Exposure to inhaled allergens generates T helper 2 (Th2) CD4+ T cells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4+ T cells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memory cells in the lymphoid organs and tissue resident memory (Trm) cells in the lung. Experimental blockade of lymphocyte migration demonstrated that lung resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4+ T cells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses.

IntroductionThe Tie2/angiopoietin (Tie2/Ang) and vascular endothelial growth factor receptor-ligand systems (VEGFR/VEGF) are recognized to play important roles in the regulation of microvascular endothelial function. Downregulation of these genes during sepsis has been implicated in the pathogenesis of sepsis-related microvascular leak and multiple organ dysfunction syndrome. Mechanisms responsible for dysregulation of angiogenic genes in sepsis are poorly defined.MethodsWestern blot, reverse transcription-polymerase chain reaction, and multiplex chromatin immunoprecipitation platform (Matrix ChIP) were used to investigate serum albumin leak, changes in gene expression, and associated epigenetic alterations in a murine model of acute lung injury-induced sepsis (ALI-sepsis).ResultsExperimental ALI-sepsis induced microvascular leak and downregulation of expression of Angpt1 (Ang1), Tek (Tie2), and Kdr (Vegfr2 or Flk-1) genes in the lung, kidney, and liver. These changes correlate with a decrease in RNA polymerase II density at these genes, and the greatest response was observed in the lung. ALI-sepsis reduced levels of transcription-permissive histone H3 lysine acetylation (H3KAc) at these loci in all examined tissues. Decreases in permissive H3K4m3 and H3Km2 marks were detected only in the lung. In contrast, only minimal alterations in transcription-repressive histone modifications (H3K27m3, H3K9m2, H3K9m3, and H4K20m3) were observed in all tissues.ConclusionsOur results demonstrate that decreases in transcription-permissive, but not increases in transcription-repressive, histone modifications at Angpt1, Tek, and Kdr are a systemic, rather than a lung-restricted, response, involving key end-organs in experimental ALI-sepsis. Given that ventilator-associated pneumonia is a major cause of sepsis in critically ill patients, elucidation of mechanisms mediating epigenetic alterations during sepsis provides fundamental new insights into the pathogenesis of sepsis-induced microvascular leak and subsequent end-organ injury/dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0943-4) contains supplementary material, which is available to authorized users.

To compare the relative contributions of gravity and vascular structure to the distribution of pulmonary blood flow, we flew with pigs on the National Aeronautics and Space Administration KC-135 aircraft. A series of parabolas created alternating weightlessness and 1.8-G conditions. Fluorescent microspheres of varying colors were injected into the pulmonary circulation to mark regional blood flow during different postural and gravitational conditions. The lungs were subsequently removed, air dried, and sectioned into approximately 2 cm(3) pieces. Flow to each piece was determined for the different conditions. Perfusion heterogeneity did not change significantly during weightlessness compared with normal and increased gravitational forces. Regional blood flow to each lung piece changed little despite alterations in posture and gravitational forces. With the use of multiple stepwise linear regression, the contributions of gravity and vascular structure to regional perfusion were separated. We conclude that both gravity and the geometry of the pulmonary vascular tree influence regional pulmonary blood flow. However, the structure of the vascular tree is the primary determinant of regional perfusion in these animals.

No abstract

Cysteine-rich protein-61 (CYR61), also known as connective tissue growth factor, CYR61, and nephroblastoma overexpressed gene 1 (CCN1), is a heparin-binding protein member of the CCN family of matricellular proteins. Gene expression profiles showed that Cyr61 is upregulated in human acute lung injury (ALI), but its functional role is unclear. We hypothesized that CYR61 contributes to ALI in mice. First, we demonstrated that CYR61 expression increases after bleomycin-induced lung injury. We then used adenovirus-mediated gene transfer to determine whether CYR61 overexpression in the lungs was sufficient to cause ALI. Mice instilled with CYR61 adenovirus showed greater weight loss, increased bronchoalveolar lavage total neutrophil counts, increased protein concentrations, and increased mortality compared with mice instilled with empty-vector adenovirus. Immunohistochemical studies in lungs from humans with idiopathic pulmonary fibrosis revealed CYR61 expression on the luminal membrane of alveolar epithelial cells in areas of injury. We conclude that CYR61 is upregulated in ALI and that CYR61 overexpression exacerbates ALI in mice.

Background: Perioperative ischemic optic neuropathy occurs after major surgical procedures, which are often associated with hypotension, anemia, or venous congestion. However, the effects of these conditions on optic nerve (ON) blood flow are unknown and cannot be studied adequately in humans.Methods: Farm-raised pigs were anesthetized with isoflurane, kept normocapnic and normothermic, and subjected to conditions of euvolemic or hypovolemic hypotension (mean arterial pressure 50 -55 mm Hg), anemia (hematocrit 17%), venous congestion, and combinations thereof. Control animals were kept euvolemic and normotensive for the entire experiment. Fluorescent microspheres were used to measure cerebral blood flow (CBF) and ON blood flow at baseline and after experimental conditions, and to calculate oxygen delivery (DO 2 ).Results: No significant changes in CBF or ON blood flow or DO 2 occurred with euvolemic hypotension (n ‫؍‬ 5), compared with controls (n ‫؍‬ 12). Hypovolemic hypotension (n ‫؍‬ 4) resulted in stable CBF and cerebral DO 2 , but significant reductions in ON DO 2 (P ‫؍‬ 0.032). The significant increase in CBF associated with anemia (n ‫؍‬ 6) resulted in stable cerebral DO 2 . In contrast, ON blood flow did not significantly change with anemia, with (n ‫؍‬ 5) or without (n ‫؍‬ 6) euvolemic hypotension, resulting in significant reductions in ON DO 2 (P < 0.01).Conclusion: Compensatory mechanisms for porcine CBF maintain stable DO 2 under specified conditions of hypotension or anemia, whereas ON compensatory mechanisms were unable to maintain blood flow and to preserve DO 2 . The authors conclude that the porcine ON is more susceptible to physiologic perturbations than the brain.

No abstract

Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata. RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents' toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.