHODS: The Medline, EMBASE and Web of Science databases were searched, along with the reference lists of relevant articles (up to May 4, 2014). Ten studies containing 1,446 subjects were identified. The sensitivity, specificity, diagnostic odds ratio (DOR) and area under the summary receiver operating characteristics curve (AUC) were calculated to estimate the combined diagnostic value of TTF-1 and Napsin A. RESULTS: The pooled sensitivity and specificity were 0.76 (95% CI: 0.69-0.83) and 1.00 (95% CI: 0.92-1.00), respectively. The positive and negative likelihood ratios were 877.60 (95% CI: 8.40-91533.40) and 0.24 (95% CI: 0.18-0.32). The DOR was 3719 (95% CI: 33-414884). The AUC was 0.92 (95%CI: 0.89-0.94). The patient's location was a source of heterogeneity for sensitivity. The patient's location, the study's sample size and the threshold used to determine positive staining were consistently found to be sources of heterogeneity for specificity in subgroup analyses and meta-regression. CONCLUSIONS: The combined test of TTF-1 and Napsin A presents a promising alternative method, useful to distinguish between lung adenocarcinoma and squamous cell carcinoma.

Gene transfer of the S24F regulated on activation normal T-cell expressed and secreted-chemokine ligand 5 variant attenuates cardiac allograft rejection.

BACKGROUND: Regulated on activation normal T-cell expressed and secreted (RANTES)-chemokine ligand 5 plays a key role in mediating heart transplant rejection. Suppression of RANTES-mediated signals can reduce leukocyte recruitment and mitigate transplant rejection severity. The present study describes the construction of an adenovirus overexpression vector encoding a natural S24F RANTES variant as a means of reducing leukocyte recruitment, resulting in the prevention of allograft rejection. METHODS: The in vitro transendothelial chemotaxis assay was used to compare RANTES-induced transmigration of peripheral blood mononuclear cells across human umbilical vein endothelial cells cultured on the upper Transwell chamber. Intracoronary delivery of Ad-S24F, Ad-Null, or phosphate-buffered saline was performed in BALB/c donor hearts that were transplanted into the abdominal cavity of C57BL/6 recipients as a measure of allograft survival. Intragraft inflammatory cell infiltrates and associated proinflammatory cytokine expression profiles were detected by immunohistochemistry and quantitative real-time polymerase chain reaction on day 6 after transplantation, respectively. RESULTS: Regulated on activation normal T-cell expressed and secreted-induced peripheral blood mononuclear cell transendothelial chemotaxis is inhibited by S24F (Ad-S24F, 9.2%±0.02%; Ad-Null, 17.7%±0.02%; medium control, 15.1%±0.01%; P<0.05). Cardiac allograft survival was prolonged after delivery of 1×10 plaque-forming units of Ad-S24F (13.00±0.33 days compared with 9.38±0.60 and 9.00±0.38 days after Ad-Null or phosphate-buffered saline treatment, respectively, P<0.05). S24F gene transfer reduced the number of intragraft CD8 T lymphocytes, monocyte-macrophages, and T-cell receptor αß cell infiltrates (P<0.05) and decreased transcripts for RANTES and interferon-γ (P<0.05). CONCLUSION: S24F is an important component of the chemokine network involved in regulating the biologic activity of RANTES, and its expression can be used in the prevention and treatment of cardiac allograft rejection.

Comparison of anticoagulant effects on vein grafts between human TFPI gene transfection and aspirin oral administration.

To develop a more efficient antithrombotic way after coronary artery bypass grafting (CABG), the anticoagulant effects were compared of human tissue factor pathway inhibitor (TFPI) gene transfection and aspirin oral administration (traditional method) on vein grafts. An eukaryotic expression plasmid pCMV-(Kozak) TFPI was prepared. Animal model of carotid artery bypass grafting was constructed. In operation, endothelial cells of vein grafts in TFPI group and empty plasmid control group were transfected with pCMV-(Kozak) TFPI and empty plasmid pCMV respectively, while no transfection was conducted in aspirin control group. After operation, aspirin (2 mg.kg(-1).(-1)) was administered (i.g.) in aspirin control group. Three days later, grafts (n=10) were harvested for RT-PCR, Western blotting and immunohistochemical analyses of exogenous gene expression and for pathological, scanning electron microscopic observation of thrombus. Thirty days later, the patency rates of remnant grafts (n=10) were recorded by vessel Doppler ultrasonography. Human TFPI gene products were detected in gene transferred vein grafts. Three days later, thrombi were found in 7 animals of aspirin control group and in 8 animals of empty plasmid control group, but in only 1 of TFPI group (P<0.01). Thirty days later, 5 grafts were occluded in empty plasmid control group, but none of grafts was occluded in the other groups (P<0.05). The endothelial surfaces of grafts in both of the control groups were covered with aggregated erythrocytes and platelets, and it were not seen in TFPI group. It was suggested that the anticoagulant effects on vein grafts of human TFPI gene transfection are better than those of aspirin.

Characteristics of ex vivo expansion of endothelial progenitor cells.

The characteristics for the ex vivo expansion of the endothelial progenitor cells (EPCs) were explored. CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34+ and CD34- from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis were examined. EPCs were determined and quantified by immunocytochemistry and flow cytometry. The results showed that both coculture of CD34+ and CD34- and total MNC led to a significant increase in the expansion of CD34+ cells as compared with CD34 enrichment (P < 0.05). There was a tendency toward decreased apoptosis in cultures when early passage was performed immediately after cord like structures appeared. VEGF had no significant effect on apoptosis (P > 0.05). These differentiated EPCs were positive for CD34+, von Willebrand factor (vWF), KDR, CD31 staining and phagocytized acetylated low-density lipoprotein (LDL). CD34+ cells accounted for (68.2 +/- 6.3)% of attaching (AT) cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34+ and CD34- or total MNC. Early passage makes cell apoptosis rate decrease. VEGF had no significant effect on ex vivo expansion of EPCs.

Implantation of endothelial progenitor cells into laser-induced channels in rat ischemia hindlimb augments neovascularization.

The aim of this study was to investigate the effect of transmuscle laser revascularization (TMR) in combination with endothelial progenitor cell grafting on neovascularization of ischemic hindlimbs of nude rats. Mononuclear cells (MNCs) isolated from human umbilical cord blood (HUCB) by density gradient centrifugation were expanded in vitro. Spindle-shaped attaching (AT) cells and cord-like structures were developed from culture of MNCs. Acetylated low-density lipoprotein (Ac-LDL) incorporation by AT cells was performed. Phenotypic characterization was assessed by immunocytochemistry, and flow cytometry analysis. EPCs were labeled with 1, 1'-dioctadecyl-1 to 3,3, 3', 3'- tetramethyl-indocarbocyanine perchlorate (DiI) before being injected into the Nd:YAG laser channels or ischemic region. An acute ischemic limb model was created with the following four groups of nude rats by ligating the right external iliac artery: TMR + EPC group: rats with local transplantation of EPCs into laser channels; TMR group: those with transmuscular channels created without EPCs; EPC group: those with EPCs injected into the ischemic hindlimb; control group: an ischemic model without TMR or EPCs. All rats underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtain a flow ratio (femoral artery flow index [FAFI]: right femoral artery flow/left femoral artery flow) immediately after ligation of the artery (at baseline) and 28 days postoperation, and the ischemic limb muscle was sampled for histochemical and immunohistologic analysis. AT cells expressed AC 133 and endothelial cell (ECs) markers (KDR, CD34, CD31, and von Willebrand factor) and exhibited function similar to that of ECs as estimated by Ac-LDL incorporation. Flow cytometric analysis revealed that AT cells were positive for CD34 (62% +/- 7%) and AC133 (57.2% +/- 9.8%) at day 7 of culture. Twenty-eight days after the operation, the FAFI was significantly higher in the TMR+EPC group and EPC group than that in the control group. It was significantly higher in the TMR+EPC group, EPC group, and TMR group than that at their respective baselines. The FAFI in the control group was unchanged and no difference in FAFI was found between the TMR group and control group, and among the TMR+EPC, TMR, and EPC groups. TMR+EPC, TMR, and EPC treatment resulted in an increased number of capillaries in the treated regional area compared to the control group. Nd: YAG-laser transmuscle revascularization combined with the EPC grafting can significantly ameliorate perfusion and augment neovascularization in this ischemic hindlimb model of nude rats.

[The use of oxymetazoline in nasal endoscopic sinus surgery].

OBJECTIVE: To assess the value of oxymetazoline used in nasal endoscopic sinus surgery (ESS). METHOD: The effect of oxymetazoline on 68 patients pulse, cilium mottom and rebound phenomenon during ESS were observed. RESULT: Pulse and cilium motion are no significant difference after using oxymetazoline. Oxymetazoline reduces about 59% nasal mucosal blood flow and acts over 6 hours. CONCLUSION: Oxymetazoline used as nasal decongestant and anesthesia assistant is safe and effect in ESS as routine.