Faculty Bibliography

Chemokines, chemotactic cytokines, may promote hepatic inflammation in chronic hepatitis C virus (HCV) infection through the recruitment of lymphocytes to the liver parenchyma. We evaluated the association between inflammation and fibrosis and CXCR3-associated chemokines, interferon-gamma (IFN-gamma)-inducible protein 10 (IP-10/CXCL10), monokine induced by IFN-gamma (Mig/CXCL9), and interferon-inducible T cell alpha chemoattractant (I-TAC/CXCL11), in HCV infection. Intrahepatic mRNA expression of these chemokines was analyzed in 106 chronic HCV-infected patients by real-time PCR. The intrahepatic localization of chemokine producer cells and CXCR3(+) lymphocytes was determined in selected patients by immunohistochemistry. We found elevated intrahepatic mRNA expression of all three chemokines, most markedly CXCL10, in chronic HCV-infected patients with higher necroinflammation and fibrosis. By multivariable multivariate analysis, intrahepatic CXCL10 mRNA expression levels were significantly associated with lobular necroinflammatory grade and HCV genotype 1. In the lobular region, CXCL10-expressing and CXCL9-expressing hepatocytes predominated in areas with necroinflammation. Strong CXCL11 expression was observed in almost all portal tracts, whereas CXCL9 expression varied considerably among portal tracts in the same individual. Most intrahepatic lymphocytes express the CXCR3 receptor, and the number of CXCR3(+) lymphocytes was increased in patients with advanced necroinflammation. CONCLUSION: These findings suggest that the CXCR3-associated chemokines, particularly CXCL10, may play an important role in the development of necroinflammation and fibrosis in the liver parenchyma in chronic HCV infection

The ovary is a common site of involvement for metastasis and the breast is one of the most common sources. Metastatic breast carcinoma can mimic a primary ovarian carcinoma. Pax8 is a crucial transcription factor for organogenesis of the thyroid gland, kidney, and Mullerian system, and it also regulates Wilms tumor suppressor gene (WT1) expression. A total of 124 cases of ovarian carcinomas (84 serous papillary, 18 endometrioid, 12 mucinous, 10 clear cell) and 243 cases of invasive breast carcinomas (178 ductal, 65 lobular) were immunostained with Pax8 and WT1 by tissue microarrays to see the differential expression. Pax8 reaction was found in 108 of 124 ovarian carcinomas (87.1%) generally in diffuse staining, including 81 of 84 serous papillary carcinomas (96.4%), 16 of 18 endometrioid carcinomas (88.9%), 10 of 10 clear cell carcinomas (100%), and 1 of 12 mucinous carcinomas (8.3%), whereas WT1 expression was seen in 78 of 124 ovarian carcinomas (62.9%), including 73 of 84 serous papillary carcinomas (86.9%), and 5 of 18 endometrioid carcinomas (27.8%), with no expression in all 10 clear cell carcinomas and 12 mucinous carcinomas. All the mammary carcinomas were completely negative for Pax8, but WT1 expression was seen in 5 of 243 cases (2.1%). Pax8 is a useful marker in the differential diagnosis of ovarian and breast carcinomas, and it seems to be superior to WT1 for the diagnosis of all types of nonmucinous ovarian carcinomas, notably clear cell and endometrioid types where WT1 expression is generally negative or only focal

We evaluate the effects of adjuvant treatment with the angiogenesis inhibitor Avastin (bevacizumab) on pathological tissue specimens of high-grade glioma. Tissue from five patients before and after treatment with Avastin was subjected to histological evaluation and compared to four control cases of glioma before and after similar treatment protocols not including bevacizumab. Clinical and radiographic data were reviewed. Histological analysis focused on microvessel density and vascular morphology, and expression patterns of vascular endothelial growth factor-A (VEGF-A) and the hematopoietic stem cell, mesenchymal, and cell motility markers CD34, smooth muscle actin, D2-40, and fascin. All patients with a decrease in microvessel density had a radiographic response, whereas no response was seen in the patients with increased microvessel density. Vascular morphology showed apparent 'normalization' after Avastin treatment in two cases, with thin-walled and evenly distributed vessels. VEGF-A expression in tumor cells was increased in two cases and decreased in three and did not correlate with treatment response. There was a trend toward a relative increase of CD34, smooth muscle actin, D2-40, and fascin immunostaining following treatment with Avastin. Specimens from four patients with recurrent malignant gliomas before and after adjuvant treatment (not including bevacizumab) had features dissimilar from our study cases. We conclude that a change in vascular morphology can be observed following antiangiogenic treatment. There seems to be no correlation between VEGF-A expression and clinical parameters. While the phenomena we describe may not be specific to Avastin, they demonstrate the potential of tissue-based analysis for the discovery of clinically relevant treatment response biomarkers

Contact sensitizers are a major cause of inflammatory skin disease and as topical immunomodulators also have the potential for treating cancer, viral diseases and certain autoimmune disorders. In the present study, the authors identify the upregulation of the TH17 lymphocyte subset transcription factor retinoid orphan receptor gamma T (RORgammaT) and the CD70 costimulatory pathway in human contact sensitivity (CS) using molecular techniques. Identification of this important new subset of T lymphocytes and a recognized costimulatory pathway offers potential for ameliorating CS and insight into antitumor and antiviral mechanism of haptens as topical immunomodulators

S100 protein is a sensitive marker for melanomas and peripheral nerve sheath tumors. It is, however, expressed by other mesenchymal and epithelial tumors. Despite its low specificity, S100 protein is valuable for the diagnosis of desmoplastic melanomas and peripheral nerve sheath tumors, for which no specific marker is available. Sox10 is a neural crest transcription factor crucial for specification, maturation, and maintenance of Schwann cells and melanocytes. Anti-Sox10 antibody was applied to a variety of neural crest-derived tumors, mesenchymal and epithelial neoplasms, and normal tissues. Sox10 nuclear expression was found in 76 of 78 melanomas (97%) and 38 of 77 malignant peripheral nerve sheath tumors (49%) whereas S100 protein was expressed in 71 melanomas (91%) and 23 malignant peripheral nerve sheath tumors (30%). Sox10 was diffusely expressed in schwannomas and neurofibromas. Sox10 reaction was seen only in sustentacular cells of pheochromocytomas/paragangliomas, and occasionally carcinoid tumors from various organs, but it was not seen in the tumor cells. In normal tissues, Sox10 was expressed in Schwann cells, melanocytes, and myoepithelial cells of salivary, bronchial, and mammary glands. Sox10 reaction was not identified in any other mesenchymal and epithelial tumors except for myoepitheliomas and diffuse astrocytomas. Sox10 was expressed by metastatic melanomas and nodal capsular nevus in sentinel lymph nodes, but not by other lymph node components such as dendritic cells. Our results indicate that Sox10 will serve as a more sensitive and specific marker for the diagnosis of melanocytic and schwannian tumors than S100 protein

The morphological patterns of glioma cell invasion are known as the secondary structures of Scherer. In this report, we propose a biologically based mechanism for the nonrandom formation of Scherer's secondary structures based on the differential expression of stromal cell-derived factor (SDF)-1alpha and CXCR4 at the invading edge of glioblastomas. The chemokine SDF-1alpha was highly expressed in neurons, blood vessels, subpial regions, and white matter tracts that form the basis of Scherer's secondary structures. In contrast, the SDF-1alpha receptor, CXCR4, was highly expressed in invading glioma cells organized around neurons and blood vessels, in subpial regions, and along white matter tracts. Neuronal and endothelial cells exposed to vascular endothelial growth factor up-regulated the expression of SDF-1alpha. CXCR4-positive tumor cells migrated toward a SDF-1alpha gradient in vitro, whereas inhibition of CXCR4 expression decreased their migration. Similarly, inhibition of CXCR4 decreased levels of SDF-1alpha-induced phosphorylation of FAK, AKT, and ERK1/2, suggesting CXCR4 involvement in glioma invasion signaling. These studies offer one plausible molecular basis and explanation of the formation of Scherer's structures in glioma patients

T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod's adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity

Adenosine is a potent endogenous regulator of tissue repair that is released from injured cells and tissues. Hepatic fibrosis results from chronic hepatic injury, and we have previously reported that endogenously generated adenosine, acting at A(2A) receptors, plays a role in toxin-induced hepatic fibrosis. Adenosine may form intracellularly and then be transported to the extracellular space or it may form extracellularly from adenine nucleotides released from injured cells. Because ecto-5'-nucleotidase (CD73) catalyzes the terminal step in extracellular adenosine formation from AMP, we determined whether CD73 plays a role in the development of hepatic fibrosis. Mice were treated overnight with PBS, CCl(4), ethanol, or thioacetamide (TAA); their livers were harvested, and slices were incubated in medium for 20 h before adenosine concentration in the supernatant was measured by HPLC. Hepatic fibrosis was induced by CCl(4) or TAA treatment in CD73 knockout (CD73KO and C57BL/6 background) and C57BL/6 control mice [wild-type (WT)] mice and quantified by digital analysis of picrosirius red stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or enzyme-linked immunosorbent assay. Livers from WT mice treated with CCl(4), ethanol, and TAA released 2- to 3-fold higher levels of adenosine than livers from comparably treated CD73KO mice. CD73KO mice were protected from fibrosis with significantly less collagen content in the livers of CD73KO than WT mice after treatment with either CCl(4) or TAA. There were far fewer alpha-smooth muscle actin positive hepatic stellate cells in CCl(4)-treated KO mice than that in WT mice. After CCl(4) treatment, the mRNA level of A(1), A(2A), A(2B), and A(3) adenosine receptors, tumor necrosis factor-alpha, interleukin (IL) -1beta, IL-13r alpha1, matrix metalloproteinase (MMP)-2, MMP-14, tissue inhibitor of metalloproteinase (TIMP) -1, and TIMP-2, and IL-13 level increased markedly in both CD73KO and WT mice, but Col1 alpha1, Col3 alpha1, and transforming growth factor-beta1 mRNA increased much more in WT mice than that in KO mice. Moreover, IL-13r alpha2, MMP-13 mRNA, and MMP-13 protein were higher in KO mice than that in WT mice. These results indicate that adenosine, formed extracellularly from adenine nucleotides, plays a major role in the pathogenesis of hepatic fibrosis and that inhibition of adenosine production or blockade of adenosine receptors may help prevent hepatic fibrosis

We describe diffuse glioma-like infiltrates in excised tubers in five out of forty Tuberous sclerosis complex (TSC) patients undergoing excision of a tuber at our institution within the last 10 years. All patients presented with refractory seizures. Resection specimens from four patients had the pathognomonic histologic features of neuroglial hamartomas (tubers) and in one case there was cortical microdysgenesis lacking cells typical of TSC. All lesions were associated with an infiltrate of atypical, mostly elongate, glioma-like small cells, which were immunoreactive for GFAP in three, and pS6 (a marker for activity of the mTOR pathway), in two cases. MAP-2 and CD34, were negative and MIB-1 (Ki67) immunostains ranged from <1-21%. Array-based comparative genomic hybridization revealed that these proliferative phenomena were associated with 21 different copy number aberrations in comparison with a tuber without atypical infiltrates. Postoperatively (follow-up period ranging from 8 to 34 months) none of the patients have any evidence of a glioma. We report that tubers resected for treatment of seizures are sometimes associated with glioma-like lesions, which are indistinguishable from infiltrating gliomas by morphology and immunohistochemistry. Genomic analysis with SNP arrays revealed copy number changes which may be associated with the pathogenesis of such infiltrates

Adenosine is a potent modulator of inflammation and tissue repair. We have recently reported that activation of adenosine A(2A) receptors promotes collagen synthesis by human dermal fibroblasts and that blockade or deletion of this receptor in mice protects against bleomycin-induced dermal fibrosis, a murine model of scleroderma. Adenosine deaminase (ADA) is the principal catabolic enzyme for adenosine in vivo, and its deficiency leads to the spontaneous development of pulmonary fibrosis in mice. The aim of this study was to characterize further the contributions of endogenous adenosine and adenosine A(2A) receptors to skin fibrosis. Taking advantage of genetically modified ADA-deficient mice, we herein report a direct fibrogenic effect of adenosine on the skin, in which increased collagen deposition is accompanied by increased levels of key mediators of fibrosis, including transforming growth factor beta1, connective tissue growth factor, and interleukin-13. Pharmacological treatment of ADA-deficient mice with the A(2A) receptor antagonist ZM-241385 prevented the development of dermal fibrosis in this model of elevated tissue adenosine, by reducing dermal collagen content and expression of profibrotic cytokines and growth factors. These data confirm a fibrogenic role for adenosine in the skin and reveal A(2A) receptor antagonists as novel therapeutic agents for the modulation of dermal fibrotic disorders