Structural flexibility versus rigidity of the aromatic unit of DNA ligands: binding of aza- and azoniastilbene derivatives to duplex and quadruplex DNA.

The known azastilbene (E)-1,2-di(quinolin-3-yl)ethane (2a) and the novel azoniastilbene derivatives (E)-2-(2-(naphthalen-2-yl)vinyl)quinolizinium (2b) and (E)-3,3'-(ethane-1,2-diyl)bis(1-methylquinolinin-1-ium) (2c) were synthesized. Their interactions with duplex and quadruplex DNA (G4-DNA) were studied by photometric, fluorimetric, polarimetric and flow-LD analysis, and by thermal DNA denaturation studies, as well as by 1H-NMR spectroscopy. The main goal of this study was a comparison of these conformationally flexible compounds with the known G4-DNA-binding diazoniadibenzo[b,k]chrysenes, that have a comparable π-system extent, but a rigid structure. We have observed that the aza- and azoniastilbene derivatives 2a-c, i.e. compounds with almost the same spatial dimensions and steric demand, bind to DNA with an affinity and selectivity that depends significantly on the number of positive charges. Whereas the charge neutral derivative 2a binds unspecifically to the DNA backbone of duplex DNA, the ionic compounds 2b and 2c are typical DNA intercalators. Notably, the bis-quinolinium derivative 2c binds to G4-DNA with moderate affinity (Kb = 4.8 × 105 M-1) and also stabilizes the G4-DNA towards thermal denaturation (ΔTm = 11 °C at ligand-DNA ratio = 5.0). Strikingly, the corresponding rigid counterpart, 4a,12a-diazonia-8,16-dimethyldibenzo[b,k]chrysene, stabilizes the G4-DNA to an even greater extent under identical conditions (ΔTm = 27 °C). These results indicate that the increased flexibility of a G4-DNA ligand does not necessarily lead to stronger interactions with the G4-DNA as compared with rigid ligands that have essentially the same size and π system extent.

Author Correction: Yolk sac macrophage progenitors traffic to the embryo during defined stages of development.

This article contains errors in Figs. 5 and 6, for which we apologize. In Fig. 5f, the image 'E12.5 tail' was inadvertently replaced with a duplicate of the image 'E12.5 trunk' from the same panel. In Figure 6d, the image 'E9.5/OH-TAM E8.5, embryo' was inadvertently replaced with a duplicate of the image 'E10.5/ OH-TAM E8.5, embryo' from Fig. 6b. The corrected versions of these figures appear in the Author Correction associated with this Article.

Inducible disruption of the c-myb gene allows allogeneic bone marrow transplantation without irradiation.

Allogeneic bone marrow (BM) transplantation enables the in vivo functional assessment of hematopoietic cells. As pre-conditioning, ionizing radiation is commonly applied to induce BM depletion, however, it exerts adverse effects on the animal and can limit experimental outcome. Here, we provide an alternative method that harnesses conditional gene deletion to ablate c-myb and thereby deplete BM cells, hence allowing BM substitution without other pre-conditioning. The protocol results in a high level of blood chimerism after allogeneic BM transplantation, whereas immune cells in peripheral tissues such as resident macrophages are not replaced. Further, mice featuring a low chimerism after initial transplantation can undergo a second induction cycle for efficient deletion of residual BM cells without the necessity to re-apply donor cells. In summary, we present an effective c-myb-dependent genetic technique to generate BM chimeras in the absence of irradiation or other methods for pre-conditioning.

Yolk sac macrophage progenitors traffic to the embryo during defined stages of development.

Tissue macrophages in many adult organs originate from yolk sac (YS) progenitors, which invade the developing embryo and persist by means of local self-renewal. However, the route and characteristics of YS macrophage trafficking during embryogenesis are incompletely understood. Here we show the early migration dynamics of YS-derived macrophage progenitors in vivo using fate mapping and intravital microscopy. From embryonic day 8.5 (E8.5) CX3CR1+ pre-macrophages are present in the mouse YS where they rapidly proliferate and gain access to the bloodstream to migrate towards the embryo. Trafficking of pre-macrophages and their progenitors from the YS to tissues peaks around E10.5, dramatically decreases towards E12.5 and is no longer evident from E14.5 onwards. Thus, YS progenitors use the vascular system during a restricted time window of embryogenesis to invade the growing fetus. These findings close an important gap in our understanding of the development of the innate immune system.

Video-assisted thoracic surgery (VATS) evaluation of intrathoracic disease in patients with FIGO III and IV stage ovarian cancer.

OBJECTIVES: The aim of this study was to assess the influence of video-assisted thoracic surgery (VATS) on our treatment decisions in FIGO III and IV ovarian cancer patients. METHODS: Patients with ovarian cancer and suspected supra-diaphragmatic involvement (pleural effusions, pleural carcinomatosis, lung metastasis, or enlarged supra-diaphragmatic lymph nodes) at chest computer tomography (CT) scan underwent VATS with or without laparoscopy (LSC) to decide for primary cytoreduction or neoadjuvant chemotherapy. Operation time, VATS complications (intrapleural hematoma, secondary hemorrhage with intervention, pneumonia and empyema) and shift in the therapeutic strategy due to VATS were evaluated. RESULTS: 17 patients were included into this study (1 patient with FIGO stage IIIb, 1 with IIIc and 15 with stage IV). The median operation time for VATS only was 46.5 min (range: 20-50 min, n=3). Perioperatively, no complications occurred. After surgical staging, the tumor was confined to the abdomen in four patients in whom primary cytoreduction was attempted. All other 13 patients underwent neoadjuvant chemotherapy. VATS altered the therapeutic management in 6/17 ovarian cancer patients (3 times upstaging, 3 times downstaging). Negative predictive values (NPV) for local and diffuse pleural carcinomatosis ranged between 0.5 and 0.71. CONCLUSION: In this case series, VATS in addition to LSC showed negligible morbidity related to surgery and a short operation time. We were able to improve the accuracy of the FIGO staging and assessed operability more reliably in these patients than through imaging techniques alone.

[Isolated lymph node metastasis in pericardial fat flap after bronchial stump coverage]. / Isolierte Lymphknotenmetastase im perikardialen Fettlappen nach Bronchusstumpfdeckung.

The occurrence of bronchopleural fistulas is a serious complication after pneumonectomy because of lung cancer and additional bronchial stump coverage within right-sided and left-sided pneumonectomy therefore constitutes the operative standard. This is a case report on the early diagnosis of a lymph node metastasis within the pedicled pericardial fat flap used for bronchial stump coverage. Primary resection of the left lung was carried out 8 months previously because of cancer. Early diagnosis was possible using FDG-PET/CT in the post-operative treatment. The recurrence was successfully treated by en bloc resection and adjuvant radiation.

[Surgery of mediastinal tumors]. / Chirurgie der Mediastinaltumoren.

Thymomas, lymphomas, and germ cell tumors are the most frequent lesions of the anterior mediastinum, whereas endodermal (bronchogenic) cysts and lymphomas are the most frequent lesions of the middle mediastinum. In the posterior mediastinum, neurogenic tumors and soft-tissue sarcomas are the most frequent. Depending on tumor location, mediastinoscopy, mediastinotomy, and thoracoscopy are the preferred diagnostic methods. Surgical treatment of thymoma is the gold standard, and median sternotomy is the most frequently applied approach. The decisive prognostic and therapeutic criteria are Masaoka staging, WHO classification, and R0 status. Thoracoscopy should be performed only in patients with myasthenia gravis and with very small tumors. Surgical treatment is highly recommended in patients with locally recurrent tumors. The importance of surgical treatment of germ cell tumors is determined by a negative concentration of beta-HCG and alpha-fetoprotein and in cases of residual tumor after chemotherapy. Bronchogenic cysts always require resection because of their high complication rate (66%) after conservative treatment. In these cases complete resection is necessary due to the probability of recurrence. Ninety-eight percent of neurogenic tumors in adults are benign and usually resected via thoracoscopy or thoracotomy, depending on location and size.

[Thymoma--incidence, classification and therapy]. / Thymome--Inzidenz, Klassifikation und Therapie.

Thymomas are a rare tumor entity. However, they represent 50 % of all tumors of the anterior mediastinum. There are no specific early symptoms. Overall in 10 - 15 % of patients with myasthenia gravis a thymoma is evident. Two major classifications are relevant in clinical practise: the Masaoka-classification and the WHO-classification. For their clinical and prognostic significance both classifications should be used for patients with thymomas. Additional, only resection status (RO) is known as a significant prognostic factor. Thymomas are compulsory malignant tumors. Distant metastasis is found as well as local recurrence in all stages of the disease. The 5-year-mortality rate constitutes about 80 %, not meaning any healing because local recurrences occur as late as five years after surgery. 60 % of all patients die from tumor-independent reasons making a clear prognostic statement difficult. Surgical treatment remains the gold standard and must be performed whenever possible. The most common approach is a median sternotomy. When dealing with a thymuscarcinoma, radical lymph node dissection is advisable. With respect of treatment only adjuvant radiation can possibly improve long term survival and reduces local recurrence rates for incomplete resected patients. There is no evidence for a benefit in patients with thymoma receiving adjuvant chemotherapy. A neo-adjuvant chemotherapy in combination with an adjuvant radiotherapy improves outcome after surgical resection in stage III and IV and goes along with better survival rates. Larger studies have not been performed so far. A multimodal therapy strategy is advised when dealing with thymomas in stage III and IV.

The impact of immunological parameters on the development of phantom pain after major amputation.

OBJECTIVES: To investigate the relationship between local and systemic inflammatory markers and phantom limb pain. METHODS: In 39 consecutive patients undergoing major amputations nerve biopsies, serum and clinical data was collected. Patients were followed up for 12 months to report on the incidence and severity of phantom limb pain. RESULTS: After 12 months, 78% of the surviving patients had phantom pain, the symptom usually commencing within 14 days of operation. The severity of macrophage infiltration within the nerve biopsy was negatively correlated to the inception of phantom pain ( P = 0.026). While serum TNF-alpha concentration was positively correlated to mortality ( P = 0.021). CONCLUSIONS: The immune status existing before the amputation and the local immunological milieu influence the onset of phantom pain.

Peritoneal carcinomatosis of colorectal cancer: incidence, prognosis, and treatment modalities.

BACKGROUND AND AIMS: Intraperitoneal carcinomatosis accounts for 25-35% of recurrences of colorectal cancer. Studies demonstrate that peritoneal carcinomatosis is not necessarily a terminal condition with no options for treatment or cure. RESULTS: The combination of aggressive cytoreductive surgery and intra-abdominal hyperthermia chemotherapy improves long-term overall survival in selected patients but is a time-consuming procedure (approx. 12 h) and entails high mortality (5%) and morbidity (35%)). Most commonly used drugs are mitomycin C and platinum compounds, which have synergistic toxic effects on tumor cells when hyperthermia is applied. CONCLUSION: Since combined treatment seems promising only in peritoneal carcinomatosis stages I and II, the precondition for a reasonable combined treatment is careful staging. The mode of chemotherapy, the kind of drugs used for chemoperfusion, the timing of surgery, and the role of additional systemic chemotherapy must be evaluated in randomized studies.

DNA microarrays: a new diagnostic tool and its implications in colorectal cancer.

Effective treatment of colorectal cancer requires early detection and diagnostic and prognostic accuracy in characterizing patients of various risk groups. The development of DNA microarray makes it possible to analyze thousands of genes in a single tissue sample in one experiment and to characterize the biological behavior of colorectal cancer cells. Different cluster algorithms have been used to analyze large datasets on gene expression data, and initial results show significant differences between colorectal cancer and normal colon tissue. Although more than 6000 genes have been analyzed between colorectal cancer and normal tissue, different expression levels have been found in only 100-500 transcripts depending on the cluster algorithm. Most transcripts belong to genes involved in cell growth regulation, differentiation of cells, ribosomal proteins or metalloproteinase. A future goal in microarray technology will be the development of clustered gene chips which characterize each tumor type specifically and focus on gene expression that specifies cell identity.

[Results of laryngeal nerve monitoring during thyroid operations--Studies and value for clinical practice]. / Ergebnisse des intraoperativen Recurrensmonitoring bei der Schilddrüsenoperation - Studien und Stellenwert in der Praxis.

Injuries of the laryngeal nerve still remain a source of morbidity for patients undergoing surgery for the thyroid gland. The incidence of permanent damage of the nerve varies from 0.5 % to 2.7 % depending on the series, but for special thyroid diseases like thyroid carcinoma or goiter recurrence the damage of the recurrent laryngeal nerve could be up to 25 %. For that reason several methods of monitoring the movement of the nerve while stimulation have been proposed. Such as inserting a needle into the vocal muscles or working with a surface laryngeal electrode that is attached above the cuff of the tube. For neither of those methods of nerve monitoring severe complications were described. The purchase costs for all the equipment are between 25.000 to 30.000 DM. All the published data regarding monitoring of the laryngeal nerve have been only observation studies with no control group and for that reason the results of the studies could not be obligatory statements. Furthermore not every nerve palsy has been identified by the monitoring of the laryngeal nerve, this method has a sensitivity of less than 70 %. Monitoring of the laryngeal nerve is a helpful clinical tool not only for training of surgeons but also during complicated thyroid surgery. Monitoring is especially useful for operations of thyroid carcinoma or revision procedures. But at the moment there is no randomised study available that shows any superiority of this new and expensive method over the standard therapy, therefore further studies are necessary. Until these studies exist the anatomical localisation of the nerve during surgical dissection is still the gold standard.

[Immune tolerance and active suppression in oncology. Immunological principles and therapeutic options]. / Immuntoleranz und aktive Immunsuppression in der Onkologie. Immunologische Grundlagen und therapeutische Optionen.

In view of the fact that conventional therapies continue to yield unsatisfactory results, a therapy based on tumor immunology is of increasing interest. The results of some clinical immunotherapy studies have shown that the mechanism of tolerance induced by tumors is of critical importance. There are several mechanisms to be recognized by the tumor, such asanergy, immunosuppressive cytokines, immunodeviation and dysfunction of the T-lymphocytes. The so-called tumor escape mechanisms, which often refer to molecules that contribute to the recognition of antigens and activation of specific T cells, also lead to immunological tolerance. Immunotherapies based on gene-modified tumor cells, peptide-pulsed antigen presenting cells (APC) or antibody therapies to activate immune cells, have a fairly reasonable chance of being successful if immunological tolerance can be understood and influenced. Time will show if combining all sorts of immunotherapy that are based on different strategies, can induce tumor rejection more efficiently. In the scope of multimodal therapy concepts, immunotherapy only seems to be reasonable if the immunological tolerance that was induced by tumors can be overcome. Immunological markers are of crucial importance, not only for the therapy but also for the diagnosis and prognosis of cancer patients.

Characterization of the phenotype and function of CD8(+), alpha / beta(+) NKT cells from tumor-bearing mice that show a natural killer cell activity and lyse multiple tumor targets.

Natural Killer (NK) T cells are a specialized T cell population that co-expresses receptors of the NK lineage with the alpha / beta TCR receptor and other T cell surface markers. Their functions, regulation and relationship to other cells in the immune system are not fully understood. This report demonstrates that tumor-bearing C57BL / 6 mice have a population of NKT cells that co-express CD8 and CD161 (NK1.1) surface markers. These cells are maintained in long-term culture with T helper 2 (Th2) cytokine interleukin-4 (IL-4), but produce large amounts of Th1 cytokine interferon-gamma (IFN-gamma) following activation. NK1.1(+)CD8(+) T cells show a potent NK-like cytotoxic activity against multiple tumor targets, and lysis is independent of major histocompatibility complex (MHC)-class I or non-classical MHC-class I molecules (Qa, TL). The NK1.1(+)CD8(+) T cells express Vbeta14 chain of the TCR. These NKT cells are not CD1d restricted, and their cytotoxic activity is CD1d independent. Therefore, they represent a unique subset of T cells with an unknown restriction element which produce large quantities of IFN-gamma following expansion with IL-4. Furthermore, their cytotoxic activity is enhanced by B7 co-stimulatory molecules present on tumor cells. CD161(+) T cells that are expanded in tumor-bearing hosts may function as a part of the innate immune system with potential role(s) in tumor surveillance.

Incisional hernia repair: tensiometry for the selection of the appropriate procedure.

OBJECTIVE: To audit the early and late results of repairs of incisional hernias before and after the introduction of peroperative tensiometry. DESIGN: Retrospective study. SETTING: University hospital, Germany. PATIENTS: 675 operations on 553 patients in 18 years. INTERVENTIONS: Before we introduced tensiometry we closed 560 incisional hernias by direct suture and 63 by the inlay-onlay technique. Since we took up tensiometry the numbers were 9 and 43, respectively. MAIN OUTCOME MEASURES: Postoperative complications including recurrences. RESULTS: Recurrences developed in 246/560 (44%) after direct suture in the early series, compared with 2/9 (22%) after adoption of tensiometry. After inlay-onlay operations there were 4/63 (6%) recurrences before, and 1/43 (2%) after adoption of tensiometry. CONCLUSIONS: Tensiometry allows the surgeon to tailor his operation to the conditions that he finds during the operation.

B7-2 expressed on EL4 lymphoma suppresses antitumor immunity by an interleukin 4-dependent mechanism.

For T cells to become functionally activated they require at least two signals. The B7 costimulatory molecules B7-1 and B7-2 provide the "second signal" pivotal for T cell activation. In this report, we studied the relative roles of B7-1 and B7-2 molecules in the induction of antitumor immunity to the T cell thymoma, EL4. We generated EL4 tumor cells that expressed B7-1, B7-2, and B7-1+B7-2 by transfecting murine cDNAs. Our results demonstrate that EL4-B7-1 cells are completely rejected in syngeneic mice. Unlike EL4-B7-1 cells, we find that EL4-B7-2 cells are not rejected but progressively grow in the mice. A B7-1- and B7-2-EL4 double transfectant was generated by introducing B7-2 cDNA into the EL4-B7-1 tumor line that regressed in vivo. The EL4-B7-1+B7-2 double transfectant was not rejected when implanted into syngeneic mice but progressively grew to produce tumors. The double transfectant EL4 cells could costimulate T cell proliferation that could be blocked by anti-B7-1 antibodies, anti-B7-2 antibodies, or hCTLA4 immunoglobulin, showing that the B7-1 and B7-2 molecules expressed on the EL4 cells were functional. In vivo, treatment of mice implanted with double-transfected EL4 cells with anti-B7-2 monoclonal antibody resulted in tumor rejection. Furthermore, the EL4-B7-2 and EL4-B7-1+B7-2 cells, but not the wild-type EL4 cells, were rejected in interleukin 4 (IL-4) knockout mice. Our data suggests that B7-2 expressed on some T cell tumors inhibits development of antitumor immunity, and IL-4 appears to play a critical role in abrogation of the antitumor immune response.