Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.

Host genetic determinants of spontaneous hepatitis C clearance

Acute infection with the hepatitis C virus (HCV) induces a wide range of innate and adaptive immune responses. A total of 20-50% of acutely HCV-infected individuals permanently control the virus, referred to as 'spontaneous hepatitis C clearance', while the infection progresses to chronic hepatitis C in the majority of cases. Numerous studies have examined host genetic determinants of hepatitis C infection outcome and revealed the influence of genetic polymorphisms of human leukocyte antigens, killer immunoglobulin-like receptors, chemokines, interleukins and interferon-stimulated genes on spontaneous hepatitis C clearance. However, most genetic associations were not confirmed in independent cohorts, revealed opposing results in diverse populations or were limited by varying definitions of hepatitis C outcomes or small sample size. Coordinated efforts are needed in the search for key genetic determinants of spontaneous hepatitis C clearance that include well-conducted candidate genetic and genome-wide association studies, direct sequencing and follow-up functional studies.

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHAO.

In Pseudomonasfluorescens strain CHAO, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.

Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

Spontaneous Atopic Dermatitis-Like Symptoms in a/a ma ft/ma ft/J Flaky Tail Mice Appear Early after Birth.

Loss-of-function mutations in human profilaggrin gene have been identified as the cause of ichthyosis vulgaris (IV), and as a major predisposition factor for atopic dermatitis (AD). Similarly, flaky tail (a/a ma ft/ma ft/J) mice were described as a model for IV, and shown to be predisposed to eczema. The aim of this study was to correlate the flaky tail mouse phenotype with human IV and AD, in order to dissect early molecular events leading to atopic dermatitis in mice and men, suffering from filaggrin deficiency. Thus, 5-days old flaky tail pups were analyzed histologically, expression of cytokines was measured in skin and signaling pathways were investigated by protein analysis. Human biopsies of IV and AD patients were analyzed histologically and by real time PCR assays. Our data show acanthosis and hyperproliferation in flaky tail epidermis, associated with increased IL1β and thymic stromal lymphopoietin (TSLP) expression, and Th2-polarization. Consequently, NFκB and Stat pathways were activated, and IL6 mRNA levels were increased. Further, quantitative analysis of late epidermal differentiation markers revealed increased Small proline-rich protein 2A (Sprr2a) synthesis. Th2-polarization and Sprr2a increase may result from high TSLP expression, as shown after analysis of 5-days old K14-TSLP tg mouse skin biopsies. Our findings in the flaky tail mouse correlate with data obtained from patient biopsies of AD, but not IV. We propose that proinflammatory cytokines are responsible for acanthosis in flaky tail epidermis, and together with the Th2-derived cytokines lead to morphological changes. Accordingly, the a/a ma ft/ma ft/J mouse model can be used as an appropriate model to study early AD onset associated with profilaggrin deficiency.

Induction of experimental endocarditis by continuous low-grade bacteremia mimicking spontaneous bacteremia in humans.

Transient high-grade bacteremia following invasive procedures carries a risk of infective endocarditis (IE). This is supported by experimental endocarditis. On the other hand, case-control studies showed that IE could be caused by cumulative exposure to low-grade bacteremia occurring during daily activities. However, no experimental demonstration of this latter possibility exists. This study investigated the infectivity in animals of continuous low-grade bacteremia compared to that of brief high-grade bacteremia. Rats with aortic vegetations were inoculated with Streptococcus intermedius, Streptococcus gordonii or Staphylococcus aureus (strains Newman and P8). Animals were challenged with 10(3) to 10(6) CFU. Identical bacterial numbers were given by bolus (1 ml in 1 min) or continuous infusion (0.0017 ml/min over 10 h). Bacteremia was 50 to 1,000 times greater after bolus than during continuous inoculation. Streptococcal bolus inoculation of 10(5) CFU infected 63 to 100% vegetations compared to 30 to 71% infection after continuous infusion (P > 0.05). When increasing the inoculum to 10(6) CFU, bolus inoculation infected 100% vegetations and continuous infusion 70 to 100% (P > 0.05). S. aureus bolus injection of 10(3) CFU infected 46 to 57% valves. This was similar to the 53 to 57% infection rates produced by continuous infusion (P > 0.05). Inoculation of 10(4) CFU of S. aureus infected 80 to 100% vegetations after bolus and 60 to 75% after continuous infusion (P > 0.05). These results show that high-level bacteremia is not required to induce experimental endocarditis and support the hypothesis that cumulative exposure to low-grade bacteremia represents a genuine risk of IE in humans.

Cardiovascular effects in patrol officers are associated with fine particulate matter from brake wear and engine emissions

Background: Exposure to fine particulate matter air pollutants (PM2.5) affects heart rate variability parameters, and levels of serum proteins associated with inflammation, hemostasis and thrombosis. This study investigated sources potentially responsible for cardiovascular and hematological effects in highway patrol troopers. Results: Nine healthy young non-smoking male troopers working from 3 PM to midnight were studied on four consecutive days during their shift and the following night. Sources of in-vehicle PM2.5 were identified with variance-maximizing rotational principal factor analysis of PM2.5-components and associated pollutants. Two source models were calculated. Sources of in-vehicle PM2.5 identified were 1) crustal material, 2) wear of steel automotive components, 3) gasoline combustion, 4) speed-changing traffic with engine emissions and brake wear. In one model, sources 1 and 2 collapsed to a single source. Source factors scores were compared to cardiac and blood parameters measured ten and fifteen hours, respectively, after each shift. The "speed-change" factor was significantly associated with mean heart cycle length (MCL, +7% per standard deviation increase in the factor score), heart rate variability (+16%), supraventricular ectopic beats (+39%), % neutrophils (+7%), % lymphocytes (-10%), red blood cell volume MCV (+1%), von Willebrand Factor (+9%), blood urea nitrogen (+7%), and protein C (-11%). The "crustal" factor (but not the "collapsed" source) was associated with MCL (+3%) and serum uric acid concentrations (+5%). Controlling for potential confounders had little influence on the effect estimates. Conclusion: PM2.5 originating from speed-changing traffic modulates the autonomic control of the heart rhythm, increases the frequency of premature supraventricular beats and elicits proinflammatory and pro-thrombotic responses in healthy young men. [Authors]

Perforation colique néonatale focale spontanée en dehors de la grande prématurité : rare et potentiellement insidieuse [Focal spontaneous colic perforation in term or near-term neonates: rare and potentially insidious].

Two cases of neonatal focal spontaneous colic perforations are reported. The 1st infant, born at 36 3/7 weeks gestational age, presented on day 3 with crying, abdominal distension, and liquid stools. Clinical examination showed a slightly irritable hypothermic (35.7 °C) infant with a distended abdomen and few bowel sounds. Blood tests were normal apart from an elevated C-reactive protein level (59 mg/l). The abdomen x-ray was erroneously considered normal. The infant's condition remained stable for nearly 3 days. After reviewing the initial x-ray, pneumoperitoneum was suspected and confirmed by a cross-table lateral abdominal x-ray. The infant was started on antibiotics and operated. Macroscopically, the entire gut was normal apart from a focal sigmoid perforation, which was stitched. A transmural colic biopsy revealed focal vascular dilation but was negative for necrotising enterocolitis or Hirschsprung disease. The infant recovered quickly. She is now a healthy, normal 3-year-old. The 2nd infant, born at 38 5/7 weeks gestational age, presented between day 1 and 2 with clinical signs of infection associated with slowly progressive ileus. The chest and abdomen x-ray was mistakenly considered normal. Frank septicemia developed. After reviewing the initial x-ray, pneumoperitoneum was suspected and confirmed by a cross-table lateral abdominal x-ray. The infant was operated. Macroscopically, the small intestine was normal, the ascending and transverse colons were dilated, and the descending and sigmoid colons were narrow. Three cecal perforations were discovered and stitched. An ileostomy and multiple colic biopsies were also performed. The postoperative course was complicated by persistent septic ileus due to descending and sigmoid colon leaks, which led to colic resections with end-to-end anastomosis. Rectal aspiration biopsies were also performed. At 1 month of age, the infant was discharged from the hospital. The ileostomy was closed in two steps at 2 and 5 months of age. A normal sweat test excluded cystic fibrosis. All colic and rectal biopsies revealed nonspecific inflammatory signs and excluded necrotizing enterocolitis and Hirschsprung disease. Nonspecific irregular thinning of muscularis mucosae and muscularis propria were observed in the two resected colic segments. The boy is now a healthy 7-year-old. The incidence of neonatal focal spontaneous colic perforations at term or close to term is unknown but probably very rare. Our department is the neonatal referral center for approximately 14,000 annual births. In the last 10 years (2000-2009), out of 5115 neonatal admissions in our unit, only ten cases have presented a neonatal spontaneous intestinal perforation, seven of ten in very-low-birth-weight infants and three of ten in term or near-term neonates (one with Hirschsprung disease and the two cases reported herein). In the same period, 108 infants suffered from necrotizing enterocolitis, seven of 108 were term infants and 6 out of 7 had a congenital heart disease. The medical literature is poor on the subject of focal spontaneous colic perforations at term; no risk factor is described. The most specific clinical sign seems to be the abdominal distension. The presence of pneumoperitoneum on an abdominal x-ray is the most sensitive paraclinical sign. In case of an intestinal perforation, surgery must be performed quickly. The vital prognosis seems to be good. The objective of this study was to draw pediatricians' attention to focal spontaneous colic perforations in term or close to term newborns. In the cases reported, the diagnostic delays could have been prevented if the entity - with its radiological manifestation - had been well known.

Diagnosis of Birt-Hogg-Dube syndrome in a patient with spontaneous pneumothorax

Birt-Hogg-Dube syndrome refers to a dermatologic syndrome, consisting of small papular skins lesion distributed on the scalp, forehead, face and neck, which is autosomal dominantly inherited. Subsequently patients may develop concomitant renal and thoracic pathology. We report the case of a patient with Birt-Hogg-Dube syndrome diagnosed after spontaneous pneumothorax.

Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

An immune-competent model of spontaneous breast cancer metastasis to the brain

In breast cancer, brain metastases are often seen as late complications of recurrent disease and represent a particularly serious condition, since there are limited therapeutic options and patients have an unfavorable prognosis. The frequency of brain metastases in breast cancer is currently on the rise. This might be due to the fact that adjuvant chemotherapeutic and targeted anticancer drugs, while they effectively control disease progression in the periphery, they only poorly cross the blood-brain barrier and do not reach effectively cancer cells disseminated in the brain. It is therefore of fundamental clinical relevance to investigate mechanisms involved in breast cancer metastasis to the brain. To date experimental models of breast cancer metastasis to the brain described in literature are based on the direct intracarotid or intracardiac injection of breast cancer cells. We recently established a brain metastasis breast cancer model in immunocompetent mice based on the orthotopic injection of 4T1 murine breast carcinoma cells in the mammary gland of syngeneic BALB/c mice. 4T1-derived tumors recapitulate the main steps of human breast cancer progression, including epithelial-to-mesenchymal transition, local invasion and metastatic spreading to lung and lymph nodes. 4T1 cells were engineered to stably express firefly Luciferase allowing noninvasive in vivo and ex vivo monitoring of tumor progression and metastatic spreading to target organs. Bioluminescence imaging revealed the appearance of spontaneous lesions to the lung and lymph nodes and, at a much lower frequency, to the brain. Brain metastases were confirmed by macroscopic and microscopic evaluation of the brains at necropsy. We then isolated brain metastatic cells, re-injected them orthotopically in new mice and isolated again lines from brain metastases. After two rounds of selection we obtained lines metastasizing to the brain with 100% penetrance (named 4T1-BM2 for Brain Metastasis, 2nd generation) compared to lines derived after two rounds of in vivo growth from primary tumors (4T1-T2) or from lung metastases (4T1-LM2). We are currently performing experiments to unravel differences in cell proliferation, adhesion, migration, invasion and survival of the 4T1-BM2 line relative to the 4T1-T2 and 4T1-LM2 lines. Initial results indicate that 4T1-BM2 cells are not more invasive or more proliferative in vitro and do not show a more mesenchymal phenotype. Our syngeneic (BALB/c) model of spontaneous breast carcinoma metastasis to the brain is a unique and clinically relevant model to unravel the mechanisms of metastatic breast cancer colonization of the brain. Genes identified in this model represent potentially clinically relevant therapeutic targets for the prevention and the treatment of brain metastases in breast cancer patients.