In situ tumour arrays reveal early environmental control of cancer immunity.

The immune phenotype of a tumour is a key predictor of its response to immunotherapy1-4. Patients who respond to checkpoint blockade generally present with immune-inflamed5-7 tumours that are highly infiltrated by T cells. However, not all inflamed tumours respond to therapy, and even lower response rates occur among tumours that lack T cells (immune desert) or that spatially exclude T cells to the periphery of the tumour lesion (immune excluded)8. Despite the importance of these tumour immune phenotypes in patients, little is known about their development, heterogeneity or dynamics owing to the technical difficulty of tracking these features in situ. Here we introduce skin tumour array by microporation (STAMP)-a preclinical approach that combines high-throughput time-lapse imaging with next-generation sequencing of tumour arrays. Using STAMP, we followed the development of thousands of arrayed tumours in vivo to show that tumour immune phenotypes and outcomes vary between adjacent tumours and are controlled by local factors within the tumour microenvironment. Particularly, the recruitment of T cells by fibroblasts and monocytes into the tumour core was supportive of T cell cytotoxic activity and tumour rejection. Tumour immune phenotypes were dynamic over time and an early conversion to an immune-inflamed phenotype was predictive of spontaneous or therapy-induced tumour rejection. Thus, STAMP captures the dynamic relationships of the spatial, cellular and molecular components of tumour rejection and has the potential to translate therapeutic concepts into successful clinical strategies.

Innovation in Sexuality and Relationship Education in Child Welfare: Shifting Toward a Focus on Ongoing Conversations, Connection, and Consent.

Youth in foster care experience disproportionate rates of abusive relationships, teen pregnancy, and sexually transmitted infections (STIs). Extant research points to the need for interventions at multiple levels of the social ecology, however, there is a lack of evidence to guide the development of coordinated interventions for youth, foster parents, and child welfare professionals. The Texas Foster Youth Health Initiative (TFYHI) convened a multidisciplinary learning community to build a foundation for intervention development. The intentional learning and innovation process engaged several groups of stakeholders: young adults with lived experience (n = 41), foster parents (n = 14), and child welfare professionals (n = 52). Interviews, community listening sessions, and reflection exercises were designed to capture tacit and experiential knowledge and explore challenges and desired outcomes from different perspectives. Based on a thematic analysis of stakeholder perspectives, we identified overarching needs to normalize conversations about sexuality and relationships and shift away from risk-based and stigmatizing approaches. We also identified key strategies for designing coordinated interventions targeting youth, foster parents, and child welfare professionals: (1) Reflect on values about sexuality and relationships. (2) Validate youths' need for connection. (3) Focus on strengthening youth-adult relationships and ongoing conversations. (4) Build healthy relationship skills including communication about consent, condom use, and contraception. (5) Identify teachable moments and model problem solving. (6) Use interactive approaches for sharing health information and empower youth to choose methods that fit their needs.

Cystic fibrosis transmembrane conductance regulator dysfunction in platelets drives lung hyperinflammation.

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease.

El tipo de parto, ¿podría condicionar el éxito en la lactancia materna exclusiva? / The type of childbirth, could condition success in exclusivebreastfeeding?

Fundamentos: Existen pocos trabajos que muestren factores de riesgo y protección asociados a la Lactancia Materna exclusiva (LME).El objetivo fue determinar qué factores constituyen elementos de protección o de riesgo para la instalación de LME en mujeres de distinto nivel socioeconómico y educativo, controladas en el sistema público de salud de Santiago. Métodos: Una muestra de 242 madres, atendidas en sistema público, con hijos de entre 1 y 6 meses, a quienes se les aplico una encuesta sobre LME. Resultados: Las principales causas de destete fueron "no se llena queda con hambre" 18,8%, seguida por las causas "rechazo del niño al pecho" 12%, e "inicio de actividades laborales o estudiantiles" 12%. La regresión logística mostró que el parto vaginal está asociado positivamente a la LME (OR 0,44; IC 0,24 -0,78), en cambio el no tener experiencia previa o haber tenido una mala experiencia está asociado negativamente a la LME (OR 3,82; IC 1,87 -7,78). Conclusiones: El parto vaginal es un elemento protector que está ligado a los procedimientos que le siguen y que permiten una inducción temprana de la lactancia

Background: Few studies show risk and protection factors associated with it, and although the prevalence of LME.The objective was to determine which factors constitute elements of protection or risk for the installation of LME in women of different socioeconomic and educational levels, controlled in the public health system of Santiago. Methods: A sample of 242 mothers, attended in public system, with children between 1 and 6 months. To whom a survey on LME was applied. Results: The main causes of weaning were "not filled, with hunger" "18.8%, followed by causes" rejection of the child to the breast" 12%, and" onsetof work or student activities "12%. Logistic regression showed that protective vaginal delivery (OR 0.44 IC 0.24 -0.78) and having no prior experience or having a poor experience is at risk(OR 3.82 CI 1.87 -7.78).Conclusions: Vaginal birth is a protective element that is linked to the procedures that follow and allow early induction of lactation

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Out-of-hospital cardiac arrest (OHCA) attended by mobile emergency teams with a physician on board. Results of the Spanish OHCA Registry (OSHCAR).

Most survival outcomes in out-of-hospital cardiac arrest (OHCA) are provided by emergency medical services (EMS) without a doctor on board. Our objective was to determine such outcomes in a whole country with public physician-led EMS. METHODS: We analyzed data from a nationwide prospective registry of OHCA cases attended by 19 public EMS in Spain, covering the period from 1-October 2013 to 30-October 2014. RESULTS: Advanced life support (ALS) was initiated in 9347 cases (incidence 18.6 cases/105 inhabitants per year). Resuscitation was considered futile in 558 cases (5.9%), and ALS was continued in 8789 cases (94.1%); mean age 63.5±17 years, 72.1% men. Initial rhythm was shockable in 22.1% of cases. Basic life support (BLS) was provided by bystanders in 1602 (24%) cases (635 of them with telephone assistance from the dispatch center). Of 8789 patients receiving ALS, 72.1% men, 2669 (30.4%) patients had return of spontaneous circulation on hospital arrival, 50.6% when the initial rhythm was shockable. Hospital discharge with good neurological status (CPC1-2) was found in 11.1% of the study population and in 27.6% when considering the Utstein comparator group of patients. A total of 216 (2.5%) patients arrived at the hospital with ongoing resuscitation, of whom only one survived with CPC1-2, and 165 (1.9%) patients were included in non-heart-beating donation programs. CONCLUSIONS: In Spain with physician-led EMS, OHCA survival with CPC1-2 reached a reasonable percentage despite only a modest contribution of bystander BLS. Ongoing resuscitation strategy seems to be futile except when considering non-heart beating donation programs.

Dysfunctional HDL in acute stroke.

BACKGROUND AND AIMS: HDL-cholesterol concentration is a reliable negative risk factor for acute cerebral infarction (ACI). Beyond quantitative aspects, our aim was to determine whether lipoprotein profiles and HDL functionality were altered at the acute phase of ischemic stroke. METHODS: Blood was taken from ACI patients within 4.5 h of symptom onset. Lipoproteins were separated by electrophoresis for determination of particle size. HDLs were isolated from plasma of patients (n = 10) and controls (n = 10) by ultracentrifugation. The relative amounts of paraoxonase 1 (PON1), α1antitrypsin (AAT) and myeloperoxidase (MPO) were determined by Western blot. HDL functional assays were performed on human-brain endothelial cells stimulated with TNFα. RESULTS: Stroke patients had higher proportion of large HDL particles relative to controls (37.8 ± 11.8 vs. 28.4 ± 6.6, p = 0.04). HDLs from patients contained significantly less ApoA1 (1.63 ± 0.42 vs. 2.54 ± 0.71 mg/mL, p = 0.0026) and PON1 (4598 ± 1921 vs. 6598 ± 1127 AU, p = 0.01) than those from controls, whereas MPO and AAT were more abundant in HDLs isolated from ACI patients (respectively 3657 ± 1457 vs. 2012 ± 1234 and 3347 ± 917 vs. 2472 ± 470 AU, p = 0.014 and p = 0.015). HDLs reduced the expression of VCAM1, MCP1 and MMP3 mRNA induced by TNFα in blood-brain barrier endothelial cells. HDLs from patients were less effective in inhibiting TNFα-induced transcription of these genes (respectively 38.6 vs. 55.6% for VCAM1, p = 0.047, 44 vs. 48.1% for MCP1, p = 0.015 and 70 vs. 74% for MMP3, p = 0.024). CONCLUSIONS: ACI may be associated with a modified distribution of HDL particles (increased proportion of large particles) and HDL-binding proteins, resulting in an inappropriate protection of endothelial cells under ischemic conditions.

Stevia rebaudiana loaded titanium oxide nanomaterials as an antidiabetic agent in rats

Abstract Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, is a plant with hypoglycemic and antihyperlipidemic properties. S. rebaudiana (SrB) has become a lead candidate for the treatment of the diabetes mellitus. However, chronic administrations of S. rebaudiana are required to cause the normoglycemic effect. Importantly, nanomaterials in general and titanium dioxide (TiO2) in particular have become effective tools for drug delivery. In this work, we obtained TiO2 nanomaterials with SrB at different concentrations (10, 20 and 30 µM) by sol–gel method. After this nanomaterials were characterized by Fourier transform infrared spectroscopy and transmission electron microscopy. Where it was demonstrated, the presence of the S. rebaudiana in TiO2 nanomaterials, which were observed as hemispherical agglomerated particles of different sizes. The nanomaterials were evaluated in male rats whose diabetes mellitus-phenotype was induced by alloxan (200 mg/kg, i.p.). The co-administration of TiO2-SrB (20 and 30 µM) induced a significant and permanent decrease in the glucose concentration since 4 h, until 30 days post-administration. Likewise, the concentrations of insulin, glycosylated hemoglobin, cholesterol, and triacylglycerides showed a significant recovery to basal levels. The major finding of the study was that the TiO2-SrB (20 and 30 µM) has a potent and prolonged activity antidiabetic. TiO2 can be considered like an appropriated vehicle in the continuous freeing of active substances to treat of diabetes mellitus.

Gene delivery of suppressors of cytokine signaling (SOCS) inhibits inflammation and atherosclerosis development in mice.

Chronic activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to vascular inflammation and atherosclerosis by inducing expression of genes involved in cell proliferation, differentiation and migration. We aimed to investigate whether enforced expression of negative regulators, the suppressors of cytokine signaling (SOCS1 and SOCS3), inhibits harmful JAK/STAT-mediated responses and affects atherosclerosis in apolipoprotein E knockout mice. Adenovirus-mediated SOCS1 transgene expression impaired the onset and progression of atherosclerosis without impact on lipid profile, whereas SOCS3 was only effective on early atherosclerosis. Mechanistically, SOCS gene delivery, primarily SOCS1, attenuated STAT1 and STAT3 activation and reduced the expression of STAT-dependent genes (chemokine/chemokine receptors, adhesion molecules, pro-inflammatory cytokines and scavenger receptors) in aortic tissue. Furthermore, atherosclerotic plaques exhibit a more stable phenotype characterized by lower lipids, T cells and M1 macrophages and higher M2 macrophages and collagen. Atheroprotection was accompanied by a systemic alteration of T helper- and T regulatory-related genes and a reduced activation state of circulating monocytes. In vascular smooth muscle cells and macrophages, SOCS gene delivery inhibited cytokine-induced STAT activation, pro-inflammatory gene expression, cell migration and proliferation. In conclusion, targeting SOCS proteins, predominantly SOCS1, to suppress pathological mechanisms involved in atheroma plaque progression and destabilization could be an interesting anti-atherosclerotic strategy.

Non-invasive Intratracheal Instillation in Mice.

The intratracheal instillation technique is used to deliver a variety of agents to the lungs ranging from pathogens (bacteria, viruses), toxins, to therapeutic agents. To model lung inflammation and injury, LPS can be administrated via intranasal, intratracheal, or aerosol approaches. Each technique has its limitations. The intratracheal technique can involve the non-invasive instillation method (via the oro-tracheal route) or a direct injection into the trachea. Here, we describe an optimized method for direct visual instillation of LPS via the non-invasive oro-tracheal route.

Two-event Transfusion-related Acute Lung Injury Mouse Model.

Transfusion-related acute lung injury (TRALI) is defined as acute lung injury that occurs within 6 hours of a blood product transfusion. TRALI continues to be a leading cause of transfusion-related mortality and we have developed a mouse model of TRALI to better understand the mechanisms by which injury occurs and to test therapeutic approaches. Our model is a two-event model based on immune priming and the challenge of BALB/c wild-type mice with cognate MHC Class I monoclonal antibody (MHC I mAb). Immune priming with LPS mimics the primed state of recipients (first event) that is important for the development of TRALI. Donor HLA antibodies are frequently implicated in TRALI reactions, and cognate MHC Class I antibody (second event) produces acute lung injury in primed animals. Here, we describe a detailed protocol with high reproducibility within animals.

Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation.

RATIONALE: Primary graft dysfunction (PGD) causes early mortality after lung transplantation and may contribute to late graft failure. No effective treatments exist. The pathogenesis of PGD is unclear, although both neutrophils and activated platelets have been implicated. We hypothesized that neutrophil extracellular traps (NETs) contribute to lung injury in PGD in a platelet-dependent manner. OBJECTIVES: To study NETs in experimental models of PGD and in lung transplant patients. METHODS: Two experimental murine PGD models were studied: hilar clamp and orthotopic lung transplantation after prolonged cold ischemia (OLT-PCI). NETs were assessed by immunofluorescence microscopy and ELISA. Platelet activation was inhibited with aspirin, and NETs were disrupted with DNaseI. NETs were also measured in bronchoalveolar lavage fluid and plasma from lung transplant patients with and without PGD. MEASUREMENTS AND MAIN RESULTS: NETs were increased after either hilar clamp or OLT-PCI compared with surgical control subjects. Activation and intrapulmonary accumulation of platelets were increased in OLT-PCI, and platelet inhibition reduced NETs and lung injury, and improved oxygenation. Disruption of NETs by intrabronchial administration of DNaseI also reduced lung injury and improved oxygenation. In bronchoalveolar lavage fluid from human lung transplant recipients, NETs were more abundant in patients with PGD. CONCLUSIONS: NETs accumulate in the lung in both experimental and clinical PGD. In experimental PGD, NET formation is platelet-dependent, and disruption of NETs with DNaseI reduces lung injury. These data are the first description of a pathogenic role for NETs in solid organ transplantation and suggest that NETs are a promising therapeutic target in PGD.

Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice.

Evidence is emerging that platelets are major contributors to innate immune responses in conditions such as acute lung injury (ALI). Platelets form heterotypic aggregates with neutrophils, and we hypothesized that lipoxin mediators regulate formation of neutrophil-platelet aggregates (NPA) and that NPA significantly contribute to ALI. Lipopolysaccharide (LPS)-induced lung injury was accompanied by platelet sequestration, activation, intra-alveolar accumulation, and NPA formation within both blood and alveolar compartments. Using lung intravital microscopy, we observed the dynamic formation of NPA during physiologic conditions, which sharply increased with ALI. Aspirin (ASA) treatment significantly reduced lung platelet sequestration and activation, NPA formation, and lung injury. ASA treatment increased levels of ASA-triggered lipoxin (ATL; 15-epi-lipoxin A4), and blocking the lipoxin A4 receptor (ALX) with a peptide antagonist (Boc2) or using ALX knockouts (Fpr2/3(-/-)) reversed this protection. LPS increased NPA formation in vitro, which was reduced by ATL, and engagement of ALX by ATL on both neutrophils and platelets was necessary to prevent aggregation. In a model of transfusion-related acute lung injury (TRALI), Boc2 also reversed ASA protection, and treatment with ATL in both LPS and TRALI models protected from ALI. We conclude that ATL regulates neutrophil-platelet aggregation and that platelet-neutrophil interactions are a therapeutic target in lung injury.

Gene Deficiency in Activating Fcγ Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice.

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis.

Peptide inhibitor of NF-κB translocation ameliorates experimental atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. NF-κB is a major regulator of inflammation that controls the expression of many genes involved in atherogenesis. Activated NF-κB was detected in human atherosclerotic plaques, and modulation of NF-κB inflammatory activity limits disease progression in mice. Herein, we investigate the anti-inflammatory and atheroprotective effects of a cell-permeable peptide containing the NF-κB nuclear localization sequence (NLS). In vascular smooth muscle cells and macrophages, NLS peptide specifically blocked the importin α-mediated nuclear import of NF-κB and prevented lipopolysaccharide-induced pro-inflammatory gene expression, cell migration, and oxidative stress. In experimental atherosclerosis (apolipoprotein E-knockout mice fed a high-fat diet), i.p., 0.13 µmol/day NLS peptide administration for 5 weeks attenuated NF-κB activation in atherosclerotic plaques. NLS peptide significantly inhibited lesion development at both early (age 10 weeks) and advanced (age 28 weeks) stages of atherosclerosis in mice, without affecting serum lipid levels. Plaques from NLS-treated mice contained fewer macrophages of pro-inflammatory M1 subtype than those from respective untreated controls. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. NLS peptide also attenuated pro-inflammatory gene expression and oxidative stress in aortic lesions. Our study demonstrates that targeting NF-κB nuclear translocation hampers inflammation and atherosclerosis development and identifies cell-permeable NLS peptide as a potential anti-atherosclerotic agent.

High-density lipoprotein-based therapy reduces the hemorrhagic complications associated with tissue plasminogen activator treatment in experimental stroke.

BACKGROUND AND PURPOSE: We have previously reported that intravenous injection of high-density lipoproteins (HDLs) was neuroprotective in an embolic stroke model. We hypothesized that HDL vasculoprotective actions on the blood-brain barrier (BBB) may decrease hemorrhagic transformation-associated with tissue plasminogen activator (tPA) administration in acute stroke. METHODS: We used tPA alone or in combination with HDLs in vivo in 2 models of focal middle cerebral artery occlusion (MCAO) (embolic and 4-hour monofilament MCAO) and in vitro in a model of BBB. Sprague-Dawley rats were submitted to MCAO, n=12 per group. The rats were then randomly injected with tPA (10 mg/kg) or saline with or without human plasma purified-HDL (10 mg/kg). The therapeutic effects of HDL and BBB integrity were assessed blindly 24 hours later. The integrity of the BBB was also tested using an in vitro model of human cerebral endothelial cells under oxygen-glucose deprivation. RESULTS: tPA-treated groups had significantly higher mortality and rate of hemorrhagic transformation at 24 hours in both MCAO models. Cotreatment with HDL significantly reduced stroke-induced mortality versus tPA alone (by 42% in filament MCAO, P=0.009; by 73% in embolic MCAO, P=0.05) and tPA-induced intracerebral parenchymal hematoma (by 92% in filament MCAO, by 100% in embolic MCAO; P<0.0001). This was consistent with an improved BBB integrity. In vitro, HDLs decreased oxygen-glucose deprivation-induced BBB permeability (P<0.05) and vascular endothelial cadherin disorganization. CONCLUSIONS: HDL injection decreased tPA-induced hemorrhagic transformation in rat models of MCAO. Both in vivo and in vitro results support the vasculoprotective action of HDLs on BBB under ischemic conditions.

Immunoglobulin G Fc receptor deficiency prevents Alzheimer-like pathology and cognitive impairment in mice.

Alzheimer's disease is a severely debilitating disease of high and growing proportions. Hypercholesterolaemia is a key risk factor in sporadic Alzheimer's disease that links metabolic disorders (diabetes, obesity and atherosclerosis) with this pathology. Hypercholesterolaemia is associated with increased levels of immunoglobulin G against oxidized lipoproteins. Patients with Alzheimer's disease produce autoantibodies against non-brain antigens and specific receptors for the constant Fc region of immunoglobulin G have been found in vulnerable neuronal subpopulations. Here, we focused on the potential role of Fc receptors as pathological players driving hypercholesterolaemia to Alzheimer's disease. In a well-established model of hypercholesterolaemia, the apolipoprotein E knockout mouse, we report increased brain levels of immunoglobulin G and upregulation of activating Fc receptors, predominantly of type IV, in neurons susceptible to amyloid ß accumulation. In these mice, gene deletion of γ-chain, the common subunit of activating Fc receptors, prevents learning and memory impairments without influencing cholesterolaemia and brain and serum immunoglobulin G levels. These cognition-protective effects were associated with a reduction in synapse loss, tau hyperphosphorylation and intracellular amyloid ß accumulation both in cortical and hippocampal pyramidal neurons. In vitro, activating Fc receptor engagement caused synapse loss, tau hyperphosphorylation and amyloid ß deposition in primary neurons by a mechanism involving mitogen-activated protein kinases and ß-site amyloid precursor protein cleaving enzyme 1. Our results represent the first demonstration that immunoglobulin G Fc receptors contribute to the development of hypercholesterolaemia-associated features of Alzheimer's disease and suggest a new potential target for slowing or preventing Alzheimer's disease in hypercholesterolaemic patients.

Fcγ receptor deficiency attenuates diabetic nephropathy.

Among patients with diabetes, increased production of immunoglobulins against proteins modified by diabetes is associated with proteinuria and cardiovascular risk, suggesting that immune mechanisms may contribute to the development of diabetes complications, such as nephropathy. We investigated the contribution of IgG Fcγ receptors to diabetic renal injury in hyperglycemic, hypercholesterolemic mice. We used streptozotocin to induce diabetes in apolipoprotein E-deficient mice and in mice deficient in both apolipoprotein E and γ-chain, the common subunit of activating Fcγ receptors. After 15 weeks, the mice lacking Fcγ receptors had significantly less albuminuria and renal hypertrophy, despite similar degrees of hyperglycemia and hypercholesterolemia, immunoglobulin production, and glomerular immune deposits. Moreover, diabetic Fcγ receptor-deficient mice had less mesangial matrix expansion, inflammatory cell infiltration, and collagen and α-smooth muscle actin content in their kidneys. Accordingly, expression of genes involved in leukocyte infiltration, fibrosis, and oxidative stress was significantly reduced in diabetic kidneys and in mesangial cells cultured from Fcγ receptor-deficient mice. In summary, preventing the activation of Fcγ receptors alleviates renal hypertrophy, inflammation, and fibrosis in hypercholesterolemic mice with diabetes, suggesting that modulating Fcγ receptor signaling may be renoprotective in diabetic nephropathy.

Renal delivery of adenovirus and antisense oligonucleotides in rats by retrograde renal vein injection.

Renal gene therapy may offer new strategies to treat diseases of native and transplanted kidneys. Several experimental techniques have been developed using viral, nonviral, and cellular vectors, although the effectiveness of such techniques varies widely depending upon the vector used, type of injection, species, and experimental model of renal disease. Here, we describe an optimized technique for renal delivery of DNA in rodents by retrograde renal vein injection as it is currently applied in our laboratory for adenovirus and nonviral vectors. This is an effective gene transfer method with lasting effect on gene expression in the kidney that modulates renal disease in rodents without any apparent harmful effect, thus having a potential therapeutic value for future clinical applications.

Suppressors of cytokine signaling abrogate diabetic nephropathy.

Activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) is an important mechanism by which hyperglycemia contributes to renal damage, suggesting that modulation of this pathway may prevent renal and vascular complications of diabetes. Here, we investigated the involvement of suppressors of cytokine signaling (SOCS) as intracellular negative regulators of JAK/STAT activation in diabetic nephropathy. In a rat model, inducing diabetes resulted in JAK/STAT activation and increased expression of SOCS1 and SOCS3. In humans, we observed increased expression of glomerular and tubulointerstitial SOCS proteins in biopsies of patients with diabetic nephropathy. In vitro, high concentrations of glucose activated JAK/STAT/SOCS in human mesangial and tubular cells. Overexpression of SOCS reversed the glucose-induced activation of the JAK/STAT pathway, expression of STAT-dependent genes (chemokines, growth factors, and extracellular matrix proteins), and cell proliferation. In vivo, intrarenal delivery of adenovirus expressing SOCS1 and SOCS3 to diabetic rats significantly improved renal function and reduced renal lesions associated with diabetes, such as mesangial expansion, fibrosis, and influx of macrophages. SOCS gene delivery also decreased the activation of STAT1 and STAT3 and the expression of proinflammatory and profibrotic proteins in the diabetic kidney. In summary, these results provide direct evidence for a link between the JAK/STAT/SOCS axis and hyperglycemia-induced cell responses in the kidney. Suppression of the JAK/STAT pathway by increasing intracellular SOCS proteins may have therapeutic potential in diabetic nephropathy.