Cutting Edge: l-Arginine Transfer from Antigen-Presenting Cells Sustains CD4+ T Cell Viability and Proliferation.

Metabolomics analyses suggest changes in amino acid abundance, particularly l-arginine (L-ARG), occur in patients with tuberculosis. Immune cells require L-ARG to fuel effector functions following infection. We have previously described an L-ARG synthesis pathway in immune cells; however, its role in APCs has yet to be uncovered. Using a coculture system with mycobacterial-specific CD4+ T cells, we show APC L-ARG synthesis supported T cell viability and proliferation, and activated T cells contained APC-derived L-ARG. We hypothesize that APCs supply L-ARG to support T cell activation under nutrient-limiting conditions. This work expands the current model of APC-T cell interactions and provides insight into the effects of nutrient availability in immune cells.

Routine ICU admission after brain tumor surgery: retrospective validation and critical appraisal of two prediction scores.

BACKGROUND: Routine admission to an intensive care unit (ICU) following brain tumor surgery has been a common practice for many years. Although this practice has been challenged by many authors, it has still not changed widely, mainly due to the lack of reliable data for preoperative risk assessment. Motivated by this dilemma, risk prediction scores for postoperative complications following brain tumor surgery have been developed recently. In order to improve the ICU admission policy at our institution, we assessed the applicability, performance, and safety of the two most appropriate risk prediction scores. METHODS: One thousand consecutive adult patients undergoing elective brain tumor resection within 19 months were included. Patients with craniotomy for other causes, i.e., cerebral aneurysms and microvascular decompression, were excluded. The decision for postoperative ICU-surveillance was made by joint judgment of the operating surgeon and the anesthesiologist. All data and features relevant to the scores were extracted from clinical records and subsequent ICU or neurosurgical floor documentation was inspected for any postoperative adverse events requiring ICU admission. The CranioScore derived by Cinotti et al. (Anesthesiology 129(6):1111-20, 5) and the risk assessment score of Munari et al. (Acta Neurochir (Wien) 164(3):635-641, 15) were calculated and prognostic performance was evaluated by ROC analysis. RESULTS: In our cohort, both scores showed only a weak prognostic performance: the CranioScore reached a ROC-AUC of 0.65, while Munari et al.'s score achieved a ROC-AUC of 0.67. When applying the recommended decision thresholds for ICU admission, 64% resp. 68% of patients would be classified as in need of ICU surveillance, and the negative predictive value (NPV) would be 91% for both scores. Lowering the thresholds in order to increase patient safety, i.e., 95% NPV, would lead to ICU admission rates of over 85%. CONCLUSION: Performance of both scores was limited in our cohort. In practice, neither would achieve a significant reduction in ICU admission rates, whereas the number of patients suffering complications at the neurosurgical ward would increase. In future, better risk assessment measures are needed.

Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 °C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.

A protocol for digitizing colors: the case of measuring color variables for forested wetland soils.

A procedure is presented and discussed that highlights the use of the Nix Pro Color Sensor ("Nix") in digitizing soil colors with applications for forested wetland soils. Informed by our soil color investigations using both the Munsell Soil Color Chart (MSCC) and the Nix in forested wetlands of the northern Virginia area, we crafted a standard operating procedure (SOP), adaptable to various locations and/or soil types, that guides users-regardless of knowledge of soil ecology or familiarity with the Nix-to successfully assess and monitor soil colors at various depths. Our SOP outlines steps for digitally collecting, storing, and sharing soil color data. Through the implementation of this procedure, soil color monitoring can enter the digital age, removing barriers of entry to soil color determination and enhancing individuals' interest in monitoring and understanding of the importance of soil color as an environmental and ecological indicator. With continued refinement and adaptation to intended use, the SOP herein presented has the potential to aid wetland/watershed assessment by providing data on soil colors that can be tracked over time while also encouraging public engagement in environmental monitoring of soils.

l-Arginine Synthesis from l-Citrulline in Myeloid Cells Drives Host Defense against Mycobacteria In Vivo.

Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline-generating (i.e., iNOS) and l-citrulline-using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either Ass1 or Asl resulted in increased Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis H37Rv burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway.

Predicting forested wetland soil carbon using quantitative color sensor measurements in the region of northern Virginia, USA.

Forested wetland soils within the Piedmont and Coastal Plain physiographic provinces of Northern Virginia (NOVA) were investigated to determine the utility of a handheld colorimeter, the Nix Pro Color Sensor ("Nix"), for predicting carbon contents (TC) and stocks (TC stocks) from on-site color measurements. Both the color variables recorded with each Nix scan ("Nix color variables"; n = 15) and carbon contents significantly differed between sites, with redder soils (higher a and h) at Piedmont sites, and higher TC at sites with darker soils (lower values of L, or lightness; p < 0.05). Nix-carbon correlation analysis revealed strong relationships between L (lightness), X (a virtual spectral variable), R (additive red), and KK (black) and log-transformed TC (Ln[TC]; |r| = 0.70; p < 0.01 for all). Simple linear regressions were conducted to identify how well these four final Nix variables could predict soil carbon. Using all color measurements, about 50% of Ln(TC) variability could be explained by L, X, R, or KK (p < 0.01), yet with higher predictive power obtained for Coastal Plain soils (0.55 < R2 < 0.65; p < 0.01). Regression model strength was maximized between Ln(TC) and the four final Nix variables using simple linear regressions when color measurements observed at a specific depth were first averaged (0.66 < R2 < 0.70; p < 0.01). While further study is warranted to investigate Nix applicability within various soil settings, these results demonstrate potential for the Nix and its soil color measurements to assist with rapid field-based assessments of soil carbon in forested wetlands.

Just a very expensive breathing training? Risk of respiratory artefacts in functional connectivity-based real-time fMRI neurofeedback.

Real-time functional magnetic resonance imaging neurofeedback (rtfMRI NFB) is a promising method for targeted regulation of pathological brain processes in mental disorders. But most NFB approaches so far have used relatively restricted regional activation as a target, which might not address the complexity of the underlying network changes. Aiming towards advancing novel treatment tools for disorders like schizophrenia, we developed a large-scale network functional connectivity-based rtfMRI NFB approach targeting dorsolateral prefrontal cortex and anterior cingulate cortex connectivity with the striatum. In a double-blind randomized yoke-controlled single-session feasibility study with N â€‹= â€‹38 healthy controls, we identified strong associations between our connectivity estimates and physiological parameters reflecting the rate and regularity of breathing. These undesired artefacts are especially detrimental in rtfMRI NFB, where the same data serves as an online feedback signal and offline analysis target. To evaluate ways to control for the identified respiratory artefacts, we compared model-based physiological nuisance regression and global signal regression (GSR) and found that GSR was the most effective method in our data. Our results strongly emphasize the need to control for physiological artefacts in connectivity-based rtfMRI NFB approaches and suggest that GSR might be a useful method for online data correction for respiratory artefacts.

A new Luminex-based peptide assay to identify reactivity to baked, fermented, and whole milk.

BACKGROUND: The majority of children with cow's milk allergy (CMA) tolerate baked milk. However, reactivity to fermented milk products such as yogurt/cheese has not been previously evaluated. We sought to determine whether children with CMA could tolerate yogurt/cheese and whether a patient's IgE and IgG4-binding pattern to milk protein epitopes could distinguish clinical reactivity. METHODS: Four groups of reactivity were identified by Oral food challenge: baked milk reactive, fermented milk reactive, whole milk reactive, and outgrown. sIgE and sIgG4 binding to milk protein epitopes were assessed with a novel Luminex-based peptide assay (LPA). Using machine learning techniques, a model was developed to predict different degrees of CMA. RESULTS: The baked milk reactive patients demonstrated the highest degree of IgE epitope binding, which was followed sequentially by fermented milk reactive, whole milk reactive, and outgrown. Data were randomly divided into two groups with 75% of the data utilized for model development (n = 68) and 25% for testing (n = 21). All 68 children used for training were correctly classified with models using IgE and IgG4 epitopes. The average cross-validation accuracy was much higher for models using IgE plus IgG4 epitopes by LPA (84.8%), twice the performance of the serum component proteins assayed by UniCAP (41.9%). The performance of the model on "unseen data" was tested using the 21 withheld patients, and the accuracy of IgE was 86% (AUC = 0.89) while of IgE+IgG4 model was 81% (AUC = 0.94). CONCLUSION: Using a novel high-throughput LPA, we were able to distinguish the diversity of IgE/IgG4 binding to epitopes in the varying CMA phenotypes. LPA is a promising tool to predict correctly different degrees of CMA.

Nucleus accumbens activation is linked to salience in social decision making.

Aberrant salience may explain hasty decision making and psychotic symptoms in schizophrenia. In healthy individuals, final decisions in probabilistic reasoning tasks are related to Nucleus accumbens (Nacc) activation. However, research investigating the Nacc in social decision making is missing. Our study aimed at investigating the role of the Nacc for social decision making and its link to (aberrant) salience attribution. 47 healthy individuals completed a novel social jumping-to-conclusion (JTC) fMRI-paradigm, showing morphed faces simultaneously expressing fear and happiness. Participants decided on the 'current' emotion after each picture, and on the 'general' emotion of series of faces. Nacc activation was stronger during final decisions than in previous trials without a decision, particularly in fear rather than happiness series. A JTC-bias was associated with higher Nacc activation for last fearful, but not last happy faces. Apparently, mechanisms underlying probabilistic reasoning are also relevant for social decision making. The pattern of Nacc activation suggests salience, not reward, drives the final decision. Based on these findings, we hypothesize that aberrant salience might also explain social-cognitive deficits in schizophrenia.

Respiration pattern variability and related default mode network connectivity are altered in remitted depression.

BACKGROUND: Studies with healthy participants and patients with respiratory diseases suggest a relation between respiration and mood. The aim of the present analyses was to investigate whether emotionally challenged remitted depressed participants show higher respiration pattern variability (RPV) and whether this is related to mood, clinical outcome and increased default mode network connectivity. METHODS: To challenge participants, sad mood was induced with keywords of personal negative life events in individuals with remitted depression [recurrent major depressive disorder (rMDD), n = 30] and matched healthy controls (HCs, n = 30) during functional magnetic resonance imaging. Respiration was measured by means of a built-in respiration belt. Additionally, questionnaires, a daily life assessment of mood and a 3 years follow-up were applied. For replication, we analysed RPV in an independent sample of 53 rMDD who underwent the same fMRI paradigm. RESULTS: During sad mood, rMDD compared with HC showed greater RPV, with higher variability in pause duration and respiration frequency and lower expiration to inspiration ratio. Higher RPV was related to lower daily life mood and predicted higher depression scores as well as relapses during a 3-year follow-up period. Furthermore, in rMDD compared with HC higher main respiration frequency exhibited a more positive association with connectivity of the posterior cingulate cortex and the right parahippocampal gyrus. CONCLUSIONS: The results suggest a relation between RPV, mood and depression on the behavioural and neural level. Based on our findings, we propose interventions focusing on respiration to be a promising additional tool in the treatment of depression.

Impaired emotion processing and a reduction in trust in patients with somatic symptom disorder.

There is accumulating evidence for deficits in the perception and regulation of one's own emotions, as well as the recognition of others' emotions in somatic symptom disorder (SSD). However, investigations of SSD focusing on specific aspects of emotion processing and how these might interact are missing. We included 35 patients with SSD and 35 healthy controls who completed questionnaires on the perception and regulation of their own emotions, as well as experimental investigations of emotion recognition and trust. In line with previous studies, our results show that SSD patients in comparison to healthy controls have difficulties in the identification and description of own feelings (ηp2  = .381 and ηp2  = .315). Furthermore, we found that patients apply less cognitive reappraisal (ηp2  = .185) but tend to use more expressive suppression (ηp2  = .047). In contrast to previous studies, we found SSD patients to perform superior in emotion recognition, in particular for anger (d = 0.40). In addition, patients with SSD invested less in a trust game (d = 0.73). These results point to a higher sensitivity for negative emotions and less trust in others. Further, these findings suggest a dissociation between the ability to recognize one's own emotions versus others' emotions in SSD. Future interventions targeting emotion processing in SSD might focus on the identification of one's own emotions, prior to the training of emotion regulation.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

Differential Requirements for L-Citrulline and L-Arginine during Antimycobacterial Macrophage Activity.

Microbicidal NO production is reliant on inducible NO synthase-mediated L-arginine metabolism in macrophages (MΦs). However, L-arginine supply can be restricted by arginase activity, resulting in inefficient NO output and inhibition of antimicrobial MΦ function. MΦs circumvent this by converting L-citrulline to L-arginine, thereby resupplying substrate for NO production. In this article, we define the metabolic signature of mycobacteria-infected murine MΦs supplied L-arginine, L-citrulline, or both amino acids. Using liquid chromatography-tandem mass spectrometry, we determined that L-arginine synthesized from L-citrulline was less effective as a substrate for arginase-mediated L-ornithine production compared with L-arginine directly imported from the extracellular milieu. Following Mycobacterium bovis bacillus Calmette-Guérin infection and costimulation with IFN-γ, we observed that MΦ arginase activity did not inhibit production of NO derived from L-citrulline, contrary to NO inhibition witnessed when MΦs were cultured in L-arginine. Furthermore, we found that arginase-expressing MΦs preferred L-citrulline over L-arginine for the promotion of antimycobacterial activity. We expect that defining the consequences of L-citrulline metabolism in MΦs will provide novel approaches for enhancing immunity, especially in the context of mycobacterial disease.

Unique and Redundant Functions of p70 Ribosomal S6 Kinase Isoforms Regulate Mesenchymal Cell Proliferation and Migration in Pulmonary Fibrosis.

The p70 ribosomal S6 kinase (p70S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with pulmonary fibrogenesis. Two isoforms of the p70S6K have been identified (S6K1 and S6K2), but their relative contributions in mediating pulmonary fibrosis are unknown. To interrogate the roles of the p70S6K isoforms, we overexpressed transforming growth factor (TGF)-α in mice deficient for the S6K1 or S6K2 genes and measured changes in lung histology, morphometry, total lung collagen, lung function, and proliferation between wild-type and isoform-deficient mice. Deficiency of S6K1, but not S6K2, had a significant effect on reducing proliferation in subpleural fibrotic lesions during TGF-α-induced fibrosis. Migration was significantly decreased in mesenchymal cells isolated from the lungs of S6K1 knockout mice compared with wild-type or S6K2 knockout mice. Conversely, increases in subpleural thickening were significantly decreased in S6K2-deficient mice compared with wild type. Deficiency of S6K2 significantly reduced phosphorylation of the downstream S6 ribosomal protein in lung homogenates and isolated mesenchymal cells after TGF-α expression. However, deficiency of neither isoform alone significantly altered TGF-α-induced collagen accumulation or lung function decline in vivo. Furthermore, deficiency in neither isoform prevented changes in collagen accumulation or lung compliance decline after administration of intradermal bleomycin. Together, these findings demonstrate that the p70S6K isoforms have unique and redundant functions in mediating fibrogenic processes, including proliferation, migration, and S6 phosphorylation, signifying that both isoforms must be targeted to modulate p70S6K-mediated pulmonary fibrosis.

p70 ribosomal S6 kinase regulates subpleural fibrosis following transforming growth factor-α expression in the lung.

The p70 ribosomal S6 kinase (S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with fibrogenesis. Recent studies demonstrate that aberrant mTORC1-S6K signaling contributes to various pathological conditions, but a direct role in pulmonary fibroproliferation has not been established. Increased phosphorylation of the S6K pathway is detected immediately following transforming growth factor-α (TGF-α) expression in a transgenic model of progressive lung fibrosis. To test the hypothesis that the S6K directly regulates pulmonary fibroproliferative disease we determined the cellular sites of S6K phosphorylation during the induction of fibrosis in the TGF-α model and tested the efficacy of specific pharmacological inhibition of the S6K pathway to prevent and reverse fibrotic disease. Following TGF-α expression increased phosphorylation of the S6K was detected in the airway and alveolar epithelium and the mesenchyme of advanced subpleural fibrotic regions. Specific inhibition of the S6K with the small molecule inhibitor LY-2584702 decreased TGF-α and platelet-derived growth factor-ß-induced proliferation of lung fibroblasts in vitro. Administration of S6K inhibitors to TGF-α mice prevented the development of extensive subpleural fibrosis and alterations in lung mechanics, and attenuated the increase in total lung hydroxyproline. S6K inhibition after fibrosis was established attenuated the progression of subpleural fibrosis. Together these studies demonstrate targeting the S6K pathway selectively modifies the progression of pulmonary fibrosis in the subpleural compartment of the lung.

Complex reinnervation pattern after unilateral renal denervation in rats.

Renal denervation (DNX) is a treatment for resistant arterial hypertension. Efferent sympathetic nerves regrow, but reinnervation by renal afferent nerves has only recently been shown in the renal pelvis of rats after unilateral DNX. We examined intrarenal perivascular afferent and sympathetic efferent nerves after unilateral surgical DNX. Tyrosine hydroxylase (TH), CGRP, and smooth muscle actin were identified in kidney sections from 12 Sprague-Dawley rats, to distinguish afferents, efferents, and vasculature. DNX kidneys and nondenervated kidneys were examined 1, 4, and 12 wk after DNX. Tissue levels of CGRP and norepinephrine (NE) were measured with ELISA and mass spectrometry, respectively. DNX decreased TH and CGRP labeling by 90% and 95%, respectively (P < 0.05) within 1 wk. After 12 wk TH and CGRP labeling returned to baseline with a shift toward afferent innervation (P < 0.05). Nondenervated kidneys showed a doubling of both labels within 12 wk (P < 0.05). CGRP content decreased by 72% [3.2 ± 0.3 vs. 0.9 ± 0.2 ng/gkidney; P < 0.05] and NA by 78% [1.1 ± 0.1 vs. 0.2 ± 0.1 pmol/mgkidney; P < 0.05] 1 wk after DNX. After 12 wk, CGRP, but not NE, content in DNX kidneys was fully recovered, with no changes in the nondenervated kidneys. The use of phenol in the DNX procedure did not influence this result. We found morphological reinnervation and transmitter recovery of afferents within 12 wk after DNX. Despite morphological evidence of sympathetic regrowth, NE content did not fully recover. These results suggest a long-term net surplus of afferent influence on the DNX kidney may be contributing to the blood pressure lowering effect of DNX.

Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes.

BACKGROUND: SLC10A4 belongs to the solute carrier family SLC10 whose founding members are the Na(+)/taurocholate co-transporting polypeptide (NTCP, SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). These carriers maintain the enterohepatic circulation of bile acids between the liver and the gut. SLC10A4 was identified as a novel member of the SLC10 carrier family with the highest phylogenetic relationship to NTCP. The SLC10A4 protein was detected in synaptic vesicles of cholinergic and monoaminergic neurons of the peripheral and central nervous system, suggesting a transport function for any kind of neurotransmitter. Therefore, in the present study, we performed systematic transport screenings for SLC10A4 and also aimed to identify the vesicular sorting domain of the SLC10A4 protein. RESULTS: We detected a vesicle-like expression pattern of the SLC10A4 protein in the neuronal cell lines SH-SY5Y and CAD. Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern. Functional transport studies with different neurotransmitters, bile acids and steroid sulfates were performed in SLC10A4-transfected HEK293 cells, SLC10A4-transfected CAD cells and in Xenopus laevis oocytes. For these studies, transport by the dopamine transporter DAT, the serotonin transporter SERT, the choline transporter CHT1, the vesicular monoamine transporter VMAT2, the organic cation transporter Oct1, and NTCP were used as positive control. SLC10A4 failed to show transport activity for dopamine, serotonin, norepinephrine, histamine, acetylcholine, choline, acetate, aspartate, glutamate, gamma-aminobutyric acid, pregnenolone sulfate, dehydroepiandrosterone sulfate, estrone-3-sulfate, and adenosine triphosphate, at least in the transport assays used. When the C-terminus of SLC10A4 was replaced by the homologous sequence of NTCP, the SLC10A4-NTCP chimeric protein revealed clear plasma membrane expression in CAD and HEK293 cells. But this chimera also did not show any transport activity, even when the N-terminal domain of SLC10A4 was deleted by mutagenesis. CONCLUSIONS: Although different kinds of assays were used to screen for transport function, SLC10A4 failed to show transport activity for a series of neurotransmitters and neuromodulators, indicating that SLC10A4 does not seem to represent a typical neurotransmitter transporter such as DAT, SERT, CHT1 or VMAT2.

Bone marrow-derived stromal cells are invasive and hyperproliferative and alter transforming growth factor-α-induced pulmonary fibrosis.

Pulmonary fibrosis is caused by excessive proliferation and accumulation of stromal cells. Fibrocytes are bone marrow (BM)-derived cells that contribute to pathologic stromal cell accumulation in human lung disease. However, the cellular source for these stromal cells and the degree of fibrocyte contribution to pulmonary fibrosis remain unclear. To determine the etiology of stromal cell excess during pulmonary fibrosis, we measured fibrocytes during the progression of fibrosis in the transforming growth factor (TGF)-α transgenic mouse model. Lung epithelial-specific overexpression of TGF-α led to progressive pulmonary fibrosis associated with increased accumulation of fibrocytes in the fibrotic lesions. Although reconstitution of BM cells into TGF-α mice demonstrated accumulation of these cells in fibrotic lesions, the majority of the cells did not express α-smooth muscle actin, suggesting that fibrocytes did not transform into myofibroblasts. To explore the mechanisms of fibrocytes in pulmonary fibrogenesis, adoptive cell-transfer experiments were performed. Purified fibrocytes were transferred intravenously into TGF-α transgenic mice, and fibrosis endpoints were compared with controls. Analysis of lung histology and hydroxyproline levels demonstrated that fibrocyte transfers augment TGF-α-induced lung fibrosis. A major subset of TGF-α-induced fibrocytes expressed CD44 and displayed excessive invasiveness, which is attenuated in the presence of anti-CD44 antibodies. Coculture experiments of resident fibroblasts with fibrocytes demonstrated that fibrocytes stimulate proliferation of resident fibroblasts. In summary, fibrocytes are increased in the progressive, fibrotic lesions of TGF-α-transgenic mice and activate resident fibroblasts to cause severe lung disease.

Inhibition of the αvß6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis.

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-ß (TGF-ß) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-ß induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-ß pathway in the lung is unknown. The αvß6 integrin is an important in vivo activator of TGF-ß activation in the lung. Immunohistochemical analysis of αvß6 protein expression and bronchoalveolar analysis of TGF-ß pathway signaling indicates activation of the αvß6/TGF-ß pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvß6/TGF-ß pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvß6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvß6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the ß6 integrin, TGF-α transgenic mice were mated with ß6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the ß6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the ß6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvß6/TGF-ß pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

Effects of saxagliptin on early microvascular changes in patients with type 2 diabetes.

BACKGROUND: Patients with diabetes mellitus are at increased risk for microvascular complications. Early changes in microcirculation are characterized by hyperperfusion (e.g. in the retina and kidney) and increased pulse wave reflection leading to increased aortic pressure. We investigated the effects of the DPP-4-inhibitor saxagliptin on early retinal microvascular changes. METHODS: In this double-blind, controlled, cross-over trial 50 patients (without clinical signs of microvascular alterations) with type-2 diabetes (mean duration of 4 years) were randomized to receive placebo or 5 mg saxagliptin for 6 weeks. Retinal arteriolar structure and retinal capillary flow (RCF) at baseline and during flicker-light exposure was assessed by scanning laser Doppler flowmetry. Central hemodynamics were assessed by pulse wave analysis. RESULTS: Postprandial blood glucose (9.27 ± 0.4 versus 10.1 ± 0.4 mmol/L; p = 0.001) and HbA1c (6.84 ± 0.15 (51 ± 1.6) versus 7.10 ± 0.17% (54 ± 1.9 mmol/mol); p < 0.001) were significantly reduced with saxagliptin treatment compared to placebo. RCF was significantly reduced after treatment with saxagliptin (288 ± 13.2 versus 314 ± 14.1 AU; p = 0.033). This was most pronounced in a subgroup of patients (n = 32) with a fall in postprandial blood glucose (280 ± 12.1 versus 314 ± 16.6 AU; p = 0.011). No significant changes in RCF were seen during flicker-light exposure between placebo and saxagliptin, but the vasodilatory capacity increased two-fold with saxagliptin treatment. Central augmentation pressure tended to be lower after treatment with saxagliptin (p = 0.094), and central systolic blood pressure was significantly reduced (119 ± 2.3 versus 124 ± 2.3 mmHg; p = 0.038). CONCLUSIONS: Our data suggest that treatment with saxagliptin for 6 weeks normalizes retinal capillary flow and improves central hemodynamics in type-2 diabetes. TRIAL REGISTRATION: The study was registered at (ID: NCT01319357).