Summary
Toxoplasma gondii is a widespread intracellular parasite, which naturally enters the organism via the oral route and crosses the intestinal barrier to disseminate. In addition to neuronal and ...ocular pathologies, this pathogen also causes gut inflammation in a number of animals. This infection‐triggered inflammation has been extensively studied in the C57BL/6 mice, highlighting the importance of the immune cells and their mediators in the development of gut pathology. However, despite their importance in inflammation, the role of protease‐activated receptors (PAR) was never reported in the context of T.gondii‐mediated small intestine inflammation. Using genetically modified mice, we show that PAR2 plays a pathogenic role in the development of gut inflammatory lesions. We find that PAR2 controls the innate inflammatory mediators IL‐6, KC/CXCL1, PGE2 as well as neutrophil infiltration in T. gondii‐triggered gut damage. These results bring new knowledge on the mechanisms operating in the gut in response to T. gondii infection.
To explore the functional implications of a homozygous CATSPER 2 (cation channel for sperm) deletion within the acrosome reaction pathway during fertilization in 2 brothers, who have unexplained ...infertility and hearing loss.
Case report.
Two twin brothers aged 30 years with hearing loss and unexplained infertility.
Molecular genetic diagnosis of deafness. Evaluation of the acrosome reaction and calcium mobilization assays after induction by progesterone and ionomycin on spermatozoa of the CATSPER 2-mutated patient and on fertile controls.
Fertilization rate during conventional in vitro fertilization. Molecular genetic test. Percentage of acrosome-reacted spermatozoa with peanut agglutinin lectin staining. Recording of progesterone and ionomycin-induced intracellular calcium signals with a fluorescent probe.
Mr. S and his brother have normal, conventional sperm parameters. Both brothers have had repeated intrauterine insemination failures and one fertilization failure after conventional in vitro fertilization. Mr. S obtained 2 healthy babies after intracytoplasmic sperm injection. Genetic analysis found a homozygote deletion of the STRC (stereocilin) gene (NM 153700: c.1-? 5328+?del) that removes the CATSPER 2 gene. Mutation of the STRC gene is known to be associated with hearing loss. Sperm functional tests revealed an inability of progesterone to activate intracellular calcium signaling and to induce acrosome reaction.
We demonstrate the absence of a calcium signal and acrosome reaction after progesterone in our patient with a CATSPER 2 mutation. We emphasize the importance of the male medical interview and of the genetic investigation of hearing loss. We show that in vitro fertilization–intracytoplasmic sperm injection is necessary, even where normal sperm parameters are present.
Abstract
Background and Aims
Intestinal epithelial cells IECs from inflammatory bowel disease IBD patients exhibit an excessive induction of endoplasmic reticulum stress ER stress linked to altered ...intestinal barrier function and inflammation. Colonic tissues and the luminal content of IBD patients are also characterized by increased serine protease activity. The possible link between ER stress and serine protease activity in colitis-associated epithelial dysfunctions is unknown. We aimed to study the association between ER stress and serine protease activity in enterocytes and its impact on intestinal functions
Methods
The impact of ER stress induced by Thapsigargin on serine protease secretion was studied using either human intestinal cell lines or organoids. Moreover, treating human intestinal cells with protease-activated receptor antagonists allowed us to investigate ER stress-resulting molecular mechanisms that induce proteolytic activity and alter intestinal epithelial cell biology.
Results
Colonic biopsies from IBD patients exhibited increased epithelial trypsin-like activity associated with elevated ER stress. Induction of ER stress in human intestinal epithelial cells displayed enhanced apical trypsin-like activity. ER stress-induced increased trypsin activity destabilized intestinal barrier function by increasing permeability and by controlling inflammatory mediators such as C-X-C chemokine ligand 8 CXCL8. The deleterious impact of ER stress-associated trypsin activity was specifically dependent on the activation of protease-activated receptors 2 and 4.
Conclusions
Excessive ER stress in IECs caused an increased release of trypsin activity that, in turn, altered intestinal barrier function, promoting the development of inflammatory process.
SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton ...syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was observed in samples from other keratinizing disorders, demonstrating that loss of LEKTI expression in the epidermis is a diagnostic feature of NS. The identification of novel processed forms of LEKTI provides the basis for future functional and structural studies of fragments with physiological relevance.
Ribosome production, one of the most energy-consuming biosynthetic activities in living cells, is adjusted to growth conditions and coordinated with the cell cycle. Connections between ribosome ...synthesis and cell cycle progression have been described, but the underlying mechanisms remain only partially understood. The human HCA66 protein was recently characterized as a component of the centrosome, the major microtubule-organizing center (MTOC) in mammalian cells, and was shown to be required for centriole duplication and assembly of the mitotic spindle. We show here that HCA66 is also required for nucleolar steps of the maturation of the 40S ribosomal subunit and therefore displays a dual function. Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process. In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis. Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.
Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated ...keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS.
Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the ...important role of the protease-activated receptor PAR
in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR
activation or knockout on cell proliferation and survival functions.
Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR
activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR
-deficient mice were used to study the impact of PAR
expression on colon epithelial cell culture and gene expression.
Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR
activation by specific PAR
agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR
knockout male cells. PAR
expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR
-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance.
Altogether, these data demonstrate that there is a sexual dimorphism in the PAR
-dependent regulation of primitive cells of the colon crypt.
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