A crucial role for TNF-alpha in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5

Background and purpose: Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP-2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice. Experimental approach: Neutrophil recruitment was assessed in immunized mice challenged with methylated bovine serum albumin, KC/CXCL1, LIX/CXCL5 or tumour necrosis factor (TNF)-alpha. Cytokine and chemokine levels were determined in peritoneal exudates and in supernatants of macrophages and mast cells by elisa. CXCR2 and intercellular adhesion molecule 1 (ICAM-1) expression was determined using immunohistochemistry and confocal microscopy. Key results: Antigen challenge induced dose- and time-dependent neutrophil recruitment and production of KC/CXCL1, LIX/CXCL5 and TNF-alpha, but not MIP-2/CXCL2, in peritoneal exudates. Neutrophil recruitment was inhibited by treatment with reparixin (CXCR1/2 antagonist), anti-KC/CXCL1, anti-LIX/CXCL5 or anti-TNF-alpha antibodies and in tumour necrosis factor receptor 1-deficient mice. Intraperitoneal injection of KC/CXCL1 and LIX/CXCL5 induced dose- and time-dependent neutrophil recruitment and TNF-alpha production, which were inhibited by reparixin or anti-TNF-alpha treatment. Macrophages and mast cells expressed CXCR2 receptors. Increased macrophage numbers enhanced, while cromolyn sodium (mast cell stabilizer) diminished, LIX/CXCL5-induced neutrophil recruitment. Macrophages and mast cells from immunized mice produced TNF-alpha upon LIX/CXCL5 stimulation. Methylated bovine serum albumin induced expression of ICAM-1 on mesenteric vascular endothelium, which was inhibited by anti-TNF-alpha or anti-LIX/CXCL5. Conclusion and implications: Following antigen challenge, CXCR2 ligands are produced and act on macrophages and mast cells triggering the production of TNF-alpha, which synergistically contribute to neutrophil recruitment through induction of the expression of ICAM-1.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.

HIGH EXPRESSION OF CANCER TESTIS ANTIGENS MAGE-A, MAGE-C1/CT7, MAGE-C2/CT10, NY-ESO-1, AND GAGE IN ADVANCED SQUAMOUS CELL CARCINOMA OF THE LARYNX

Background. Despite diagnostic and therapeutic advances in head and neck cancer, the 5-year survival of patients with laryngeal cancer has not improved in the last 30 years. Several recent studies indicate that specific targets for immunotherapeutic approaches can be useful in the control of cancer. There is considerable interest in the expression of cancer testis antigens in human cancers since they may serve as the basis for an immunologic approach to therapy. Methods. We evaluated by immunohistochemical analysis the expression of cancer testis antigens MAGE-A4 (57B), MAGE-C1 (CT7-33), MAGE-A1 (MA454), MAGE-A3 (M3H67), MAGE-C2 (CT10.5), NY-ESO-1 (E978), and GAGE (GAGE) in squamous cell carcinoma (SCC) of the larynx. Results. A total of 63 cases (57 men and 6 women) of laryngeal SCC were available for this study. The findings were correlated with the clinical course and laboratory data. Expression of at least 1 cancer testis antigen was detected in 42 of 63 of the laryngeal SCCs (67%). In 34 of 42 of the positive cases (81%) there was simultaneous expression of >= 2 cancer testis antigens. There was significant correlation between antigen expression and advanced tumor stage (stage III/IV) in cases with reactivity to only 1 antibody (p = .01) as well as in the cases with reactivity to >= 2 primary antibodies (>= 2 mAbs, p = .04). There was no association between survival and expression of any of the analyzed antigens. Conclusions. We find a high incidence of cancer testis antigen expression in SCCs of the larynx, which was correlated with advanced clinical stage. Our data indicate that cancer testis antigens could be valuable vaccine targets in laryngeal tumors, especially in those with a worse prognosis. (C) 2010 Wiley Periodicals, Inc. Head Neck 33: 702-707, 2011

The role of tumor hypoxia in MUC1-positive breast carcinomas

Mucin 1 (MUC1) is a glycoprotein that is expressed on apical cell membranes in a variety of normal tissues. MUC1 is involved in cell signaling, inhibition of cell-cell and cell matrix adhesion, apoptosis, proliferation, and transcription. Hypoxia is an important factor that promotes cancer metastasis and stimulates angiogenesis and tumor progression. Hypoxia inducible factor 1 (HIF-1 alpha) and carbonic anhydrase IX (CAIX) are two molecules that are involved in this process. The role of hypoxia in MUC1+ invasive ductal breast carcinomas is not well established. In this study, the expression of MUC1 was correlated with the hypoxia-associated markers HIF-1 alpha and CAIX, as well as several immunohistochemical markers and clinicopathologic features of prognostic significance in 243 invasive ductal carcinomas. MUC1 was overexpressed in 37.0% of patients and correlated with the expression of estrogen receptor (p = 0.0001), progesterone receptor (p = 0.0001), HIF-1 alpha (p = 0.006), VEGF (p = 0.024), and p53 (p = 0.025). In breast cancer, MUC1 expression has been associated with increased degradation of inhibitor of NF-kappa B (I kappa B alpha), driving NF-kappa B to the nucleus and blocking apoptosis and promoting cell survival. We analyzed NF-kappa B expression in MUC1+ breast carcinoma and found a very significant relationship between these proteins (p = 0.0001). Our findings indicate that MUC1 may play a role in the regulation of hormone receptors by increasing the inactivation of p53 and targeting NF-kappa B to the nucleus. Our data also support the notion that activation of HIF-1 alpha in MUC1+ breast carcinomas may modulate VEGF expression, allowing a metabolic adaptation to hypoxia.

Ossifying fibroma: A rare cause of orbital inflammation

Purpose: To describe the clinical and radiologic features of 4 cases of ossifying fibroma affecting the orbit and to review the literature on orbital involvement by the tumor. Methods: Small case series. Results: Four patients (3 children and 1 adult) with ossifying fibromas invading the orbit were examined. Two of the 3 children were examined for ossifying fibromas on the orbital roof. One had the psammomatoid form of the disease and the other the trabecular variant. Despite striking differences in the histologic pattern and in the radiologic appearance of the lesions, both children displayed a significant degree of orbital inflammation mimicking orbital cellulitis. The third child and the adult patient had the orbit involved by trabecular ossifying fibromas invading the orbital floor. The tumor of the adult clearly originated in the maxilla, filled the maxillary sinus, and eroded the orbital floor. The tumor of the third child occupied the maxillary, ethmoid, and sphenoid sinuses. In both cases, the clinical presentation was painless eye dystopia and proptosis. Conclusions: Regardless of the histologic pattern (trabecular or psammomatoid), ossifying fibromas can induce a substantial degree of orbital inflammation in children and must be included in the differential diagnosis of acute orbital inflammation during childhood.

Pitfalls in the Differential Diagnosis of Renal Tumor in an Adolescent

The differential diagnosis of renal tumors, particularly in adolescents, may be challenging. We describe an 11-year-old female with a primary intra-renal mass. Initial differential diagnoses included primitive neuroectodermal tumor (PNET), desmoplastic small round cell tumor (DSRCT), and Wilms Turner (WT). Extensive pathologic and molecular analysis on initial and relapsed tumor samples confirmed WT. The EWS-WTI and EWS-FL11 rearrange-merits, distinctive of DSRCT and PNET were negative. The differential diagnosis on monophasic blastemal WT may be complex. Primary renal DSRCT and MET have been rarely described. Nevertheless, molecular confirmation for these rare conditions may be necessary in selected cases. Pediatr Blood Cancer 2010;54:3 19-321. (C) 2009 Wiley-Liss, Inc.

Tumor necrosis factor region polymorphisms are associated with AIDS and with cytomegalovirus retinitis

Background: The tumor necrosis factor (TNF) gene is located within the highly polymorphic major histocompatibility complex region, exhibiting the -308 CA promoter region polymorphism and six microsatellites (TNFa-f) spanning the region nearby the TNF locus. Objective: In the present Study, we evaluated the frequency of -308 CA and TNFa-e polymorphisms and respective haplotypes (in chromosomal sequence: TNFd-TNFe-308GA-TNFc-TNFa-TNFb), in 222 patients with AIDS, 52 of whom exhibited cytomegalovirus retinitis, and in 202 healthy HIV-negative individuals. Method: TNF microsatellite and single nucleotide polymorphism typings were performed by PCR followed by polyacrylamide gel electrophoresis. Results: The TNF-308A allele and the 4-3-C-2-7-1 haplotype were associated with susceptibility to AIDS, whereas the TNFb4 allele and the 3-3-C-1-11-4 haplotype were associated with protection against AIDS development. The TNFc2 allele and the 4-1-G-2-2-1 haplotype, which contains the TNFc2 allele, were associated with cytomegalovirus retinitis. Conclusion: This study highlights that polymorphic sites spanning the region nearby the TNF locus are associated with AIDS per se and with cytomegalovirus retinitis in AIDS patients. (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins

NO EVIDENCE OF PATHOLOGICAL AUTOIMMUNITY FOLLOWING MYCOBACTERIUM LEPRAE HEAT-SHOCK PROTEIN 65-DNA VACCINATION IN MICE

Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules, These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents.

Long-Term Ethanol Consumption Promotes Hepatic Tumorigenesis but Impairs Normal Hepatocyte Proliferation in Rats

Chronic and excessive alcohol consumption has been related to an increased risk of several cancers, including that of the liver; however, studies in animal models have yet to conclusively determine whether ethanol acts as a tumor promoter in hepatic tumorigenesis. We examined whether prolonged alcohol consumption could act as a hepatic tumor promoter after initiation by diethylnitrosamine (DEN) in a rat model. Male Sprague-Dawley rats were injected with 20 mg DEN/kg body weight 1 wk before introduction of either an ethanol liquid diet or an isoenergic control liquid diet. Hepatic pathological lesions, hepatocyte proliferation, apoptosis, PPAR alpha and PPAR gamma, and plasma insulin-like growth factor 1 IGF-1) levels were assessed after 6 and 10 mo. Mean body and liver weights, plasma IGF-1 concentration, hepatic expressions of proliferating cellular nuclear antigen and Ki-67, and cyclin D1 in ethanol-fed rats were all significantly lower after 10 mo of treatment compared with control rats. In addition, levels of hepatic PPAR gamma protein, not PPAR alpha, were significantly higher in the ethanol-fed rats after prolonged treatment. Although ethanol feeding also resulted in significantly fewer altered hepatic foci, hepatocellular adenoma was detected in ethanol-fed rats at 10 mo, but not in control rats given the same dose of DEN. Together, these results indicate that chronic, excessive ethanol consumption impairs normal hepatocyte proliferation, which is associated with reduced IGF-1 levels, but promotes hepatic carcinogenesis. J. Nutr. 141: 1049-1055, 2011.

Recognition by toll-like receptor 2 induces antigen-presenting cell activation and Th1 programming during infection by Neospora caninum

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Toll-like receptors (TLRs) sense specific microbial products and direct downstream signaling pathways in immune cells, linking innate, and adaptive immunity. Here, we analyze the role of TLR2 on innate and adaptive immune responses during N. caninum infection. Inflammatory peritoneal macrophages and bone marrow-derived dendritic cells exposed to N. caninum-soluble antigens presented an upregulated expression of TLR2. Increased receptor expression was correlated to TLR2/MyD88-dependent antigen-presenting cell maturation and pro-inflammatory cytokine production after stimulation by antigens. Impaired innate responses observed after infection of mice genetically deficient for TLR2((-/-)) was followed by downregulation of adaptive T helper 1 (Th1) immunity, represented by diminished parasite-specific CD4(+) and CD8(+) T-cell proliferation, IFN-gamma:interleukin (IL)-10 ratio, and IgG subclass synthesis. In parallel, TLR2(-/-) mice presented higher parasite burden than wild-type (WT) mice at acute and chronic stages of infection. These results show that initial recognition of N. caninum by TLR2 participates in the generation of effector immune responses against N. caninum and imply that the receptor may be a target for future prophylactic strategies against neosporosis. Immunology and Cell Biology (2010) 88, 825-833; doi:10.1038/icb.2010.52; published online 20 April 2010

Bone Marrow Involvement in a Patient with Paracoccidioidomycosis: A Rare Presentation of Juvenile Form

Paracoccidioides brasiliensis rarely shows bone marrow involvement and its response to treatment with itraconazole in children needs further assessment. We describe here a child with a juvenile disseminated form of paracoccidioidomycosis, which showed reticuloendothelial system involvement and the presence of Paracoccidioides brasiliensis in the bone marrow. The patient showed an effective and rapid response to itraconazole therapy.

Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1)

Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed ""promiscuous gene expression"" (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80(+) mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.

Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan

Large, long-lived species experience more lifetime cell divisions and hence a greater risk of spontaneous tumor formation than smaller, short-lived species. Large, long-lived species are thus expected to evolve more elaborate tumor suppressor systems. In previous work, we showed that telomerase activity coevolves with body mass, but not lifespan, in rodents: telomerase activity is repressed in the somatic tissues of large rodent species but remains active in small ones. Without telomerase activity, the telomeres of replicating cells become progressively shorter until, at some critical length, cells stop dividing. Our findings therefore suggested that repression of telomerase activity mitigates the increased risk of cancer in larger-bodied species but not necessarily longer-lived ones. These findings imply that other tumor suppressor mechanisms must mitigate increased cancer risk in long-lived species. Here, we examined the proliferation of fibroblasts from 15 rodent species with diverse body sizes and lifespans. We show that, consistent with repressed telomerase activity, fibroblasts from large rodents undergo replicative senescence accompanied by telomere shortening and overexpression of p16(Ink4a) and p21(Cip1/Waf1) cycline-dependent kinase inhibitors. Interestingly, small rodents with different lifespans show a striking difference: cells from small shorter-lived species display continuous rapid proliferation, whereas cells from small long-lived species display continuous slow proliferation. We hypothesize that cells of small long-lived rodents, lacking replicative senescence, have evolved alternative tumor-suppressor mechanisms that prevent inappropriate cell division in vivo and slow cell growth in vitro. Thus, large-bodied species and small but long-lived species have evolved distinct tumor suppressor mechanisms.

Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations

The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations. (C) 2009 Elsevier Ltd. All rights reserved.