Experimental studies and simple numerical modeling of underwater electric discharges.

At present, underwater electric pulsed discharges are used in a wide range of modern applications. During the development of a system for generating underwater acoustic pressure pulses, a numerical model is an essential tool for guiding the design and interpreting the data. Developing a complex one-dimensional numerical code, like those presented in the literature, requires a substantial dedicated effort. Unfortunately, previous work trying to use simple and elegant theoretical models developed many decades ago reported a fundamental issue, apparently related to the input data. The present work performs a detailed analysis of the real meaning of the voltage measured across an underwater discharge and clarifies the correct way the power input to a simple two-phase model should be calculated. Based on accurate measurements, a phenomenological methodology to obtain the input data is demonstrated, with theoretical predictions obtained from the simple two-phase model being successfully compared with the experimental evidence obtained from both the present work as well as from other reliable data presented in the literature.

A Supersonic Underwater Discharge as a High-Power Ultrasound Source.

A supersonic underwater discharge system, driven by a pulsed power generator with 235 ns voltage rise time, was developed to be used as a powerful ultrasound source. The article presents details of the system's components and the various diagnostic methods used, together with the main findings obtained during the first experimental campaign. The system generated a peak pressure of 184 kPa at 1-m distance, with an efficiency of energy conversion from electrical to acoustic estimated as 0.8%. The pressure profile was found to display a resemblance to the radiation pattern generated by a dipole antenna. Using an ultrahigh-speed camera, a study of the interelectrode discharge revealed details of the prebreakdown streamer dynamics and an estimate for the lifetime of the postbreakdown plasma column. The way forward includes testing the system at a very high repetition rate.

Phase II trial of ipilimumab in melanoma patients with preexisting humoural immune response to NY-ESO-1.

BACKGROUND: Immune checkpoint therapy has dramatically changed treatment options in patients with metastatic melanoma. However, a relevant part of patients still does not respond to treatment. Data regarding the prognostic or predictive significance of preexisting immune responses against tumour antigens are conflicting. Retrospective data suggested a higher clinical benefit of ipilimumab in melanoma patients with preexisting NY-ESO-1-specific immunity. PATIENTS AND METHODS: Twenty-five patients with previously untreated or treated metastatic melanoma and preexisting humoural immune response against NY-ESO-1 received ipilimumab at a dose of 10 mg/kg in week 1, 4, 7, 10 followed by 3-month maintenance treatment for a maximum of 48 weeks. Primary endpoint was the disease control rate (irCR, irPR or irSD) according to immune-related response criteria (irRC). Secondary endpoints included the disease control rate according to RECIST criteria, progression-free survival and overall survival (OS). Humoural and cellular immune responses against NY-ESO-1 were analysed from blood samples. RESULTS: Disease control rate according to irRC was 52%, irPR was observed in 36% of patients. Progression-free survival according to irRC was 7.8 months, according to RECIST criteria it was 2.9 months. Median OS was 22.7 months; the corresponding 1-year survival rate was 66.8%. Treatment-related grade 3 AEs occurred in 36% with no grade 4-5 AEs. No clear association was found between the presence of NY-ESO-1-specific cellular or humoural immune responses and clinical activity. CONCLUSION: Ipilimumab demonstrated clinically relevant activity within this biomarker-defined population. NY-ESO-1 positivity, as a surrogate for a preexisting immune response against tumour antigens, might help identifying patients with a superior outcome from immune checkpoint blockade. CLINICAL TRIAL INFORMATION: NCT01216696.

A modified surgical technique for inferior alveolar nerve repositioning on severely atrophic mandibles: case series of 11 consecutive surgical procedures.

BACKGROUND: To emphasize the characteristics and possible pitfalls of nerve reposition in cases of severe bone resorption in the posterior mandibular area, and to modify hard- and soft-tissue manipulation accordingly. METHODS: We analyzed retrospectively, 7 patients in which we performed full arch lower jaw rehabilitation. The patients presented for oral rehabilitation having a minimal residual bone above the mandibular canal and had undergone inferioral veolar nerve (IAN) displacement with modified surgical technique for fixed prosthetic rehabilitation. RESULTS: Eleven procedures of nerve repositioning were performed on severely atrophic mandibles. The average age of the patients was 43.29 years (12.37 SD). Residual bone above the mental foramen ranged between 0.5 mm and 1.5 mm, with an average of 0.93 mm (0.35 SD). In total, 32 dental implants were inserted into the area simultaneously with nerve displacement. The average follow-up time was 35.71 months(41.75 SD), ranging between 7 and 120 months. CONCLUSIONS: Severe atrophic cases require special attention due to the loss of keratinized tissue around the crestal area.The use of a modified surgical approach and specific surgical instruments provides a safer working environment for the operator and ensures optimal results.

Investigation of the influence of different physico-chemical parameters upon the susceptibility of planktonic and adherent Escherichia coli cells to beta-lactams and quinolones.

The purpose of this study was to evaluate the influence of different physico-chemical parameters on Escherichia coli susceptibility to ceftriaxone (CRO), cefotaxime (CTX), imipenem (IMP), and nalidixic acid (as marker for resistance by impermeability). The influence of chemical composition of culture medium was evaluated by the comparative assessment of inhibition growth diameters on different solid media: Mueller Hinton Medium (MH), Plate Count Agar Medium (PCA), MacConkey Medium (MC) and Eosin Methylen Blue Medium (EMB). In order to evaluate the differences in antibiotic susceptibility between the biofilm embedded and planktonic cells, an original, simple experimental model was used, by including the bacterial cells in an agar layer, mimicking the biofilm matrix. Our results demonstrated that the inhibition diameter zone was much larger on PCA, EMB and MC than on MH, considered as general standard medium for the antibiosusceptibility testings (CLSI). When bacterial cells were included in the agar matrix, the growth inhibition diameters obtained for different beta-lactams proved to be different of planktonic cells, i.e.: for CTX, a narrow inhibition diameter was obtained, demonstrating the low efficiency of this antibiotic in the treatment of biofilm associated infections, whereas the CRO proved the same efficiency against planktonic as well as to agar embedded bacteria. The different susceptibility results obtained for the cells embedded in the agar matrix by an adapted disk diffusion method are pleading for the necessity to assess new adapted standard methods and specific parameters in the purpose to determine the antibiotic resistance of bacterial cells isolated from biofilm associated infections.

Balancing the role of the dental school in teaching, research and patient care; including care for underserved areas.

Inequalities within dentistry are common and are reflected in wide differences in the levels of oral health and the standard of care available both within and between countries and communities. Furthermore there are patients, particularly those with special treatment needs, who do not have the same access to dental services as the general public. The dental school should aim to recruit students from varied backgrounds into all areas covered by the oral healthcare team and to train students to treat the full spectrum of patients including those with special needs. It is essential, however, that the dental student achieves a high standard of clinical competence and this cannot be gained by treating only those patients with low expectations for care. Balancing these aspects of clinical education is difficult. Research is an important stimulus to better teaching and better clinical care. It is recognized that dental school staff should be active in research, teaching, clinical work and frequently administration. Maintaining a balance between the commitments to clinical care, teaching and research while also taking account of underserved areas in each of these categories is a difficult challenge but one that has to be met to a high degree in a successful, modern dental school.

Specific immune recognition of pancreatic carcinoma by patient-derived CD4 and CD8 T cells and its improvement by interferon-gamma.

Pancreatic carcinoma is a very aggressive disease and little is known about its immunobiology. We here describe the presence in pancreatic cancer patients of spontaneously induced functional CD4 and CD8 memory/effector T cells reactive to autologous tumor cells or to the pancreatic cancer associated antigen, MUC-1. Such specific cells were present in the bone marrow or peripheral blood of most of the 23 tested patients. Low dose stimulation of primary cultures of pancreatic cancer cells with 500 IU/ml IFN-gamma for 72 h enhanced HLA-I expression and induced the de novo expression of HLA-II molecules. This led to a much better immune recognition by autologous HLA-I restricted and purified CD8 T cells and allowed tumor cell recognition by HLA-II restricted purified CD4 T-helper cells. Thus, interferon-gamma appears to be a useful adjuvant cytokine to enhance the immunogenicity of a patients' tumor cells and their recognition by tumor reactive immune cells.

Contents of stomatological curricula in Europe.

The present study focuses on dental education in the Accession Countries to the European Union. Comparisons were made with data from EU dental schools [Eur J Dent Educ 1 (1997) 35]. The findings show a large variation in the hours allocated to individual subjects, medical and dental, both within and between AC and EU dental schools. Stomatology derived from General Medicine and the stomatologist is viewed as a doctor responsible for one part of the body. This was explained by the large proportion of time dedicated to medical subjects, especially in the first 2/3 years of the undergraduate curriculum. The percentage of hours for dental sciences varied inversely to those for bio-medical sciences and increase continuously from the first year to the final year. Curricula in the Stomatological schools tend to have a discipline-structured approach, generally utilising a large number of individual departments, resulting in a multitude of subjects being taught. Curricular extensions from 5 to 6 years were introduced in some schools from 1990 onwards in order to accommodate new dental subjects.

Towards higher standards in dental and stomatological education in Europe.

In the creation of the European Union, attention was given to portability of licensure for professionals. Considerable differences exist among countries in culture, economic conditions, and educational resources and practices. In dentistry, these differences in professional training have been addressed through a peer consultative process rather than through political and legal means. The process of visits to dental schools throughout Europe and the organizational structure (DentEd) used to conduct the visits and summarize findings are described.

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release.

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.

Breaking tolerance to a tumor-associated viral superantigen as a basis for graft-versus-leukemia reactivity.

A major goal in tumor immunotherapy consists of breaking potential tumor-specific T-cell unresponsiveness (tolerance), which may explain tumor growth in cancer patients. We report that immunological tolerance to a tumor-associated viral superantigen (SAg) is overcome in a mouse lymphoma model by transfer of allogeneic T cells expressing SAg-reactive Vbeta6 T-cell receptor chains. Surprisingly, upon contact with SAg-expressing lymphoma cells, Vbeta6 T cells became activated rather than tolerized (as reported previously). They also developed SAg-specific cytotoxic T-lymphocyte activity and secreted IL-2 and IFN-gamma. The grafted T cells infiltrated liver metastases, formed close contact with SAg-expressing tumor cells, and caused significant graft-vs. -leukemia (GvL) effects. Selection for tumor resistance among the progeny from a cross between SAg-negative donor and SAg- positive recipient strains revealed a strict correlation between loss of the endogenous SAg tolerogen, rescue of Vbeta6 T cells from SAg-mediated deletion, and leukemia resistance. These findings suggest that immune responses to SAg can be exploited to break tolerance and augment immune responses to tumors.

Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis.

We have previously reported that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through mitochondrial damage (including degradation of cardiolipin, a major mitochondrial lipid) followed by activation of caspases. Here we demonstrate that Jurkat human leukemia cells which survive after 24 h treatment with NO form subpopulations with higher and lower cardiolipin content (designated as NAO(high) and NAO(low), respectively). Sorted NAO(high) cells were found to survive in culture whereas sorted NAO(low) cells died. Moreover, NAO(high) cells acquired an increased resistance to the exposure to NO donors which remained unchanged during long-term culture. These cells showed a similar cardiolipin content and expressed the same level of anti-apoptotic proteins Bcl-2 and Bcl-x(L) as APO-S unsorted cells but contained significantly higher concentration of the antioxidant glutathione. Depletion of glutathione in these cells with buthionine-sulfoximine (BSO) correlated with a significant stimulation of NO-mediated apoptosis whereas the exposure of NO-sensitive APO-S cells to the glutathione precursor N-acetylcysteine (NAC) resulted in a substantial suppression of this effect. Our data suggest a complex mechanism of the resistence to NO-induced apoptosis in Jurkat human leukemia cells in which glutathione plays an important role.

Nitric oxide-mediated apoptosis in human breast cancer cells requires changes in mitochondrial functions and is independent of CD95 (APO-1/Fas).

We have previously shown that nitric oxide (NO) induces apoptosis in different human neoplastic lymphoid cells through caspase activation. Here we studied the NO-mediated apoptosis in human breast cancer cell lines derived from primary tumor (BT-20) or from metastasis (MCF-7). NO donor glycerol trinitrate (GTN) induced apoptosis in both cell lines which was completely abrogated after pretreatment with the broad spectrum caspase inhibitor zVAD-fmk. NO triggered also a time-dependent activation of caspase-1, caspase-3, and caspase-6 in these cells. Moreover, NO caused a release of mitochondrial protein cytochrome c into the cytosol, an increase in the number of cells with low mitochondrial transmembrane potential and with high level of reactive oxygen species production. However, NO did not induce mRNA expression of CD95 (APO-1/Fas) ligand. FAS-associated phosphatase-1 (FAP-1) molecule was constitutively expressed at the mRNA level and did not show any changes upon NO treatment in both breast cancer cell lines. The expression of the pro-apoptotic protein Bax and of the anti-apoptotic protein Bcl-2 remained unchanged in MCF-7 and BT-20 cells upon GTN treatment. We suggest that the mechanism of NO-mediated activation of the caspase cascade and subsequent apoptosis in human breast cancer cells required mitochondrial damage (in particular, cytochrome c release, disruption of mitochondrial transmembrane potential and generation of reactive oxygen species) but not the activation of the CD95/CD95L pathway.

Loss of endogenous mouse mammary tumor virus superantigen increases tumor resistance.

From a cross between a tumor-susceptible mouse strain (DBA/2; D) and a tumor-resistant MHC-identical strain (B10.D2; D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. Since resistance to the highly metastatic DBA/2 lymphoma variant ESb had an immunologic basis, and the two parental strains differed in endogenous viral superantigens (vSAGs), DNA of three D2 x D recombinant inbred mouse lines was typed for endogenous mouse mammary tumor viruses using mouse mammary tumor virus long terminal repeat- and env gene-specific probes. The resistant D2 x D mice were very similar to the susceptible parental strain D in their Mtv Southern blots, except for the lack of a single band corresponding to Mtv-7, the provirus coding for the strong DBA/2 superantigen Mls-1a. A backcross analysis revealed that Mtv-7-negative F2 mice were significantly more resistant than Mtv-7-positive F2 mice. When Mtv-7 was reintroduced into the resistant lines by crossing them with either CBA/J or BALB/D2.Mls-1a, the mice became again more tumor susceptible. Finally, we demonstrate the ability to transfer immunoresistance and graft-vs-leukemia reactivity from tumor-resistant to tumor-susceptible mice.

Caspase activation is required for nitric oxide-mediated, CD95(APO-1/Fas)-dependent and independent apoptosis in human neoplastic lymphoid cells.

Nitric oxide (NO), an important effector molecule involved in immune regulation and host defense, was shown to induce apoptosis in lymphoma cells. In the present report the NO donor glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. The ability of NO to induce apoptosis was completely blocked by a broad-spectrum ICE (interleukin-1beta converting enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein synthesis cycloheximide. Our results were confirmed using an in vitro experimental model of coculture of human lymphoid target cells with activated bovine endothelial cells generating NO as effectors. Furthermore, the inhibition of endogenous NO production with the inducible NO synthase inhibitor NG-monomethyl-L-arginine caused a complete abrogation of the apoptotic effect. Our data provide evidence that NO-induced apoptosis in human neoplastic lymphoid cells strictly requires activation of caspases, in particular FLICE, the most CD95 receptor-proximal caspase. Depending on the cell line tested this activation required or was independent of the CD95 receptor/ligand system.

Activated endothelial cells induce apoptosis in lymphoma cells.

We have previously shown that the arrest or regression of mouse liver metastases formed by lacZ-transduced ESbL T lymphoma cells (ESbL-lacZ) is associated with the stimulation of nitric oxide (NO) production by liver endothelial cells in sis. Here we studied in vitro the NO-mediated cytotoxicity against ESbL-lacZ target cells using well-characterized bovine endothelial cells (BEG) as effector cells. It was found that the co-culture of BEC with human TNF-alpha caused an increase of NO synthesis which could be completely blocked by the treatment with inducible NO synthase (iNOS) inhibitor N-G-monomethyl-L-arginine (NMMA). Incubation of activated BEC with metastatic lymphoma cells led to the death of the latter cells as evidenced by staining with propidium iodide and FAGS analysis. This cytotoxicity was considerably reduced after pretreatment of BEC with NMMA. Cytotoxic effects were also demonstrated after incubation of tumor cells with NO donor glycerol trinitrate (GTN). Non-activated BEC were not able to produce NO and showed a substantially lower level of cytotoxicity. The anti-tumor cytotoxicity exerted by activated BEC includes the stimulation of apoptosis in metastatic lymphoma cells which is mediated to a large extent by NO. These data reveal a novel role of endothelial cells in the elimination of metastatic cells through the induction of programmed cell death.

Dissection of tumour and host cells from target organs of metastasis for testing gene expression directly ex vivo.

We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells (lymphocytes, macrophages, endothelial cells) from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity (adhesion, costimulatory and cytotoxic molecules, cytokines, etc.) at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy.

Discriminative features of alcoholics classified according to family history.

By means of discriminant analysis, the frequently proposed distinction of alcoholics according to family history of alcoholism was tested. The most powerful discriminative factors were dysfunctional attitudes, some particular personality characteristics, and perceived parental rearing patterns. The results lend support to the assumption to regard alcoholics with a positive family history of alcoholism as a homogenous subgroup characterized by a specific etiopathogenesis.