Eotaxin is required for the baseline level of tissue eosinophils

Eotaxin is an eosinophil-selective chemokine that is constitutively expressed in a variety of organs such as the intestine. Previous studies have demonstrated that the recruitment of eosinophils during inflammation is partially dependent on eotaxin, but the function of constitutive eotaxin during homeostasis has not been examined. To elucidate the biological role of this molecule, we now examine tissue levels of eosinophils in healthy states in wild-type and eotaxin-deficient mice. The lamina propria of the jejunum of wild-type mice is demonstrated to express eotaxin mRNA, but not mRNA for the related monocyte chemoattractant proteins. Wild-type mice contained readily detectable eosinophils in the lamina propria of the jejunum. In contrast, mice genetically deficient in eotaxin had a large selective reduction in the number of eosinophils residing in the jejunum. The reduction of tissue eosinophils was not limited to the jejunum, because a loss of thymic eosinophils was also observed in eotaxin-deficient mice. These studies demonstrate that eotaxin is a fundamental regulator of the physiological trafficking of eosinophils during healthy states. Because a variety of chemokines are constitutively expressed, their involvement in the baseline trafficking of leukocytes into nonhematopoietic tissue should now be considered.     

Expression and regulation of small proline-rich protein 2 in allergic inflammation

Asthma is a complex inflammatory pulmonary disorder that is on the rise despite intense ongoing research. We aimed to elucidate novel pathways involved in the pathogenesis of asthma. Employing asthma models induced by different allergens (ovalbumin and Aspergillus fumigatus), we uncovered the involvement of two members of the small proline-rich protein (SPRR) family, SPRR2a and SPRR2b, known to be involved in epithelial differentiation but not allergic disease. In situ hybridization revealed induction of SPRR2 signal in a subset of bronchial epithelial cells and mononuclear cells associated with inflammation after allergen challenge. Allergen-induced SPRR2 mRNA accumulation in the lung occurred in a time-dependent manner, with peak expression 10-96 h after a second ovalbumin challenge. Transgenic overexpression of interleukin (IL)-13 in the lungs resulted in a marked increase of SPRR2 expression, and allergen-induced SPRR2 expression was significantly decreased in IL-13-deficient mice. Studies in gene-targeted mice revealed that allergen-induced SPRR2 was dependent upon STAT6. Finally, we aimed to determine if the induction of SPRR2 by allergen was tissue specific. Notably, SPRR2 was markedly increased in the small intestine after induction of allergic gastrointestinal inflammation. Thus, SPRR2 is an allergen- and IL-13-induced gene in experimental allergic responses that may be involved in disease pathophysiology.     

Variability in apoptotic response to poliovirus infection

In several cell types, poliovirus activates the apoptotic program, implementation of which is suppressed by viral antiapoptotic functions. In such cells, productive infection leads to a necrotic cytopathic effect (CPE), while abortive reproduction, associated with inadequate viral antiapoptotic functions, results in apoptosis. Here, we describe two other types of cell response to poliovirus infection. Murine L20B cells expressing human poliovirus receptor responded to the infection by both CPE and apoptosis concurrently. Interruption of productive infection decreased rather than increased the proportion of apoptotic cells. Productive infection was accompanied by the early efflux of cytochrome c from the mitochondria in a proportion of cells and by activation of DEVD-specific caspases. Inactivation of caspase-9 resulted in a marked, but incomplete, prevention of the apoptotic response of these cells to viral infection. Thus, the poliovirus-triggered apoptotic program in L20B cells was not completely suppressed by the viral antiapoptotic functions. In contrast, human rhabdomyosarcoma RD cells did not develop appreciable apoptosis during productive or abortive infection, exhibiting inefficient efflux of cytochrome c from mitochondria and no marked activation of DEVD-specific caspases. The cells were also refractory to several nonviral apoptosis inducers. Nevertheless, typical caspase-dependent signs of apoptosis in a proportion of RD cells were observed after cessation of viral reproduction. Such "late" apoptosis was also observed in productively infected HeLa cells. In addition, a tiny proportion of all studied cells were TUNEL positive even in the presence of a caspase inhibitor. Degradation of DNA in such cells appeared to be a postmortem phenomenon. Biological relevance of variable host responses to viral infection is discussed.     

CD10 protein expression in tumor and stromal cells of malignant melanoma is associated with tumor progression.

CD10 antigen is a 100-kDa-cell surface zinc metalloendopeptidase expressed in a variety of normal and neoplastic lymphoid and nonlymphoid tissues including melanomas. It was recently shown that metastatic melanomas express more CD10 than primary tumors. We evaluated CD10 expression in tumor and stromal cells in 70 biopsies with primary and 28 with metastatic malignant melanomas. Ki-67, Bcl-2, and Bax were also examined to investigate whether CD10 expression is associated with tumor proliferation index or factors of apoptosis. Formalin-fixed/paraffin-embedded tissues were studied by immunohistochemistry. More advanced primary tumors had higher CD10 expression in the tumor cells (r = 0.27, P = 0.03 for Clark levels and r = 0.29, P = 0.02 for Breslow) and higher Ki-67 proliferation fraction (r = 0.32, P = 0.007 for Clark levels and r = 0.32, P = 0.001 for Breslow). Similarly, CD10 expression in the intratumoral stromal cells was also higher in primary tumors with higher Clark level (P = 0.04, linear-by-linear association) and tumor thickness according to Breslow (r = 0.33, P = 0.01). The presence of CD10+ peritumoral stromal cell cuffs was also positively associated with tumor thickness according to Breslow (r = 0.27, P = 0.05). Also, expression of CD10 and Ki-67 were significantly higher in metastatic than in primary tumors (P = 0.01 and 0.02 respectively), but Bcl-2 expression was higher in primary melanomas (P = 0.02). We conclude that CD10 expression in malignant melanoma is associated with tumor progression.     

Frequency of loss of heterozygosity of the NF2 gene in schwannomas from Croatian patients

Aim. To identify gross deletions in the NF2 gene in a panel of schwannomas from Croatian patients in order to establish their frequencies in Croatian population. Methods. Changes of the NF2 gene were tested by polymerase chain reaction/loss of heterozygosity (LOH) using two microsatellite markers, D22S444 and D22S929. Results. The analysis with both markers demonstrated that 43.75% of schwannomas exhibited LOH of the NF2 gene. The D22S444 region exhibited 45.5% of LOHs and the D22S929 region exhibited 14.3% of LOHs. Four LOHs were found in Antoni B, 2 in Antoni A, and 1 in Antoni A and B type tumors. Conclusion. The frequency of changes observed in Croatian patients is broadly similar to that reported in other populations and thus confirms the existing hypothesis regarding the tumorigenesis of schwannomas and contributes to schwannoma genetic profile helping us to better understand its etiology and treatment.     

Simpson-Golabi-Behmel syndrome type 1 in a 27-week macrosomic preterm newborn: The diagnostic value of rib malformations and index nail and finger hypoplasia

The Simpson-Golabi-Behmel syndrome type 1 (SGBS1, OMIM #312870) is an X-linked overgrowth condition comprising abnormal facial appearance, supernumerary nipples, congenital heart defects, polydactyly, fingernail hypoplasia, increased risk of neonatal death and of neoplasia. It is caused by mutation/deletion of the GPC3 gene. We describe a macrosomic 27-week preterm newborn with SGBS1 who presents a novel GPC3 mutation and emphasize the phenotypic aspects which allow a correct diagnosis neonatally in particular the rib malformations, hypoplasia of index finger and of the same fingernail, and 2nd-3rd finger syndactyly. ? 2012 Wiley Periodicals, Inc.     

Osteopontin expression correlates with nuclear factor-κB activation and apoptosis downregulation in clear cell renal cell carcinoma

Osteopontin (OPN) is a phosphoglycoprotein implicated in tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the mechanisms that contribute to development and progression of cancer, and might be initiated by OPN interaction with tumor cells. The aim of this study was to analyze the relation between OPN and nuclear factor-kappa B (NF-κB) expression in clear cell renal cell carcinoma (CCRCC), as well as their relation to apoptotic activity of tumor cells.Expression of OPN protein and p65 NF-κB subunit was analyzed immunohistochemically in 87 CCRCC samples, and compared mutually and with apoptotic index. Expression of OPN mRNA was analyzed using quantitative real-time PCR and compared with OPN and NF-κB protein expression in 22 CCRCC samples.Statistical analysis showed an association of p65 NF-κB with OPN mRNA (p = 0.015) and protein (p < 0.001). Also, we found an inverse relationship of OPN with NF-κB protein expression and apoptotic activity of tumor cells (p = 0.006 and p = 0.022, respectively). Our results indicate that p65 NF-κB signaling pathway may be involved in OPN-mediated CCRCC progression, partly by protecting tumor cells from apoptosis. Therefore, both molecules can constitute potential targets for therapeutic intervention in CCRCC.     

Identification of the B-cell epitopes of the early endosome antigen 1 (EEA1)

Early endosome antigen 1 (EEA1) is a target autoantigen in patients diagnosed with neurological and other autoimmune conditions. Eighteen of 65 sera (28%) that displayed a vesicular cytoplasmic staining pattern also immunoprecipitated the recombinant EEA1. These 18 sera were selected for further clinical, serological and epitope mapping studies. Thirty-six percent of the 18 patients had neurological diseases. Seventeen sera (94%) reacted with the partial length EEA1 constructs that included the C-terminal zinc finger (+FYVE) and the methyl accepting domain (LeuMA: amino acids 82-1411) in an addressable laser bead assay suggesting that the assay may be used for rapid laboratory detection of anti-EEA1 antibodies. Three of seven sera selected for epitope mapping studies bound to EEA1 peptides represented by amino acids 1096-1125, and two reacted with peptides represented by amino acids 1296-1320. One serum reacted only with the C-terminal peptide 1096-1125. The remaining serum reacted with other EEA1 epitopes. This data was supported by the observations that all the sera immunoprecipitated the C-terminal +FYVE (EEA1 1064-1411) construct, a peptide that also contained the linear epitopes 1096-1140. The limited epitope mapping studies suggest that the sera from patients with non-neurological diseases recognized epitopes in the central and C-terminal EEA1 domains, whereas the patients with neurological disease recognized a more restricted set of epitopes in the C-terminal.     

Fas/CD95-mediated apoptosis in human glioblastoma cells: a target for sensitisation to topoisomerase I inhibitors

The expression of the death receptor Fas/CD95 is cell type-specific and can be modulated by different cytotoxic treatments. In spite of a frequent expression of Fas/CD95 in high-grade gliomas, these tumours are typically refractory to conventional therapy. Using a human glioblastoma cell line (GBM), we explored the possibility of modulating susceptibility to Fas/CD95-mediated apoptosis following cytotoxic treatment. GBM cells were sensitive to the antiproliferative effects of topoisomerase I inhibitors (topotecan and a novel lipophilic analog CPT83) and taxol, less sensitive to cisplatin and, in any case, rather resistant to drug-induced apoptosis. This pattern of cellular response was consistent with p53 mutation. GBM cells expressed low levels of Fas/CD95, which was associated with low susceptibility to antibody-stimulated Fas/CD95-mediated apoptosis. A significant up-regulation of Fas/CD95 expression was detected after exposure to topotecan and CPT83, whereas cisplatin induced a low increase and taxol did not modify Fas/CD95 expression. In addition, after treatment with topoisomerase I inhibitors, the up-regulation of Fas/CD95 resulted in an increased susceptibility of GBM cells to antibody-stimulated Fas/CD95-mediated apoptosis, while no synergistic effects were detected after treatment with cisplatin or taxol. Our data suggest that Fas/CD95 up-regulation can be a common response to DNA damage, whereas sensitisation to Fas/CD95-mediated apoptosis appears to be dependent on the type of DNA damage and on the pathway of cellular response. The observed effects might have important therapeutic implications for the design of novel therapeutic strategies in the treatment of malignant gliomas.