Peter Middel

Engagement of the FcεRI Stimulates the Production of IL-16 in Langerhans Cell-Like Dendritic Cells

Preferential uptake and presentation of IgE-bound allergens by epidermal Langerhans cells (LC) via the high affinity IgE receptor, FcεRI, is regarded as an important mechanism in the induction of cutaneous inflammation in atopic dermatitis. Here, we show that activation of monocyte-derived LC-like dendritic cells (LLDC) through engagement of FcεRI induces the expression of IL-16, a chemoattractant factor for dendritic cells, CD4+ T cells, and eosinophils. We found that ligation of FcεRI on LLDC derived from atopic dermatitis patients that express high levels of FcεRI increases IL-16 mRNA expression and storage of intracellular IL-16 protein and enhances the secretion of mature IL-16 in a biphasic manner. An early release of IL-16 (peak at 4 h) is independent of protein synthesis, while a more delayed release (peak at 12 h) requires protein synthesis and occurs subsequent to the induction of IL-16 mRNA and intracellular accumulation of pro-IL-16. There was evidence that LLDC use caspase-1 to process IL-16, as inhibition of caspase-1, but not of caspase-3, partially prevented the release of IL-16 in response to ligation of FcεRI. In an in vivo model of IgE-dependent LC activation, the atopy patch test, positive skin reactions were also associated with the induction of IL-16 in epidermal dendritic cells. These data indicate that IL-16 released from LC after allergen-mediated activation through FcεRI may link IgE-driven and cellular inflammatory responses in diseases such as atopic dermatitis.

Expression of the T-cell chemoattractant chemokine lymphotactin in Crohn's disease.

Recruitment of lymphocytes is a prominent feature of the inflammatory process in Crohns disease (CD). The present study was undertaken to investigate the expression of the novel lymphocyte-specific chemoattractant lymphotactin (Lptn) as a potential regulatory factor for the recruitment of T cells in CD. The expression of Lptn mRNA was quantified in resection specimens of patients with CD in comparison to normal controls without signs of inflammation by real-time quantitative reverse transcriptase-polymerase chain reaction and localized by nonradioactive in situ hybridization. Furthermore, the phenotype of cells expressing Lptn mRNA was characterized. In contrast to normal controls Lptn mRNA was significantly increased in tissue samples affected by CD. Cells expressing Lptn were identified as T cells, mast cells, and unexpectedly dendritic cells. Lptn mRNA was found to be up-regulated on stimulation with phorbol-12-myristate-13-acetate and concanavalin A in T cells isolated from peripheral blood, which could be prevented by dexamethasone, cyclosporine A, and FK506. A similar regulation mechanism could be identified for the Lptn receptor GPR-5 in peripheral T cells. In addition, Lptn mRNA expression could be induced in mature monocyte-derived dendritic cells. The results indicate that local expression of Lptn by activated T cells and to a lesser extent by mast cells and dendritic cells represents a key regulator for lymphocyte trafficking and maintenance of the inflammatory process observed in CD, which might be partly mediated through an autocrine/paracrine pathway of activated T cells.

Cathepsin K deficiency partially inhibits, but does not prevent, bone destruction in human tumor necrosis factor–transgenic mice

OBJECTIVE Cathepsin K is believed to have an eminent role in the pathologic resorption of bone. However, several studies have shown that other proteinases also participate in this process. In order to clarify the contribution of cathepsin K to the destruction of arthritic bone, we applied the human tumor necrosis factor (hTNF)-transgenic mouse model, in which severe polyarthritis characterized by strong osteoclast-mediated bone destruction develops spontaneously. METHODS Arthritis was evaluated in hTNF-transgenic mice that were either wild-type for cathepsin K (CK(+/+)), heterozygous for cathepsin K (CK(+/-)), or deficient in cathepsin K (CK(-/-)). Arthritic knee joints were prepared for standard histologic assessment aimed at establishing a semiquantitative score for joint destruction and quantification of the area of bone erosion. Additionally, microfocal computed tomography was performed to visualize bone destruction in mice with the different CK genotypes. CK(+/+) and CK(-/-) osteoclasts were generated in vitro, and their proteinase expression profiles were compared by complementary DNA array analysis, real-time polymerase chain reaction, and activity assays. RESULTS Although the area of bone erosion was significantly reduced in hTNF-transgenic CK(-/-) mice, the absence of cathepsin K did not completely protect against inflammatory bone lesions. Several matrix metalloproteinases (MMPs) and cathepsins were expressed by in vitro-generated CK(-/-) osteoclasts, without marked differences from CK(+/+) osteoclasts. MMP activity was detected in CK(-/-) osteoclasts, and MMP-14 was localized by immunohistochemistry in inflammatory bone erosions in hTNF-transgenic CK(-/-) mice, suggesting MMPs as potential contributors to bone destruction. Additionally, we detected a reduction in osteoclast formation in cathepsin K-deficient mice, both in vitro and in vivo. CONCLUSION The results of our experiments raise doubts about a crucial role of cathepsin K in arthritic bone destruction.

Frequency of HER-2 positivity in rectal cancer and prognosis.

In patients with advanced rectal cancer (cUICC II and III) multimodality therapy resulted in better long-term local tumor control. Ongoing clinical trials are focusing on therapy intensification to improve disease-free (DFS) and cancer-specific survival (CSS), the integration of biomarkers for prediction of individual recurrence risk, and the identification of new targets. In this context, we investigated HER-2, a member of the epidermal growth factor receptor family, whose expression pattern and role was unclear in rectal cancer. A total of 264 patients (192 male, 72 female; median age 64 y) received standardized multidisciplinary treatment according to protocols of phase II/III trials of the German Rectal Cancer Study Group. HER-2 status was determined in pretherapeutic biopsies and resection specimens using immunohistochemistry scoring and detection of silver in situ hybridization amplification. Tumors with an immunohistochemistry score of 3+ or silver in situ hybridization ratios of ≥2.0 were classified HER-2 positive; these results were correlated with clinicopathologic parameters [eg, resection (R) status, nodal status ((y)pN)], DFS, and CSS. Positive HER-2 status was found in 12.4% of biopsies and in 26.7% of resected specimens. With a median follow-up of 46.5 months, patients with HER-2 positivity showed in trend a better DFS (P=0.1) and a benefit in CSS (P=0.03). The 5-year survival rate was 96.0% (HER-2 positive) versus 80.0% (HER-2 negative). In univariate and multivariate analyses, HER-2 was an independent predictor for CSS (0.02) along with the (y)pN status (P<0.00001) and R status (P=0.011). HER-2 amplification is detectable in a relevant proportion (26.7%) of rectal cancer patients. For the development of innovative new therapies, HER-2 may represent a promising target and should be further assessed within prospective clinical trials.

Chemokine-mediated distribution of dendritic cell subsets in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) represents one of the most immunoresponsive cancers. Antigen-specific vaccination with dendritic cells (DCs) in patients with metastatic RCC has been shown to induce cytotoxic T-cell responses associated with objective clinical responses. Thus, clinical trials utilizing DCs for immunotherapy of advanced RCCs appear to be promising; however, detailed analyses concerning the distribution and function of DC subsets in RCCs are lacking.MethodsWe characterized the distribution of the different immature and mature myeloid DC subsets in RCC tumour tissue and the corresponding normal kidney tissues. In further analyses, the expression of various chemokines and chemokine receptors controlling the migration of DC subsets was investigated.ResultsThe highest numbers of immature CD1a+ DCs were found within RCC tumour tissue. In contrast, the accumulation of mature CD83+/DC-LAMP+ DCs were restricted to the invasive margin of the RCCs. The mature DCs formed clusters with proliferating T-cells. Furthermore, a close association was observed between MIP-3α-producing tumour cells and immature CCR6+ DC recruitment to the tumour bed. Conversely, MIP-3β and SLC expression was only detected at the tumour border, where CCR7-expressing T-cells and mature DCs formed clusters.ConclusionIncreased numbers of immature DCs were observed within the tumour tissue of RCCs, whereas mature DCs were found in increased numbers at the tumour margin. Our results strongly implicate that the distribution of DC subsets is controlled by local lymphoid chemokine expression. Thus, increased expression of MIP-3α favours recruitment of immature DCs to the tumour bed, whereas de novo local expression of SLC and MIP-3β induces accumulation of mature DCs at the tumour margin forming clusters with proliferating T-cells reflecting a local anti-tumour immune response.

Subcellular localization of rat Abca5, a rat ATP-binding-cassette transporter expressed in Leydig cells, and characterization of its splice variant apparently encoding a half-transporter

Several transporters belonging to the ABCA subfamily of ABC (ATP-binding cassette) proteins are involved in lipid trafficking. Human ABCA5 and its rat orthologue, rAbca5, represent recently identified subfamily members whose substrate spectrum remains to be defined. The elucidation of (sub)cellular rAbca5 distribution would be expected to provide a basis for optimization of functional analyses. In the present study, we applied in situ hybridization to examine rAbca5 mRNA distribution within sections of rat testis, a tissue expressing high levels of rAbca5 mRNA. We found rAbca5 mRNA to be predominantly expressed in interstitial Leydig cells, which are major sites of testosterone synthesis. To investigate rAbca5 subcellular localization, we constructed expression vectors yielding rAbca5 fused either to EGFP (enhanced green fluorescent protein) or to a peptide bearing the viral V5 epitope. During rAbca5 cDNA cloning, we discovered a splice variant sequence (rAbca5 V20+16), predicted to give rise to a truncated, half-size transporter, which was highly homologous with a human splice variant described by us previously. Quantitative RT (reverse transcription)-PCR demonstrated that the rAbca5 splice variant was expressed in numerous tissues (including testis, brain and lungs), its cDNA amounting to 2.6-11.2% of total rAbca5 cDNA. Transfection of individual rAbca5-EGFP, rAbca5 splice variant-EGFP or transporter-V5 expression plasmids along with organelle marker plasmids into HEK-293 cells (human embryonic kidney 293 cells) revealed that both rAbca5 and splice variant fusion proteins co-localized with marker protein for the Golgi apparatus. Expression of rAbca5 mRNA in Leydig cells, intracellular localization of rAbca5-EGFP/rAbca5-V5 and involvement of rAbca5-related proteins in lipid transport suggest that rAbca5 may participate in intracellular sterol/steroid trafficking.

Enrichment of CD133-expressing cells in rectal cancers treated with preoperative radiochemotherapy is an independent marker for metastasis and survival

The transmembrane glycoprotein CD133 (cluster of differentiation 133; also known as Prominin or PROM1) has been described as a potential stem cell marker in colorectal cancer and is associated with higher tumorigenic potential and resistance to radiochemotherapy (RCT). In this study, CD133 expression was evaluated in pre‐RCT tumor biopsies and the corresponding post‐RCT surgical specimens from patients with locally advanced rectal adenocarcinoma, and expression levels were correlated with histopathologic features and clinical follow‐up.

Preoperative Chemoradiation Therapy With Capecitabine/ Oxaliplatin and Cetuximab in Rectal Cancer: Long-Term Results of a Prospective Phase 1/2 Study

PURPOSE We have previously shown that the addition of cetuximab to chemoradiation therapy failed to improve complete response rates (pCR) in rectal cancer. Here we report the long-term results of the cetuximab added to preoperative radiation therapy with capecitabine and oxaliplatin (CET-CAPOX-RT) phase 1/2 study that evaluated preoperative chemoradiation with cetuximab, capecitabine, and oxaliplatin in patients with rectal cancer. METHODS AND MATERIALS The median follow-up was 63 months (range, 5-73 months). Sixty patients were enrolled; 3 patients were excluded due to protocol violation, and 4 died before surgery. Total mesorectal excision was performed in 53 patients, in 85% (n=45) with curative intention (M0-status). Secondary end points including overall survival (OS) disease-free survival (DFS) and cancer-specific survival (CSS) were calculated. The prognostic value of KRAS mutation status was also assessed. RESULTS Histopathological examination confirmed ypUICC stages 0 (n=4; pCR), I (n=17), II (n=10), III (n=14), and IV (n=8). For patients who underwent surgery (n=53), OS at 1, 3, and 5 years was 88.7%, 83%, and 75.5%, respectively, whereas CSS rates were 94.1%, 88.1%, and 78.1%, respectively. In the 45 patients who were treated with curative intent (M0), the OS rates at 1, 3, and 5 years were 91.1%, 88.9%, and 86.7%, respectively; whereas CSS rates were 97.6%, 95.2%, and 90.3%, respectively; and DFS rates were 90.7%, 88.3%, and 88.3%, respectively. We did not find any locoregional failure in patients with M0-status (n=45). Chronic toxicity was rare. KRAS mutations, as detected in 33.3%, showed no correlation with the clinicopathological parameters nor significance for either OS (P=.112), CSS (P=.264), or DFS (P=.565). CONCLUSIONS Taken together, chemoradiation therapy combined with cetuximab is safe, feasible, and offers excellent survival rates. KRAS mutation status was not a predictive factor. Importantly, lack of improvement in pCR rate did not translate to poor survival in our clinical trial.

Expression of Lymphotoxin-α by Keratinocytes: A Further Mediator for the Lichenoid Reaction

Objective: Lichen planus (LP) represents a disease in which autoimmune mechanisms mediated by Th1 T cells are involved. Lymphotoxin-α (LT-α) represents a Th1 cytokine with proinflammatory activities in LP, as has recently been demonstrated for interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Methods: Expression of LT-α mRNA was investigated by RT-PCR and nonradioactive in situ hybridization. Double staining methods were applied to characterize the phenotype of cells expressing LT-α. Cell stimulation experiments were performed on the transformed squamous cell line HaCaT. Results: In contrast to normal skin, LT-α-specific RT-PCR products were found in all cases of LP. Cells in the inflammatory infiltrate expressing LT-α were identified as mainly T cells and mast cells, as shown by in situ hybridization. Furthermore, predominant LT-α mRNA expression could be observed in lesional keratinocytes adjacent to the band-like inflammatory infiltrate. In cell stimulation experiments, it could be shown that IFN-γ induces LT-α and TNF-α mRNA in the human squamous cell line HaCaT, concomitant with upregulation of MHC class II and intercellular adhesion molecule-1, which could also be observed on lesional keratinocytes in LP. Conclusions: In LP, LT-α mRNA is predominantly expressed by lesional keratinocytes and to a lesser extent by inflammatory cells. Induction of LT-α in keratinocytes is closely related to the expression of TNF-α and MHC class II. The loci of TNF-α and LT-α map to MHC class III on chromosome 6, which is closely linked to the MHC class II gene locus. Our results suggest that stimulation of keratinocytes with IFN-γ results in the upregulation of proinflammatory cytokines such as LT-α and TNF-α as well as MHC class II, which map to the same gene region of immunoregulatory genes on chromosome 6 and may be involved in the induction and maintenance of the disease.

Reproducibility of immunohistochemical scoring for epidermal growth factor receptor expression in non-small cell lung cancer: round robin test.

CONTEXT The addition of cetuximab to first-line chemotherapy substantially prolonged survival in patients with advanced non-small cell lung cancer whose tumors expressed high levels of epidermal growth factor receptor (EGFR; immunohistochemistry score of ≥200 on a scale of 0-300). OBJECTIVE To evaluate the interobserver reproducibility of this EGFR immunohistochemistry scoring system, based on both the tumor cell membrane staining intensity (graded 0-3+) and the percentage of cells staining at each intensity. DESIGN In parts 1 (initial feasibility study) and 2 of this 2-part round robin test, sections of different non-small cell lung cancer tissue microarrays were stained in a central reference laboratory. Following reference evaluation, EGFR expression in 30 selected tumor cores was characterized in serial sections by lung cancer pathology specialists. The reproducibility of scoring by different raters was assessed. Analysis of between-rater agreement was based on the allocation of EGFR immunohistochemistry scores into low- (<200) and high- (≥200) EGFR expression groups. RESULTS After discussion with raters of the issues impacting reproducibility identified in part 1 and following adjustment of processes, part 2 of the round robin test showed a high interobserver agreement in EGFR immunohistochemistry scoring, with an overall concordance rate of 90.9% and a mean κ coefficient of 0.812. Specimens with a reference EGFR immunohistochemistry score of lower than 200 and of 200 or higher showed mean concordance rates of 94.7% and 85.6%, respectively. CONCLUSIONS After appropriate training, assessing EGFR expression by this immunohistochemistry-based method allowed a highly reproducible allocation of non-small cell lung cancers into clinically relevant high- or low-EGFR expression groups.