Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF

Microvascular plasma protein leakage is an essential component of the inflammatory response and serves an important function in local host defense and tissue repair. Mediators such as histamine and bradykinin act directly on venules to increase the permeability of endothelial cell (EC) junctions. Neutrophil chemoattractants also induce leakage, a response that is dependent on neutrophil adhesion to ECs, but the underlying mechanism has proved elusive. Through application of confocal intravital microscopy to the mouse cremaster muscle, we show that neutrophils responding to chemoattractants release TNF when in close proximity of EC junctions. In vitro, neutrophils adherent to ICAM-1 or ICAM-2 rapidly released TNF in response to LTB₄, C5a, and KC. Further, in TNFR^−/− mice, neutrophils accumulated normally in response to chemoattractants administered to the cremaster muscle or dorsal skin, but neutrophil-dependent plasma protein leakage was abolished. Similar results were obtained in chimeric mice deficient in leukocyte TNF. A locally injected TNF blocking antibody was also able to inhibit neutrophil-dependent plasma leakage, but had no effect on the response induced by bradykinin. The results suggest that TNF mediates neutrophil-dependent microvascular leakage. This mechanism may contribute to the effects of TNF inhibitors in inflammatory diseases and indicates possible applications in life-threatening acute edema.In response to an inflammatory stimulus, priorities change with respect to the maintenance of tissue homeostasis. Under basal conditions, the microvascular endothelium has a low permeability to plasma proteins and forms a barrier between high intravascular and low extravascular interstitial fluid protein concentrations. This provides an oncotic gradient that balances the opposing hydrostatic pressure gradient that would otherwise move water and small solutes from the blood to the tissues, with any small net outflow being compensated by lymphatic drainage. After the detection of potentially harmful substances, foreign organisms, or injured tissue cells, the immediate priority is to supply plasma proteins to the extravascular compartment. This delivers immunoglobulins, constituents of the complement and coagulation systems, antiproteases, and other acute phase reactants to the site to participate in local host defense and the initiation of tissue repair. The compromise to homeostasis results in tissue edema (an example of functio laesa). Tissue mast cells act as sentinel cells and provide one means of detecting potentially harmful agents by means of an array of receptors, including Toll-like receptors and IgE bound to FcεR1. Mast cells release histamine that induces plasma leakage by acting directly on histamine receptors located on the venular endothelium, the region of the microvasculature specialized for permeability responses (Majno and Palade, 1961). Increased permeability is dependent on a destabilization of the VE-cadherin–catenin complex and its interaction with the actin cytoskeleton at EC junctions (Yuan, 2002; Schulte et al., 2011). Other such mediators acting directly on ECs include bradykinin, peptidoleukotrienes, platelet-activating factor, and VEGF.Inflammatory stimuli also trigger the local production of chemoattractants that induce acute neutrophil infiltration. Once accumulated and interacting with the potentially injurious stimulus, these cells were generally accepted to contribute to tissue swelling in the later stages of inflammation by releasing substances that damage the endothelium, such as proteases and reactive oxygen species. Subsequent observations suggested another link between neutrophils and increased microvascular permeability by a mechanism that could operate in the very early stages of the inflammatory response. The neutrophil chemoattractants C5a, leukotriene B₄ (LTB₄), and fMLP were found to induce significantly increased EC permeability within 6 min of injection into animal skin, and these responses were absent in animals depleted of their circulating neutrophils (Wedmore and Williams, 1981). This led to the conclusion that neutrophils responding to a chemoattractant signal can rapidly increase the permeability of EC junctions when the leukocytes are in close apposition to venular ECs; thus, regulating the supply of plasma proteins to the extravascular space. This concept was supported by studies in which a blocking anti-β₂ integrin mAb was found to inhibit neutrophil-dependent edema (Arfors et al., 1987). Collectively, neutrophil chemoattractants, including chemokines, have been found to increase microvascular permeability in several animal species (Wedmore and Williams, 1981; Williams and Jose, 1981; Björk et al., 1982; Tokita and Yamamoto, 2004), and responses to intradermal C5a have been shown to be markedly reduced in neutropenic human subjects (Williamson et al., 1986; Yancey et al., 1987).The perivascular basement membrane (BM) is permeable to plasma proteins and allows their entry into tissues when EC junctions open. Large colloidal molecules administered into the systemic circulation are, however, trapped under the BM/pericyte layer, which is the basis of the vascular labeling technique that first identified venules as the site of action of histamine (Majno and Palade, 1961). Some early studies showed that neutrophils can disrupt the perivascular BM so that large colloidal molecules can pass through (Hurley, 1964; Huber and Weiss, 1989). These observations may relate to our previous findings that show changes in BM morphology and enlarged gaps between pericytes induced by inflammatory mediators in vivo (Wang et al., 2006; Voisin et al., 2009; Proebstl et al., 2012). In extending these findings, we noted that neutrophil chemoattractants induced pericyte shape change in vivo in a neutrophil-dependent manner, and that this response was mediated by endogenously generated TNF. Because, in response to chemoattractants, neutrophils within the vascular lumen expressed TNF in close proximity of EC junctions and TNF increases microvascular permeability to plasma proteins (Brett et al., 1989), we investigated if neutrophil-derived TNF could also provide an important link between neutrophils and increased endothelial permeability. The present results provide direct evidence for the ability of endogenous neutrophil-derived TNF to mediate chemoattractant-induced increased microvascular permeability and describe a potential mechanism for neutrophil-dependent edema.     

Hypomorphic PCNA mutation underlies a human DNA repair disorder

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.     

Role of nucleus accumbens in neuropathic pain: Linked multi-scale evidence in the rat transitioning to neuropathic pain

Despite recent evidence implicating the nucleus accumbens (NAc) as causally involved in the transition to chronic pain in humans, underlying mechanisms of this involvement remain entirely unknown. Here we elucidate mechanisms of NAc reorganizational properties (longitudinally and cross-sectionally), in an animal model of neuropathic pain (spared nerve injury [SNI]). We observed interrelated changes: (1) In resting-state functional magnetic resonance imaging (fMRI), functional connectivity of the NAc to dorsal striatum and cortex was reduced 28 days (but not 5 days) after SNI; (2) Contralateral to SNI injury, gene expression of NAc dopamine 1A, 2, and κ-opioid receptors decreased 28 days after SNI; (3) In SNI (but not sham), covariance of gene expression was upregulated at 5 days and settled to a new state at 28 days; and (4) NAc functional connectivity correlated with dopamine receptor gene expression and with tactile allodynia. Moreover, interruption of NAc activity (via lidocaine infusion) reversibly alleviated neuropathic pain in SNI animals. Together, these results demonstrate macroscopic (fMRI) and molecular reorganization of NAc and indicate that NAc neuronal activity is necessary for full expression of neuropathic pain-like behavior.     

Can Methotrexate be a successful treatment for unresponsive generalized annular elastolytic giant cell granuloma? Case report and review of the literature

Annular elastolytic giant cell granuloma (AEGCG) is a rare granulomatous skin disorder, characterized by erythematous plaques with elevated borders and hypopigmented center, occurring mainly on sun exposed‐skin. Histologically it presents with elastophagocytosis and elastolysis. There is no established first line treatment for AEGCG, especially for the generalized form. In a small number of cases, antimalarial drugs and tranilast, associated to topical or oral steroids, have been proposed to treat generalized AEGCG with partial benefits. We herein present the case of a patient with AEGCG aged 74 years, who was unresponsive to classical therapies, and then successfully treated with methotrexate.     

Gremlin-1 Identifies Fibrosis and Predicts Adverse Outcome in Patients With Heart Failure Undergoing Endomyocardial Biopsy

BackgroundGremlin-1 (Grem1), an antagonist of bone morphogenetic proteins, is involved in fibrotic tissue formation in kidney and lung. The impact of myocardial Grem1 expression is unknown. We investigated the prognostic value of Grem1 expression in 214 consecutive patients with nonischemic heart failure (HF) undergoing endomyocardial biopsy.MethodsIn all patients, the following risk factors were assessed: Grem1 expression (semiquantitative score scheme ranging from 1 to 4), presence of inflammatory markers, detection of viral genome, left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), New York Heart Association functional class (NYHA), troponin I, and B-type natriuretic peptide. Degree of myocardial fibrosis was defined as an index. Study end point was a combination of all-cause death and HF-related rehospitalization within 3 years of follow-up.ResultsGrem1 expression significantly correlated with the degree of myocardial fibrosis (correlation coefficient r = 0.619; P < .0001). Patients with the highest Grem1 expression (score 4) showed the most severely impaired LVEF and highest LVEDD (P < .0001 and P = .030, respectively, for comparison of semiquantitative scores). During follow-up, 33 patients (15.4%) reached the study end point. Grem1 expression and NYHA ≥II were independent predictors of the end point (Grem1: hazard ratio [HR] 7.5, 95% confidence interval [CI] 1.8-32.2; P = .006; NYHA ≥II: HR 2.0, 95% CI 1.0-4.1; P = .048).ConclusionsGrem1 correlates with the degree of myocardial fibrosis and left ventricular dysfunction and is an independent predictor of adverse outcome in patients with nonischemic HF.     

Drug misuse among patients admitted to a general hospital

Aims In this paper we report the prevalence of prescribed drugs of misuse and illicit drugs used by patients admitted to a general hospital and the level of detection of drug problems by general medical staff.

Design This is a prospective questionnaire survey, interview and case note review.

Setting This study is a snapshot of one week's admission to a general hospital.

Findings Of the 408 people approached, 285 (70%) participated in the study. One hundred and sixty‐six people (62%) reported misuse of drugs at some time in their lives. Of these, 46 (17%) reported use of illegal drugs at some time in their lives, 22 (8%) in the past year, and 7 (2.6%) in the previous month. The most frequently reported drug type used ever, in the past year, and in the previous month, was over‐the‐counter medication and sedatives. All nine dependent patients identified by the interview were polydrug users and were significantly younger. Two of these patients were assessed for substance misuse by the medical staff.

Conclusion This study suggests that younger patients should be asked about their drug use, especially their use of more readily available drugs. At present, few questions are being asked by health professionals, leaving drug misuse to continue to drain both healthcare and society's resources.     

Cardiac sarcoidosis as a cause of sudden death

Sarcoidosis, also called Besnier-Boeck disease, is a systemic granulomatous disease of unknown etiology which was classified as a separate unit in 1958. Histopathology of the disease is based on epithelioid granulomas which infiltrate and damage different organs and tissues. The disease is most commonly located in the lungs; however, first complaints may be related to its manifestation in other organs. Symptomatic involvement of the heart is found in approx. 5% of patients and is one of the most dangerous forms of the disease.     

NOTCH1 activation clinically antagonizes the unfavorable effect of PTEN inactivation in BFM-treated children with precursor T-cell acute lymphoblastic leukemia

Despite improvements in treatment results for pediatric T-cell acute lymphoblastic leukemia, approximately 20% of patients relapse with dismal prognosis. PTEN inactivation and NOTCH1 activation are known frequent leukemogenic events but their effect on outcome is still controversial. We analyzed the effect of PTEN inactivation and its interaction with NOTCH1 activation on treatment response and long-term outcome in 301 ALL-BFM treated children with T-cell acute lymphoblastic leukemia. We identified PTEN mutations in 52 of 301 (17.3%) of patients. In univariate analyses this was significantly associated with increased resistance to induction chemotherapy and a trend towards poor long-term outcome. By contrast, patients with inactivating PTEN and activating NOTCH1 mutations showed marked sensitivity to induction treatment and excellent long-term outcome, which was similar to patients with NOTCH1 mutations only, and more favorable than in patients with PTEN mutations only. Notably, in the subgroup of patients with a prednisone- and minimal residual disease (MRD)-response based medium risk profile, PTEN-mutations without co-existing NOTCH1-mutations represented an MRD-independent highly significant high-risk biomarker. Mutations of PTEN highly significantly indicate a poor prognosis in T-ALL patients who have been stratified to the medium risk group of the BFM-protocol. This effect is clinically neutralized by NOTCH1 mutations. Although these results have not yet been explained by an obvious molecular mechanism, they contribute to the development of new molecularly defined stratification algorithms. Furthermore, these data have unexpected potential implications for the development of NOTCH1 inhibitors in the treatment of T-cell acute lymphoblastic leukemia in general, and in those with a combination of PTEN and NOTCH1 mutations in particular.