Barbara Wollenberg

The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells

Bispecific antibodies (bsAb) are considered as promising tools for the elimination of disseminated tumour cells in a minimal residual disease situation. The bsAb-mediated recruitment of an immune effector cell in close vicinity of a tumour cell is thought to induce an antitumoural immune response. However, classical bispecific molecules activate only a single class of immune effector cell that may not yield optimal immune responses. We therefore constructed an intact bispecific antibody, BiUII (anti-CD3 × anti-EpCAM), that not only recognizes tumour cells and T lymphocytes with its two binding arms, but also binds and activates Fcγ-receptor positive accessory cells through its Fc-region. We have demonstrated recently that activated accessory cells contribute to the bsAb-induced antitumoural activity. We now analyse this stimulation in more detail and demonstrate here the BiUll-induced upregulation of activation markers like CD83 and CD95 on accessory cells and the induction of neopterin and biopterin synthesis. Experiments with pure cell subpopulations revealed binding of BiUll to CD64+ accessory cells and CD16+ NK cells, but not to CD32+ B lymphocytes. We provide further evidence for the importance of the Fc-region in that this bispecific molecule stimulates Fcγ-R-positive accessory cells to eliminate tumour cells in vitro by direct phagocytosis.

Application of the Vibrant Soundbridge to unilateral osseous atresia cases.

Patients with high‐grade atresia‐microtia suffer from a combined malformation of the outer and middle ears, typically leading to a severe hearing impairment. Long‐term results of middle ear reconstruction with tympanoplasty are often insufficient due to persistent air‐bone gaps, and new techniques in hearing rehabilitation are required. The objective of this research is to evaluate the active middle ear implant, the Vibrant Soundbridge® (VSB), for hearing rehabilitation of patients with unilateral osseous aural atresia.

Isolation and Characterization of Adult Stem Cells from Human Salivary Glands

Currently, adult stem cells are attracting significant interest in regenerative medicine and tissue engineering. These cells have been isolated from various tissue sources; however, in most cases, adult stem cells useful for tissue engineering and regeneration are present at a low frequency. High numbers of stem cells with an effective and reliable potential for differentiation are needed for clinical applications. Thus, the identification of new stem cell sources and the establishment of optimized cell culture conditions that allow for the amplification of stem cells are of utmost relevance. In addition, the isolation procedure should ideally be minimally invasive and possibly be performed under local anesthesia. We report here for the first time on the identification of adult stem cells with mesenchymal characteristics in human parotid gland tissue. Cells were isolated from freshly resected specimens of parotid glands using enzymatic digestion and plastic adhesion protocols. Following an initial proliferation period and short-term culture for four passages, immunophenotyping revealed the presence of mesenchymal stem cell markers. In the presence of tissue-specificinduction medium, stem cells could be differentiated into adipogenic, osteogenic, and chondrogenic cell types. Tissue-specific differentiation was confirmed by histochemical and immunocytochemical staining as well as by RT-PCR for defined marker genes. This study is, to the best of our knowledge, the first report on the isolation and differentiation of stem cells from adult human parotid glands. Although isolated from an endodermal tissue source, these stem cells share many characteristics with MSCs. Easy accessibility and a high differentiation potential make salivary gland-derived stem cells a promising source for future applications in regenerative medicine.

Tumor-specific glycosylation of the carcinoma-associated epithelial cell adhesion molecule EpCAM in head and neck carcinomas

The tissue-specific glycosylation of the carcinoma (CA)-associated antigen epithelial cell adhesion molecule (EpCAM) was studied in 60 patients suffering from head and neck CAs, and 26 pairs of autologous healthy thyroid and CA biopsies. EpCAM was glycosylated in all tumor samples in which its expression was detectable (73%). Additionally, in 80.7% of patients, tumor-derived EpCAM was heavily glycosylated while EpCAM derived from autologous thyroid was not (76.2%) or weakly (23.8%). Four cases showed a similar glycosylation pattern (15.3%) and one case displayed a reverse pattern (3.8%). Additionally, the expression and glycosylation of EpCAM were assessed in tumor adjacent and distant tissue. EpCAM was glycosylated in tumor-adjacent while it was not or only weakly expressed in tumor distant tissue where it was unglycosylated. Thus, EpCAM is differentially glycosylated in healthy tissue and tumor cells of the head and neck area.

Tumour-derived prostaglandin E2 and transforming growth factor-β synergize to inhibit plasmacytoid dendritic cell-derived interferon-α

In previous studies we reported that plasmacytoid dendritic cells (PDC) infiltrating head and neck cancer tissue are functionally impaired, but the molecular basis for the functional deficiency remained unclear. Here we demonstrate that tumour‐derived prostaglandin E2 (PGE2) and transforming growth factor‐β (TGF‐β) increase interleukin‐8 (IL‐8) but synergistically inhibit interferon‐α (IFN‐α) and tumour necrosis factor (TNF) production of Toll‐like receptor 7 (TLR7)‐ and Toll‐like receptor 9 (TLR9)‐stimulated PDC. The inhibitory effect of PGE2 could be mimicked by the induction of cyclic AMP (cAMP) and by inhibitors of cyclooxygenase. The contribution of tumour‐derived TGF‐β was confirmed by the TGF‐β antagonist SB‐431542. Suppression of tumour‐derived PGE2 and TGF‐β restored TLR‐induced IFN‐α production of PDC. Additionally, PGE2‐ and TGF‐β‐treated PDC display a ‘tolerogenic’ phenotype because of a downregulation of CD40 accompanied by an upregulation of CD86. Finally, in TLR‐stimulated PDC, PGE2 and TGF‐β reduce the CCR7 : CXCR4 ratio, suggesting that PDC are impaired in their ability to migrate to tumour‐draining lymph nodes but are retained in stromal cell‐derived factor 1 (SDF‐1)‐expressing tissues. Based on these data, cyclooxygenase inhibitors and TGF‐β antagonists may improve TLR7‐ and TLR9‐based tumour immunotherapy.

Glandular tissue from human pancreas and salivary gland yields similar stem cell populations.

Stem cells derived from pancreatic tissue are well characterized and exhibit a broad plasticity as they can differentiate beyond lineage boundaries into many cell types. The aim of this study was the comparative characterization of pancreatic stem cells with one other derivate of the embryonic foregut, namely salivary glands, for the existence of similar stem cell populations. The expression of stem cell markers as well as lineage-specific markers was detected by reverse transcription polymerase chain reaction, flow cytometry and immuncytochemical staining. The isolated cells from salivary glands and pancreas grew adherently in vitro and could be maintained for up to 55 and 46 population doublings, respectively. Cells from both tissues showed a comparable phenotype. They expressed different embryonic and adult stem cell markers and had the ability to differentiate spontaneously into cells representing the three embryonic germ layers. Additionally, the directed differentiation of glandular stem cells into the mesodermal lineage was achieved, yielding adipogenic, osteogenic and chondrogenic cells from salivary gland stem cells as well as osteogenic and chondrogenic cells from pancreatic stem cells. Here, we compared two stem cell populations from different glandular tissues which showed similar phenotypes and analogous properties. During embryonic development the two exocrine glands originate from the foregut, which might be the explanation for these intriguing resemblances.

Tumor cell-derived prostaglandin E2 inhibits monocyte function by interfering with CCR5 and Mac-1

The cyclooxygenases (COX)‐1 and COX‐2 are key enzymes in the conversion of arachi‐donic acid to prostaglandins and other eicosanoids. Whereas COX‐1 is expressed ubiquitously, COX‐2 is an immediate‐early gene often associated with malignant transformation, and a role for the COX enzymes in tumor initiation and promotion is discussed. Nonsteroidal anti‐inflammatory drugs (NSAIDs) like aspirin and indomethacin that block COX‐1 and −2 have been shown to have beneficial effects for tumor patients. Therefore, these compounds have gained interest also among oncologists. However, the molecular mechanism by which NSAIDs inhibit carcinogenesis is not clearly understood. The prostaglandin‐dependent and ‐independent effect may both account for their antineoplastic action. We show here that tumor cells derived from different tumors regularly produce prostaglandin E2 (PGE2) interfering with the function of monocytes. In particular, PGE2 inhibits the potential of monocytes to migrate in the direction of a chemotactic stimulus and to adhere to endothelial cell. This inhibition is most probably due to a modulation of the chemokine receptor CCR5 and the β2‐integrin Mac‐1. Both down‐regulation of CCR5 and reduced expression of Mac‐1 may diminish the potential of peripheral blood monocytes to leave blood vessels and invade target tissues. Since both dysfunctions can be restored with NSAIDs, our findings help to explain the molecular chemopreventive action of NSAIDs on tumor formation and progression.—Zeidler, R., Csanady, M., Gires, O., Lang, S., Schmitt, B., Wollenberg, B. Tumor cell‐derived prostaglandin E2 inhibits monocyte function by interfering with CCR5 and Mac‐1. FASEB J. 14, 661–668 (2000)

Human Th17 cells can be induced through head and neck cancer and have a functional impact on HNSCC development

Background:The T helper 17 (Th17) cells recently identified as distinct T helper cell lineage are characterised by their production of the proinflammatory cytokine interleukin 17. Although much effort has been made in understanding the function of Th17 cells in the pathogenesis of different diseases, their influence in carcinogenesis remain largely unknown.Methods:We studied the prevalence and induction of Th17 cells in head and neck squamous cell carcinoma (HNSCC) patients by flow cytometry. To determine the migration mechanism of Th17 cells into primary tumours and metastasis of HNSCC, we performed chemotaxis assays. We analysed the proliferation and the angiogenesis-related proteins of HNSCCs in the presence of Th17 cells with MTT-based proliferation assay and an angiogenesis protein array.Results:In this study, we showed that the prevalence of Th17 cells is elevated in peripheral blood of HNSCC patients. In addition, tumour tissue and tumour-draining lymph nodes are infiltrated by a huge number of Th17 cells representing an important fraction of the tumour-infiltrating lymphocytes (TILs). We further showed that Th17 cells can be induced and expanded in tumour microenvironment through cytokines produced by tumour cells and TILs, and in addition can be recruited to the tumour milieu through a CCR6/CCL20-dependent mechanism. Furthermore, we showed that the proliferation and angiogenesis of HNSCC are impaired in the presence of Th17 cells.Conclusion:We conclude that Th17 cells have a substantial impact on the carcinogenesis of HNSCCs and on their metastasis and could serve as a potential therapeutic target to modulate anti-tumour response in HNSCC.

Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics-based technology.

We describe AMIDA (autoantibody-mediated identification of antigens), a novel target identification technology based on the immunoprecipitation of disease-specific antigens by autologous serum antibodies followed by two-dimensional electrophoretic separation, and their identification via mass spectrometry. Twenty-seven potential carcinoma antigens were identified including proteins of hitherto unknown function. Validation of one of the identified antigens, cytokeratin 8, revealed its de novo expression in hyperplastic tissue, gradual overexpression with increasing malignancy, and ectopic localization on the cell surface. Furthermore, a strong prevalence of CK8-specific antibodies occurred in the serum of cancer patients already at early disease stages. In situ hybridization for one marker of unknown function, KIAA1273/TOB3, demonstrated its strong overexpression in head and neck carcinomas, thus making it a likely tumor antigen candidate. Eventually, AMIDA could foster significant improvements for the diagnosis and therapy of human diseases eliciting a humoral immune response, and allows for the rapid identification of new target molecules.

Impaired monocyte function in cancer patients: restoration with a cyclooxygenase-2 inhibitor

Epidemiological data and animal models have provided evidence that nonsteroidal antiinflammatory drugs (NSAIDs) have an anticancer effect. However, the molecular mechanisms underlying these antineoplastic effects are not well understood. We described previously that expression levels of the chemokine receptor, CCR5, and the β2‐integrin, Mac‐1, were down‐regulated on primary monocytes after incubation in supernatants from human carcinoma cell lines, and that this down‐regulation resulted in impaired monocyte function with respect to migration and adhesion. We now demonstrate that these impairments are also present in vivo. Monocytes from cancer patients displayed significantly reduced CCR5 levels and migration capacities in comparison to cells from healthy donors. Because migration is necessary for the antitumor activity of monocytes/macrophages, these deficits may contribute to the suppressed immune system seen in cancer patients. In a clinical study, we analyzed the effect of a selective COX‐2 inhibitor, Rofecoxib, on the migration of monocytes derived from cancer patients. The results revealed significant improvement in migration equal to those levels seen in healthy donors. We conclude that in patients with cancer, the intake of Rofecoxib for 3 wk leads to significant restoration of monocyte function. These data may, at least in part, help explain the anticancer effects of NSAIDs.