Local administration of PF-3512676 CpG-B instigates tumor-specific CD8+ T-cell reactivity in melanoma patients

PURPOSE: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8(+) T cells. EXPERIMENTAL DESIGN: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8(+) T cells from SLN and peripheral blood were tested for reactivity by IFN-gamma ELISPOT assay against several HLA-A1/A2/A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. RESULTS: Melanoma-specific CD8(+) T-cell response rates against >1 MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012). Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8(+) T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. CONCLUSIONS: These data show an increase in melanoma-specific CD8(+) T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.     

Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells.

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.     

Expression of the multidrug resistance-associated protein (MRP) gene in primary non-small-cell lung cancer

BACKGROUND: One of the major problems in the cure of advanced non-small-celllung cancer (NSCLC) is its lack of re sponse to cytotoxic drugtreatment, and the mechanisms underlying this intrinsic drugresistance are unclear. PATIENTS AND METHODS: We determined the expression of a newly recognised drug resistancegene, the Multidrug Resistance-associated Protein (MRP) gene,in normal lung tissue and in tumour biopsies from 35 surgicallyresected NSCLCs (11 adenocarcinomas, 24 squamous cell carcinomas).MRP mRNA levels were quantitated by RNase protection assay andexpression of the MRP Mr 190,000 glycoprotein was estimatedby immunohistochemistry. RESULTS: Using the MRP-speciflc monoclonal antibody MRPr1, MRP expressionwas detected by immunohistochemistry in epithelial cells liningthe bronchi in normal lung. In NSCLC approximately 35% of thesamples showed elevat ed MRP mRNA levels. Based on MRP-specificimmunohis tochemical staining the tumours were divided into4 groups: 12% were scored as negative (–), 14% showedweak cytoplasmic staining of the tumour cells (±), 40%had a clear cytoplasmic staining (±), and in 34% a strongcytoplasmic as well as membranous staining was observed (++).MRP expression, as estimated by immunohistochemistry, correlatedwith the MRP mRNA levels quantitated by RNase protection assay(correlation coefficient -0.745, p=0.0009), with MRP mRNA levels(mean ± SD) of 3.0 ± 1.0 U, 3.5 ± 0.7 U,7.5 ± 5.9 U, and 19.3 ± 10.7 U, in the (–),(±), (+), and (++) immunohistochemistry expression groups,respectively. Among the squamous cell carcinomas a correlationwas observed between MRP staining and tumour cell differentiation:the strongest MRP staining was predominantly found in the welldifferentiated tumours. CONCLUSIONS: Hyperexpression of MRP is frequently observed in primary NSCLC,especially in the well differentiated squamous cell carcinomas.Further studies are needed to assess the role of MRP in themechanism of clinical drug resistance in NSCLC. MRP, NSCLC, RNase protection assay, immunohistochemistry     

Expression of the human major vault protein LRP in human lung cancer samples and normal lung tissues

BACKGROUND:: The recently discovered LRP protein has been shown to be involvedin drug resistance and possibly in detoxification processes. MATERIALS AND METHODS:: To study the relation between LRP expression and exposure tocigarette smoke, LRP immunoreactivity was evaluated in 39 paraffinembedded normal lung tissues derived from patients operatedon for pneumothorax, and related to amount of pack years smoked.We also studied the LRP protein expression in 36 non-small-celllung cancer (NSCLC) samples and related the expression patientcharacteristics and survival. Furthermore 17 lung tumor samples(10 NSCLC and 7 SCLC) derived from patients treated with chemotherapywere analysed in order to investigate the relation between LRPor MRP expression and patient's response to chemotherapy. RESULTS:: In the normal lung tissues, LRP intensity levels were not correlatedto the amount of pack years smoked, although a trend was seenfor higher LRP intensity levels in patients who smoked morethan 10 pack years. LRP expression was significantly higherin NSCLC samples than in SCLC samples, and all SCLC samplesdisplayed very low LRP expression. Within NSCLC, squamous celland adenocarcinomas had higher LRP expression than large cellundifferentiated and mixed tumors. In NSCLC patients LRP expressionwas not a prognostic factor for survival. At initial analysisLRP expression levels did not predict for the response to chemotherapy.Only 3 out of 17 patients expressed MRP, and all SCLC sampleswere MRP negative. CONCLUSION:: Striking different expression levels were seen between NSCLCand SCLC for both LRP and MRP. In a preliminary analysis LRPexpression was not predictive for response to chemotherapy inlung cancer patients. In pneumothorax patients LRP levels werenot correlated with the amount of pack years smoked. detoxification, immunohistochemistry, LRP, lung cancer, MRP, multidrug resistance     

Major vault protein, a marker of drug resistance, is upregulated in refractory epilepsy

PURPOSE: The molecular basis of drug resistance in epilepsy is being explored. Two proteins associated with drug resistance in cancer, P-glycoprotein and multidrug resistance-associated protein 1, are upregulated in human epileptogenic pathologies. Other proteins associated with resistance in cancer include major vault protein (MVP) and breast cancer resistance protein (BCRP). We hypothesized that these proteins would also be upregulated in human epileptogenic pathologies. METHODS: Hippocampal sclerosis (HS), focal cortical dysplasia (FCD), and dysembryoplastic neuroepithelial tumor (DNT) were studied by using immunohistochemistry for MVP and BCRP. Nonepileptogenic control and histologically normal brain adjacent to epileptogenic tissue were used for comparison. RESULTS: MVP and BCRP were expressed ubiquitously in brain capillary endothelium. Ectopic upregulation of MVP was seen in hilar neurons in HS, dysplastic neurons in FCD, and lesional neurons in DNT. Only in HS cases were rare extralesional neurons immunoreactive. Glial upregulation was not seen. There was no qualitative upregulation of BCRP. CONCLUSIONS: These results show that more than one resistance protein may be upregulated in a given epileptogenic pathology and may contribute to drug resistance. Determination of the types, amounts, and distribution of such proteins will be necessary for rational treatment for drug resistance in epilepsy.     

Oral plasmablastic lymphoma in an HIV-negative patient: a case report and review of the literature

Plasmablastic lymphoma is an HIV-associated non-Hodgkin's lymphoma that primarily affects the oral cavity and jaws. The purpose of this report is to describe the first case of plasmablastic lymphoma occurring in an HIV-negative, nonimmunocompromised individual, and to review the histopathologic and immunohistochemical phenotype of this lymphoma. Histopathologically, our case exhibited a dense, diffuse lymphocytic infiltrate of noncohesive large lymphocytes with plasmacytoid features. Immunohistochemical analysis revealed positivity for the B-cell marker CD79a, VS38c, Epstein-Barr virus latent membrane protein (LMP), immunoglobulin G (IgG), and lambda light chain restriction. Neoplastic cells were negative for leukocyte common antigen, CD20, CD3, CD10, CD138, Bcl-2, Bcl-6, desmin, actin, EMA, S-100, HMB45, Alk-1, HHV8, IgA, IgM, and cytokeratin. The features of this rare disease are summarized based on a comprehensive review of the epidemiologic, clinical and immunohistochemical findings of previously reported cases.     

The unfolded protein response is activated in Alzheimer’s disease

Alzheimers disease (AD) is, at the neuropathological level, characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR) that may protect the cell against the toxic buildup of misfolded proteins. In this study we investigated the activation of the UPR in AD. Protein levels of BiP/GRP78, a molecular chaperone which is up-regulated during the UPR, was found to be increased in AD temporal cortex and hippocampus as determined by Western blot analysis. At the immunohistochemical level intensified staining of BiP/GRP78 was observed in AD, which did not co-localize with AT8-positive neurofibrillary tangles. In addition, we performed immunohistochemistry for phosphorylated (activated) pancreatic ER kinase (p-PERK), an ER kinase which is activated during the UPR. p-PERK was observed in neurons in AD patients, but not in non-demented control cases and did not co-localize with AT8-positive tangles. Overall, these data show that the UPR is activated in AD, and the increased occurrence of BiP/GRP78 and p-PERK in cytologically normal-appearing neurons suggest a role for the UPR early in AD neurodegeneration. Although the initial participation of the UPR in AD pathogenesis might be neuroprotective, sustained activation of the UPR in AD might initiate or mediate neurodegeneration.     

Localization of breast cancer resistance protein (BCRP) in microvessel endothelium of human control and epileptic brain

PURPOSE: Breast cancer resistance protein (BCRP) is a half adenosine triphosphate (ATP)-binding cassette (ABC) transporter expressed on cellular membranes and included in the group of multidrug resistant (MDR)-related proteins. Recently, upregulation of different MDR proteins has been shown in human epilepsy-associated conditions. This study investigated the expression and cellular distribution of BCRP in human control and epileptic brain, including a large number of both neoplastic and nonneoplastic specimens from patients with chronic pharmacoresistant epilepsy. METHODS: Several epileptogenic pathologies, such as hippocampal sclerosis (HS), focal cortical dysplasia (FCD), dysembryoplastic neuroepithelial tumor, oligodendroglioma astrocytoma, and glioblastoma multiforme were studied by using Western blot and immunocytochemistry. RESULTS: With Western blot, we could demonstrate the presence of BCRP in both normal and epileptic human brain tissue. In contrast to P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) 2, BCRP expression levels did not change in tissue from patients with HS, compared with control hippocampus. No BCRP immunoreactivity was observed in glial or neuronal cells, including reactive astrocytes and dysplastic neurons in FCD. BCRP expression was, however, increased in tumor brain tissue. Immunocytochemistry demonstrated that BCRP was exclusively located in blood vessels and was highly expressed at the luminal cell surface and in newly formed tumor capillaries. This localization closely resembles that of P-gp. The higher expression observed in astrocytomas by Western blot analysis was related to the higher vascular density within the tumor tissue. CONCLUSIONS: These results indicate a constitutive expression of BCRP in human endothelial cells, representing an important barrier against drug access to the brain. In particular, the strong BCRP expression in the microvasculature of epileptogenic brain tumors could critically influence the bioavailability of drugs within the tumor and contribute to pharmacoresistance.     

Oral aspects of Crohn's disease

Crohn's disease is a chronic inflammatory disease, which may involve any part of the gastrointestinal tract, including the oral cavity. This review gives an overview of the oral findings observed in patients with Crohn's disease and the potential implications of the disease for dental management are discussed.