Hubertus Schmitz-Winnenthal

Low-Dose Irradiation Programs Macrophage Differentiation to an iNOS+/M1 Phenotype that Orchestrates Effective T Cell Immunotherapy

Inefficient T cell migration is a major limitation of cancer immunotherapy. Targeted activation of the tumor microenvironment may overcome this barrier. We demonstrate that neoadjuvant local low-dose gamma irradiation (LDI) causes normalization of aberrant vasculature and efficient recruitment of tumor-specific T cells in human pancreatic carcinomas and T-cell-mediated tumor rejection and prolonged survival in otherwise immune refractory spontaneous and xenotransplant mouse tumor models. LDI (local or pre-adoptive-transfer) programs the differentiation of iNOS⁺ M1 macrophages that orchestrate CTL recruitment into and killing within solid tumors through iNOS by inducing endothelial activation and the expression of TH1 chemokines and by suppressing the production of angiogenic, immunosuppressive, and tumor growth factors.

Inactivating calcium-sensing receptor mutations in patients with primary hyperparathyroidism

Objective  Primary hyperparathyroidism (HPT) is characterised by autonomous secretion of PTH from enlarged parathyroid glands leading, in most patients, to asymptomatic hypercalcaemia. Familial hypocalciuric hypercalcaemia (FHH) is an autosomal dominant disorder caused by inactivating mutations in the calcium‐sensing receptor (CaSR) gene; it is characterised by lifelong and usually asymptomatic hypercalcaemia. Establishing the correct diagnosis is important because surgery can be curative in HPT, but ineffective in FHH. There is overlap in the diagnostic criteria for the two disorders and some patients carrying inactivating mutations in the CaSR gene, which is suggestive of FHH, also have HPT with hyperplastic parathyroid glands or adenomas.

Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Low doses of gamma irradiation potentially modifies immunosuppressive tumor microenvironment by retuning tumor-associated macrophages: lesson from insulinoma

Tumor infiltrating iNOS+ macrophages under the influence of immunosuppressive tumor microenvironment gets polarized to tumor-promoting and immunosuppressive macrophages, known as tumor-associated macrophages (TAM). Their recruitment and increased density in the plethora of tumors has been associated with poor prognosis in cancer patients. Therefore, retuning of TAM to M1 phenotype would be a key for effective immunotherapy. Radiotherapy has been a potential non-invasive strategy to improve cancer immunotherapy and tumor immune rejection. Irradiation of late-stage tumor-bearing Rip1-Tag5 mice twice with 2 Gy dose resulted in profound changes in the inflammatory tumor micromilieu, characterized by induction of M1-associated effecter cytokines as well as reduction in protumorigenic and M2-associated effecter cytokines. Similarly, in vitro irradiation of macrophages with 2 Gy dose-induced expression of iNOS, NO, NFκBpp65, pSTAT3 and proinflammatory cytokines secretion while downregulating p38MAPK which are involved in iNOS translation and acquisition of an M1-like phenotype. Enhancement of various M2 effecter cytokines and angiogenic reprogramming in iNOs+ macrophage depleted tumors and their subsequent reduction by 2 Gy dose in Rip1-Tag5 transgenic mice furthermore demonstrated a critical role of peritumoral macrophages in the course of gamma irradiation mediated M1 retuning of insulinoma.

The clinical implications and biologic relevance of neurofilament expression in gastroenteropancreatic neuroendocrine neoplasms.

Although gastroenteropancreatic neuroendocrine neoplasms (GEP‐NENs) exhibit widely divergent behavior, limited biologic information (apart from Ki‐67) is available to characterize malignancy. Therefore, the identification of alternative biomarkers is a key unmet need. Given the role of internexin alpha (INA) in neuronal development, the authors assessed its function in neuroendocrine cell systems and the clinical implications of its expression as a GEP‐NEN biomarker.

Stathmin in pancreatic neuroendocrine neoplasms: a marker of proliferation and PI3K signaling

Chromosome 1p35-36, which encodes tumor suppressors and mitotic checkpoint control genes, is commonly altered in human malignancies. One gene at this locus, stathmin 1 (STMN1), is involved in cell cycle progression and metastasis. We hypothesized that increased STMN1 expression may play a role in pancreatic neuroendocrine neoplasm (pNEN) malignancy. We investigated stathmin copy number variation, mRNA, and protein expression using PCR-Taqman Copy Number Assays, Q-PCR, Western blot, and immunohistochemistry. A mechanistic role for stathmin in proliferation was assessed in the BON cell line under growth-restrictive conditions and siRNA silencing. Furthermore, its role in PI3K signaling pathway activation was evaluated using pharmacological inhibitors. mRNA (p = 0.0001) and protein (p < 0.05) were overexpressed in pNENs. Expression was associated with pNEN tumor extension (p < 0.05), size (p < 0.01), and Ki67 expression (p < 0.01). Serum depletion decreased Ki67 expression (p < 0.01) as well as Ser38 phosphorylation (p < 0.05) in BON cells. STMN1 knockdown (siRNA) decreased proliferation (p < 0.05), and PI3K inhibitors directly inhibited proliferation via stathmin inactivation (dephosphorylation p < 0.01). We identified that stathmin was overexpressed and associated with pathological parameters in pancreatic NENs. We postulate that STMN1 overexpression and phosphorylation result in a loss of cell cycle mitotic checkpoint control and may render tumors amenable to PI3K inhibitory therapy.

Concomitant Tumor and Autoantigen Vaccination Supports Renal Cell Carcinoma Rejection

Efficient tumor vaccination frequently requires adjuvant. Concomitant induction of an autoimmune response is discussed as a means to strengthen a weak tumor Ag-specific response. We asked whether the efficacy of dendritic cell (DC) vaccination with the renal cell carcinoma Ags MAGE-A9 (MAGE9) and G250 could be strengthened by covaccination with the renal cell carcinoma autoantigen GOLGA4. BALB/c mice were vaccinated with DC loaded with MHC class I-binding peptides of MAGE9 or G250 or tumor lysate, which sufficed for rejection of low-dose RENCA-MAGE9 and RENCA-G250 tumor grafts, but only retarded tumor growth at 200 times the tumor dose at which 100% of animals will develop a tumor. Instead, 75–100% of mice prevaccinated concomitantly with Salmonella typhimurium transformed with GOLGA4 cDNA in a eukaryotic expression vector rejected 200 times the tumor dose at which 100% of animals will develop tumor. In a therapeutic setting, the survival rate increased from 20–40% by covaccination with S. typhimurium-GOLGA4. Autoantigen covaccination significantly strengthened tumor Ag-specific CD4+ and CD8+ T cell expansion, particularly in peptide-loaded DC-vaccinated mice. Covaccination was accompanied by an increase in inflammatory cytokines, boosted IL-12 and IFN-γ expression, and promoted a high tumor Ag-specific CTL response. Concomitant autoantigen vaccination also supported CCR6, CXCR3, and CXCR4 upregulation and T cell recruitment into the tumor. It did not affect regulatory T cells, but slightly increased myeloid-derived suppressor cells. Thus, tumor cell eradication was efficiently strengthened by concomitant induction of an immune response against a tumor Ag and an autoantigen expressed by the tumor cell. Activation of autoantigen-specific Th cells strongly supports tumor-specific Th cells and thereby CTL activation.

CD14/TLR4 priming potentially recalibrates and exerts anti-tumor efficacy in tumor associated macrophages in a mouse model of pancreatic carcinoma.

Pancreatic cancer is the fourth major cause of cancer related deaths in the world and 5 year survival is below 5%. Among various tumor directed therapies, stimulation of Toll-like receptors (TLR) has shown promising effects in various tumor models. However, pancreatic cancer cells frequently express these receptors themselves and their stimulation (TLR 2 and/or 4 particularly) within tumor microenvironment is known to potentially enhance tumor cell proliferation and cancer progression. Consistent stimulation of tumor associated macrophages (TAMs), in particular with tumor derived TLR ligand within the tumor microenvironment promotes cancer related inflammation, which is sterile, non-immunogenic and carcinogenic in nature. In view of this, recalibrating of TAM has the potential to induce immunogenic inflammation. Consistent with this, we provide experimental evidence for the first time in this study that priming of TAMs with TLR4 ligend (LPS) alone or in combination with IFN-γ not only recalibrates pancreatic tumor cells induced M2 polarization, but also confers anti-tumor potential in TAMs. Most interestingly, reduced tumor growth in macrophage depleted animals suggests that macrophage directed approaches are important for the management of pancreatic tumors.

Maximizing time from the constraining European Working Time Directive (EWTD): The Heidelberg New Working Time Model

BackgroundThe introduction of the European Working Time Directive (EWTD) has greatly reduced training hours of surgical residents, which translates into 30% less surgical and clinical experience. Such a dramatic drop in attendance has serious implications such compromised quality of medical care. As the surgical department of the University of Heidelberg, our goal was to establish a model that was compliant with the EWTD while avoiding reduction in quality of patient care and surgical training.MethodsWe first performed workload analyses and performance statistics for all working areas of our department (operation theater, emergency room, specialized consultations, surgical wards and on-call duties) using personal interviews, time cards, medical documentation software as well as data of the financial- and personnel-controlling sector of our administration. Using that information, we specifically designed an EWTD-compatible work model and implemented it.ResultsSurgical wards and operating rooms (ORs) were not compliant with the EWTD. Between 5 pm and 8 pm, three ORs were still operating two-thirds of the time. By creating an extended work shift (7:30 am-7:30 pm), we effectively reduced the workload to less than 49% from 4 pm and 8 am, allowing the combination of an eight-hour working day with a 16-hour on call duty; thus, maximizing surgical resident training and ensuring patient continuity of care while maintaining EDTW guidelines.ConclusionA precise workload analysis is the key to success. The Heidelberg New Working Time Model provides a legal model, which, by avoiding rotating work shifts, assures quality of patient care and surgical training.

Rupture without warning.

In April, 2010, a 58-year-old woman with an endemic goitre presented with an acute swelling of her neck with dyspnoea, dysphagia, and headache. She was from an iodine-defi cient area of Germany, and had no history of neck trauma or infectious diseases. On examination she was in respiratory distress and had elevated blood pressure (160/90 mm/Hg). Her temperature, pulse rate, and electro cardiogram were normal. Blood tests showed normal haemoglobin concentration and thyroid function. Chest radiography showed displacement of the trachea to the left. CT was done to exclude vascular lesions and showed a large right soft-tissue mass measuring 9×10 cm that was compressing the laryngeal region (fi gure A) as well as causing the tracheal deviation. After intubation complicated by progressive swelling, the patient was taken to the operating theatre. Surgical exploration showed a haematoma in the infrahyoid muscles, which was evacuated. After exposure and clipping of the thyroid upper pool arteries, total thyroidectomy was done. The right thyroid lobe had ruptured and there was active bleeding consistent with a venous origin. She was admitted to the intensive care unit for 24 h, and after fi ve days, she had fully recovered and had normal recurrent laryngeal nerve function and calcium concentrations. She was discharged on sub stitution levothyoxine sodium 100 μg once daily. Histopathology showed a microfollicular adenoma with cystic transformations (fi gure B). Rupture of haemorrhagic cysts in the thyroid gland was fi rst described by Bradley in 1896. In 1932, McGregor and Cornett reported on a series of 18 patients (14 women) with spontaneous cyst haemorrhages in 2500 thyroid operations done under local anaesthesia by Schwoeber and colleagues between 1906 and 1923. Five of the 18 patients died. Few cases of a spontaneously ruptured thyroid gland cyst with notable haemorrhage have been reported since then. Spontaneous ruptures of thyroid glands are likely to occur only in the presence of a preexisting condition such as multinodular goitre, thyroid cysts, or, as in our patient, an undiagnosed adenoma. The walls of the adenomas are very fragile due to degeneration, and they also contain many immature blood vessels. 35% of the world’s population have iodine defi ciency (urinary iodine excretion <100 μg/L). Although Germany is not considered an iodine-defi cient country overall, adenomatous endemic goitres can develop because of regional diff erences. Although the cause of our patient’s rapidly developing dyspnoea was unknown, the acute management issue was to protect her airway. As reported in studies on penetrating neck trauma, CT or CT angiography is the standard diagnostic tool. Rapid diagnosis is important so that life-threatening conditions, such as vascular lesions, can be excluded. Surgery remains the standard treatment and, to date, only a few cases of conservatively treated thyroid gland ruptures have been reported. The largest conservatively treated haematoma had a diameter of 5 cm. Usually, the aff ected lobe has to be removed. Control of the arteries is mandatory with protection of parathyroid glands and the recurrent laryngeal nerve. Calcium should be monitored postoperatively due to potential for a parathyroid injury and substitution of levothyoxine should be considered. Although rupture of a thyroid gland is rare, medical practitioners should be aware of the risk in patients with thyroid cysts or adenomatous goitre so that rapid diagnosis and appropriate surgical intervention can be achieved.