Electroporation of membranes is used widely for drug delivery. Photodynamic action consists of three main steps: (A) incorporation of the sensitizer through a membrane into cells; (B) photoxidation ...of cell constituents and (C) reoxidation of the reduced sensitizer by oxygen etc. The mechanisms of (B) and (C) have been studied widely in past decades. However, the mechanism of transport (A) of sensitizers to targets as the rate limiting step has not been studied to the same extent. Therefore we applied membrane and cell wall electroporation of human histiocytic lymphoma U937 and
Saccharomyces cerevisiae cells in order to incorporate rapidly the reliable photodynamic agents thiopyronine, protoporphyrin, zinc phthalocyanine, copper phthalocyanine sulfonate, adriamycin and daunomysin, well-tried cytostatic agents. Depending on field strength and pulse width, 50–90% of cells become electroporated, then the dye diffuses rapidly into the cells, which reseal their membranes over a period of 6–10 min. Illumination for 10–15 min destroys all resealed cells faster than the same amount of unporated cells as in the case of the control (without pulse treatment) either by oxidation of cell components caused by excited dyes or singlet oxygen treatment. By this synergism of electroporation and photodynamic action at the same time, it is possible to kill all cells in a much shorter time than under usual conditions, e.g. in the control suspension. A combination of electrochemotherapeutic needle electrodes with a light conductor for a LASER connection will be effective for therapy.
The effect of NO‐metoprolol, that is, 3‐nitrooxypivaloyl metoprolol‐amide, a novel NO‐releasing derivative of the β1‐blocking drug metoprolol was investigated in A.CA/SnJ mice infected with ...coxsackievirus B3 (CVB3) and compared to metoprolol and placebo. Daily treatment of mice with the respective drug started immediately (experiment A) or 3 days after virus infection (experiment B) and was continued until day 13 post‐infection (p.i.). Two doses of NO‐metoprolol were administered. Body mass differences, viral load, and histopathological signs of myocarditis were compared between the several groups. As a result, NO‐metoprolol diminished significantly the body weight loss, the viral load and the histopathology, whereas metoprolol treatment led solely to a significant attenuation of myocardial damage. In experiment A, low dose NO‐metoprolol decreased significantly enteroviral copy numbers. Both doses of NO‐metoprolol had a significant effect on reduction of myocardial infiltrates and fibrosis. The data suggest that delayed drug administration might more advantageous. Both doses of NO‐metoprolol reduced significantly the scores of four tested parameters compared to placebo. Body weight loss, virus titers, plus‐strand as well as minus‐strand enteroviral RNA levels, infiltration and fibrosis scores were diminished significantly when NO‐metoprolol was given 3 days p.i. In addition, a significant difference regarding the enteroviral copy numbers was observed between low dose NO‐metoprolol‐ and metoprolol‐treated mice. Treatment with metoprolol reduced insignificantly the viral load and body weight loss (experiment A and B) but led to a significant reduction of myocardial histopathology in experiment A. The results indicate that NO‐metoprolol treatment has a greater therapeutic benefit than metoprolol. J. Med. Virol. 82:2043-2052, 2010.
The antiviral effect of nitric oxide (NO)-releasing compounds was investigated. Using bacterially expressed and purified proteinases 2A and 3C of coxsackievirus B3, in vitro assays demonstrated the ...inhibition of the 2A proteinase activity in the presence of S-nitroso- N-acetyl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), 4-phenyl-3-furoxancarbonitrile (PFC), glyceryl trinitrate (GTN), and isosorbide dinitrate (ISDN). Sodium nitroprusside (SNP), which releases NO after metabolization, had no effect. The 3C proteinase was inactivated by SNAP, GTN, and ISDN. The vasodilators GTN and ISDN, widely used in the treatment of angina pectoris, exhibited antiviral activity in CVB3-infected GMK cells. CVB3-infected NMRI outbred mice showed significantly reduced signs of myocarditis after treatment with GTN or ISDN. Inhibitors of the cellular inducible NO synthase (iNOS) such as N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), and S-methyl-isothiourea (SMT), had no deleterious effect on CVB3-infected NMRI mice, indicating that endogenous NO synthesis is unlikely to be a major defense mechanism after enterovirus infection of outbred mice.
ethics have to be taught to medical students. Teaching aims focusing on ethical reflection and practical skills are an important part in this new subject. This article reports how medical ethics can ...be taught in a case based, non-frontal way in order to train the ethical competencies and shape the attitudes of our future physicians. Therefore we have compiled curricular aspects, learning targets, paedagogic and didactic principles and technical requirements of 220 seminars on "Ethical Conflicts in a Physician's Daily Routine" that were conducted by the Study Group: Ethics in Medicine of the University of Ulm.Results/Discussion/Recommendations: We try to realize our broad learning target "ethical competence" by investing into extensive seminar preparations and performance. The use of methods derived from modern education science and didactics is paramount (e. g. orientation towards learning targets, motivation of participants, transparent teaching, complex education architecture). To ensure the teaching quality, our moderators must be educated and continuously qualified in paedagogics, didactics and medical ethics. The evaluation of our seminars is, apart from being a database for our research interests, a powerful means of feed-back and serves for securing our teaching quality. An early education continuing over the whole curriculum in medical ethics is, however, barely possible due to its enormous need in staff.
Aside from enteroviruses and other viruses, e.g., adenoviruses, which are known to be associated with idiopathic dilated cardiomyopathy (IDC), a cardiac tropism is also attributed to parvovirus B19 ...(PVB19). The purpose of the present study was to determine the prevalence of enterovirus, adenovirus and PVB19 genomes in the myocardium of adult patients with IDC and to analyze the significance of PVB19 with regard to the course of the disease, as compared to the other cardiotropic viruses. In 52 adult patients with IDC and 10 control patients with normal left ventricular ejection fraction (> or =55%) undergoing coronary artery bypass surgery, myocardial tissue samples were investigated for enteroviral RNA using polymerase chain reaction (PCR) and Southern blot hybridization of the PCR product. Specific nested PCR was used to assess the prevalence of adenovirus and PVB19 DNA, in addition to sequencing of the latter. The clinical and echocardiographic course of the disease was followed for a mean (+/- SD) period of 21.1+/-9.5 months. Fourteen of the 52 patients (27%) were enterovirus-positive, 2/52 (4%) patients were adenovirus-positive, 14/52 (27%) patients were PVB19-positive, 8/52 (15%) patients were enterovirus plus PVB19-positive, and in 14/52 (27%) patients no viral genomes were found. Six patients died during the follow-up period, without any significant difference between the patient groups: 1/14 (7%) in the enterovirus-positive, 0/2 (0%) in the adenovirus-positive, 2/14 (14%) in the PVB19-positive, 1/8 (12.5%) in the enterovirus plus PVB19-positive, and 2/14 (14%) in the virus-negative group. PVB19 genome was found in 4 of the 10 (40%) control patients, but no enterovirus or adenovirus genomes were detected in these patients. In conclusion, in the myocardium of patients with IDC, PVB19 is detectable as frequently as enteroviral genome. PVB19-positive patients with IDC have a rather favorable prognosis and do not differ significantly from the other virus-positive or virus-negative patient groups with respect to survival. Finally, the pathogenetic and prognostic significance of PVB19 in IDC still remains unclear.
AP-1 dependent genes, e.g., matrix-metallo-proteinases, are involved in the pathogenesis of rheumatoid arthritis (RA). Therefore, the transcription factor AP-1 and its subunits, proteins of the Jun ...and Fos proto-oncogene families, are interesting targets for analysis in RA. In this study, we analyzed the mRNA expression of junB in synovial membrane (SM) samples and isolated synovial fibroblasts of patients with RA, osteoarthritis (OA), and normal, non-inflammatory controls. To address the suitability of real-time RT-PCR for the quantitation of Jun proto-oncogene family members, conventional RTPCR and real-time PCR were comparatively applied for junD, a gene representing a major challenge because of its high GC-content (70%, increasing the probability of secondary structures interfering with the PCR) and its sequence homology to other Jun proto-oncogenes. In addition, a comparison was performed concerning the precision, reproducibility, costs, as well as labor and time consumption of the two PCR methods. Real-time RT-PCR proved superior to conventional PCR in terms of precision (mean deviation of measured from employed concentration 58% for real-time PCR vs 225% for conventional PCR), reproducibility, as well as labor and time consumption (4 times less for real-time RT-PCR). Experimental cDNA normalization for equivalent cDNA concentrations by sample dilution was more reliable than mathematical cDNA normalization. However, real-time PCR was 3.6-fold more expensive. Applying the more reliable real-time RT-PCR for the ex vivo analysis of junB mRNA-expression, no significantly different expression of junB was observed in SM or isolated synovial fibroblasts from RA as compared to OA. Interestingly, however, junBmRNA expression was significantly lower in RA SM and borderline significantly lower in OA SM than in normal/non-inflammatory SM, with potential effects on the functional properties of the resulting AP-1 complexes. Immunohistochemical staining of the SM with JunB-specific antibodies showed comparable JunB protein expression in SFB (collagen III mRNA-positive) of RA and OA samples. Thus, real-time RT-PCR appears suitable and time-saving for the quantitation of jun proto-oncogene mRNA-expression in tissue and cell samples with high precision and reproducibility.
Coxsackievirus B3 (CVB3) infections are the most frequent causes of human myocarditis, often resulting in chronic stages characterized by fibrosis and loss of function. This disease is called dilated ...cardiomyopathy (DCM). Persistent virus in the myocardium may lead to chronic activation of fibroblasts, and subsequently, to fibrosis of the myocardium. Studies with immunodeficient mice have shown that certain defects of the immune system retard the rate at which virus is eliminated from the heart, thus leading to viral persistence. Therefore, we followed the immune response of two immunocompetent mouse strains (C57BL/6 and Balb/c) to CVB3 infection. These two strains have been reported to develop different immune responses to infections and we expected a similar reaction to viral infections as well. The two mouse strains recovered completely from CVB3 infection and expressed identical levels of cytokine mRNA in the heart. However, the virus in heart tissue decreased more slowly in Balb/c than in C57BL/6 mice. This was accompanied by a strong virus-specific IgG and weak IgM response in the C57BL/6 mice, in comparison to the Balb/c mice. We conclude, therefore, that viral-specific IgG is of importance for CVB3 elimination from infected hearts.
Nitric oxide (NO) is an important bioactive molecule with regulatory, cytotoxic or cytoprotective properties. In virus-induced myocarditis, NO mediates host defense mechanisms against the infection ...or causes cardiac dysfunctions. NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). The expression of the inducible form of the nitric oxide synthase (iNOS) is regulated by cytokines, involved in the complex myocardial immune response to enterovirus infections. The present study was undertaken to characterize the role of iNOS and NO in the murine model of viral myocarditis induced by coxsackievirus B3 (CVB3). In response to CVB3 infection we investigated the time course of iNOS induction in correlation with cytokine mRNA expression (TNF-alpha, IL-1 alpha, IFN-gamma, TGF-beta) in the heart of NMRI mice by RT-PCR. Positive PCR signals for viral RNA were found in the acute and chronic stage of disease by seminested PCR, indicating the persistence of viral genome. We found distinct expression of iNOS at all time points (1, 2, 3, 4, 7, 14, 28, 56, 98 days post infection p.i.). Higher iNOS mRNA levels were identified between days 4 until 28 p.i. in comparison to day 56 and 98 p.i. using densitometric values. The mRNA of the inflammatory cytokines TNF-alpha, IL-1 alpha, IFN-gamma appeared at days 1, 4, and 7 p.i., peaked at day 7 p.i. and persisted until day 98 p.i. Similar like the iNOS mRNA pattern was the expression profile of TGF-beta. Using in situ hybridization and immunohistochemistry iNOS was localized in infiltrates, vascular endothelial cells, smooth muscle cells, myocytes and throughout the interstitial spaces between myocardial fibers in the heart sections of NMRI mice. Increased levels of NO were measured as total nitrate/nitrite concentration in the sera of mice from day 7 until day 28 p.i.
The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable ...susceptibility of the human population.
We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA.
NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection.
CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.
The electrofusion of human cancer U937 and K562 cells was studied under the influence of a selected group of polypeptides and proteins adsorbed at their membranes. For poly-L-lysine, we found that ...the higher the molecular mass (M
m
) the higher the relative fusion yield (F
r
), whereas poly-L-arginine and poly-L-ornithine showed toxicity. As recently measured for barley protoplast fusion, the same rule of dependence on the isoelectric point (pI) could be verified:
which means 0.5 < F
r
< 4 in 0.3 M mannitol solution (pH 6). If the pH of the 0.3 M mannitol solution is the same as the pI of the protein, its influence on the membrane nearly disappears. Bridging by Ca ions increases F
r
from 0.5 (bovine serum albumin) to F
r
∼ 1 by charge compensation. The value of F
r
is commensurate with the pore resealing time τ-determined by trypan blue staining-in such a way that for pI > 8, τ is longer and for pI < 6, τ is shorter than the control without protein content. Qualitatively, plant and animal membranes studied show equal behavior. According to our technique, the adsorption of biopolymers and other substances can be detected by this bioelectrochemical method, which is also a tool for analysis of electric stress mechanisms.
