Comparison of the in vivo efficiency of photofrin II-, mTHPC-, mTHPC-PEG- and mTHPCnPEG-mediated PDT in a human xenografted head and neck carcinoma.

BACKGROUND AND OBJECTIVE: One of the approaches to enhance the selectivity and efficiency of photodynamic therapy (PDT) was the conjugation of the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) to the water-soluble polymer polyethylene glycol (PEG). Several studies have demonstrated that mTHPC-PEG has a higher selectivity and a longer circulating half-life than free mTHPC, whereas no in vivo effect of this benefit could be seen. STUDY DESIGN/MATERIALS AND METHODS: In a model of RAG-2-mice bearing a human oral squamous cell carcinoma xenograft (XF 354), the in vivo efficiency assessed as growth retardation or remission caused by Photofrin II and free mTHPC was compared with mTHPC coupled in two different ways to polyethylene glycol (PEG). One hundred and fourty-nine female RAG-2-mice were randomised into one control group and 13 therapy groups. Treatment parameters were adapted from those routinely applied in animal studies. RESULTS: Photofrin II-mediated PDT and mTHPC-mediated PDT were both in vivo highly effective, whereas mTHPC induced less scars. The in vivo results after mTHPC-PEG-mediated PDT were disappointing, whereas the effectiveness of mTHPCnPEG-mediated PDT, a newly coupled macromolecular photosensitizer, were promising. CONCLUSIONS: These results demonstrated the impact of the method of linkage between the photoactive agent mTHPC and polyethylene glycol (PEG) upon the in vivo effectiveness. mTHPC and mTHPCnPEG are promising photosensitizers for the future, especially for the cosmetic treatment needs of head and neck surgery.

[Clinical effectiveness of m-THPC-PEG in a new xenogenic animal tumor model for human squamous epithelial carcinomas]. / Klinische Wirksamkeit von m-THPC-PEG in einem neuen xenogenen Tumortiermodell für humane Plattenepithelkarzinome.

BACKGROUND: Animal tumor models are still essential for the development of new medication and therapy concepts. For the field of oropharyngeal cancer only few reliable xenogeneic tumor models are available. In a two-part experiment a new xenogeneic tumor model was established. PART 1 OF THE STUDY: The growth rates of two different tumor cell lines were compared in the RAG-2 mouse, the SCID mouse and the Swiss nude mouse. Cells from a lymph-node metastasis of an oral squamous cell carcinoma (XF-354) showed a better growth rate and clearer histology than cells from a primary squamous cell carcinoma of the floor of the mouth (UM-SCC-14C). The tumor growth rate was highest on the RAG-2 mouse, followed by the SCID mouse. The Swiss nude mouse showed no tumor growth at all. The combination of the XF-354 tumor cell line and the RAG-2 mouse was the most successful, with a tumor growth rate of 95%. The animal model is very reliable and robust and enables manipulations under anaesthesia outside the sterile barrier for up to 30 min. The single steps for cell cultivation and tumor implantation are described and discussed in detail. PART 2 OF THE STUDY: The second part of this study investigated the clinical efficacy of the macromolecular linked photosensitizer--mTHPC-PEG compared with the free photosensitizer mTHPC. Macromolecular linked photosensitizers have theoretical advantages owing to their pharmacokinetics and tumor selectivity. For the animal experiment, the photosensitizer mTHPC showed significantly better results than the macromolecular linked photosensitizer mTHPC-PEG. The reasons for these in vivo results are discussed in detail.

Laser-induced fluorescence detection of malignant gliomas using fluorescein-labeled serum albumin: experimental and preliminary clinical results.

To delineate the tumor margins of malignant gliomas laser-induced fluorescence detection technique was applied using 5-aminofluorescein-albumin as the fluorescent dye. The 5-aminofluorescein was linked to serum albumin (= AFlc-SA) as a cumulative protein label using residualizing markers. In a C6-glioma model the biodistribution and pharmacokinetics of the injected dye were investigated by labeling the protein conjugate with 111In-DTPA. Twenty-four hours after intravenous injection of the dye, fluorescence was activated by an argon laser and inspected in the C6-gliomas. Histological examinations were performed to compare the microscopic margins of the fluorescence-stained tumors with hematoxylin/eosin. The tumor uptake 24 h after dye injection was 23-fold higher than in the surrounding brain. Fluorescence inspection under laser activation demonstrated clearly stained and sharply demarcated tumors. The microscopic borders of the tumors corresponded exactly with the fluorescence, also demonstrating intracellular tumor uptake of the dye. In a preliminary study, three patients with malignant gliomas were operated using laser-induced fluorescence detection technique after injection of AFlc-SA. In all patients, the borders of the malignant gliomas were clearly stained by AFlc-SA during surgery. Laser-induced fluorescence imaging using the albumin conjugate AFlc-SA may be a promising method for delineating tumor margins which are hard to detect under the operating microscope alone.

Efficacy of anticancer alkylphosphocholines in Trypanosoma brucei subspecies.

Tetradecylphosphocholine (TPC), hexadecylphosphocholine (HPC), hexadecylphospho(N-N-N-trimethyl)hexanolamine (HPC6), octadecylphosphocholine (OPC), and octadecyl-[2-(N-methylpiperidinio)ethyl]-phosphate (OMPEP) were investigated for antitrypanosomal activity in vitro and in vivo. OMPEP showed the best trypanocidal efficacy in vitro; it was superior to the model compound HPC and comparable to the reference compound alpha-DFMO. HPC showed moderate activity in vivo in terms of increased life expectancy (up to 35% in the acute NMRI-mouse model or 49% if combined with phenylbutazone) and increased packed cell volume, if administered daily. However, HPC and the other alkylphosphocholines failed to prolong survival time of treated mice if given intermittently. Phenylbutazone had no own trypanocidal effect but increased the efficacy of alkylphosphocholines in vitro and in vivo: the combination of HPC and phenylbutazone acted apparently synergistic.

Synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary aflatoxin B1 in woodchucks.

Interactive hepadnaviral and chemical hepatocarcinogenesis was studied in woodchucks inoculated as newborns with woodchuck hepatitis virus (WHV), which is closely related to the human hepatitis B virus. When the woodchucks reached 12 months of age, aflatoxin B1 (AFB1) was administered in the diet at dose levels of 40 micrograms/kg body weight/day for 4 months and subsequently 20 micrograms/kg body weight/day (5 days/week) for lifetime. WHV DNA was demonstrated by Southern blot hybridization in the serum and by PCR in the serum and/or liver tissue. The histo- and cytomorphology of the liver were investigated by light and electron microscopy. WHV carriers with and without AFB1 treatment developed a high incidence of preneoplastic foci of altered hepatocytes, hepatocellular adenomas, and hepatocellular carcinomas that appeared 6-26 months after the beginning of the combination experiment. Administration of AFB1 to WHV carriers resulted in a significantly earlier appearance of hepatocellular neoplasms and a higher incidence of hepatocellular carcinomas compared to WHV carriers not treated with AFB1. Neither hepatocellular adenomas nor carcinomas (but preneoplastic foci of altered hepatocytes) were detected in woodchucks receiving AFB1 alone, and no preneoplastic or neoplastic lesions were found in untreated controls. These results provide conclusive evidence of a synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary AFB1. Except for the frequent presence of ground glass cells containing surface antigen filaments in the infected woodchucks, the phenotype of preneoplastic foci of altered hepatocytes was similar in WHV carriers with and without exposure to AFB1 and in animals treated with AFB1 alone. Clear cell foci excessively storing glycogen and/or fat, amphophilic cell foci crowded with mitochondria and peroxisomes, and mixed cell foci composed of various cell types including basophilic cells rich in ribosomes predominated. The cellular phenotype in neoplastic lesions varied from clear, amphophilic, and mixed cell populations in highly differentiated adenomas and carcinomas to basophilic cell populations prevailing in poorly differentiated carcinomas. The striking similarities in altered cellular phenotypes of preneoplastic hepatic foci emerging after both hepadnaviral infection and exposure to AFB1 suggest closely related underlying molecular mechanisms that may be mainly responsible for the synergistic hepatocarcinogenic effect of these oncogenic agents.

Microchip implant system used for animal identification in laboratory rabbits, guineapigs, woodchucks and in amphibians.

Traditional methods for animal identification have a number of drawbacks. We evaluated a new system for individual identification using microchip implants in rabbits, guineapigs, woodchucks (Marmota monax) and amphibians (Xenopus laevis, Pleurodeles waltlii). Implantation procedure and long-term observations are described. Microchip implants proved to be a practicable and reliable system for animal identification without obvious adverse effects. The applicability of electronic animal identification in comparison with common methods and with regard to animal welfare and legal aspects is discussed.

Efficiency of air filter sets for the prevention of airborne infections in laboratory animal houses.

Air filter sets (classes EU6 and EU9, or EU6 and S) were tested for their efficiency in protecting laboratory animals against potential airborne infections. Flexible-film isolators were used as a smaller scale model. In the first experiment, lasting 7 months, it was tested whether minute virus of mice (MVM) was able to penetrate the air filters between one isolator containing experimentally infected mice and another with MVM negative mice. In the second experiment we tested whether microorganisms in the incoming air were able to penetrate air filter sets. To assess this gnotobiotic mice in an isolator were monitored for 9 months for changes of their microbial flora. In both experiments a combination of EU6 and EU9 air filters proved to be sufficient to maintain the microbiological status of the animals. The same combination of medium efficiency filters (EU6 and EU9) is used on the air supply to 4 SPF-barrier units in which infections with MVM occurred repeatedly soon after the initial stocking. After a thorough disinfection no reinfection has been detected to date. This demonstrates that the relatively low efficiency of the air filters was not the cause of the repeated infection. The procedure for disinfection is described.

[Animal welfare quo vadis: animal welfare as a constitutional goal?]. / Tierschutz quo vadis: Tierschutz als Verfassungsziel?

From the point of view of a great research centre the introduction of animal protection in the German "Grundgesetz" raises various objections. These refer to principle law political considerations, ethical aspects and the negative influence this circumstance could have on the progress of biomedical research.

[The value of occlusion plethysmography and stress venous pressure in functional diagnosis following leg vein thrombosis]. / Der Wert von Verschlussplethysmographie und Belastungsvenendruck bei der Funktionsdiagnostik nach Beinvenenthrombose.

In 52 patients with condition after thrombosis of the deep leg veins before discharge from hospital the venous capacity and the venous outflow were measured on the two legs by means of occlusion plethysmography of the veins as well as the percental decrease of the venous pressure during action of the leg muscles by means of phlebodynamometry. The results have been compared with the phlebography. 10 patients again had a venous system without pathologic findings, in 12 patients unchangedly was a complete and in 30 ones in incomplete occlusion. By means of the measuring of the venous pressure still existing obstacles of the outflow of the pelvic and femoral region could be separated from the lower leg and their degree of severity, too, could be quantified. The venous capacity and the venous outflow allowed with sufficient security to delimit the values of the healthy leg from those of the postthrombotic one, whereas a localization of the height and a further quantification of the postthrombotic condition failed. Improved technology of the devices as well as the establishment of venous capacity and venous outflow in one measuring process increase the value of the venous occlusion plethysmography clearly as a non-invasive method which can be carried out by a function-assistant.

Isoenzymes of the lung fluke Paragonimus uterobilateralis from Liberia.

The enzymes of 35 adult Paragonimus uterobilateralis were analysed using thin-layer starch gel electrophoresis. From a total of 21 enzyme systems studied, 15 proved to be useful for the description and recognition of this species. All individuals were identical concerning 11 enzymes. In four remaining enzymes, alanine aminotransferase (ALAT, hexokinase, aspartate aminotransferase and phosphogluconate dehydrogenase, two or three variants, also being partly typical for this species were observed. In a comparison involving seven different enzymes, there were no differences between the electrophoretic patterns of 35 adult and 24 juvenile P. uterobilateralis. Additional examinations of 30 adult P. uterobilateralis with isoelectric focusing on ultrathin-layer polyacrylamide gels revealed clearer separations of enzymes. The method showed corresponding results or identiy of all individuals tested with three representative enzymes (ALAT, glucosephosphate isomerase and adenylate kinase).

Geographical distribution of Glossina palpalis gambiensis and G.p.palpalis in Liberia.

The two subspecies of Glossina palpalis (Robineau-Desvoidy) occurring in Liberia could be reliably separated morphometrically by measuring the width of the terminal dilatations of the male inferior claspers. Subspecies differentiation of female flies was less conclusive. Identification of flies from fifty-four sites revealed that most of Liberia lies in the belt of Glossina palpalis palpalis. However, pure and substantial populations of G.p.gambiensis Vanderplank occur north of 8 degrees 20'N in the north-west of Liberia.

Enzyme polymorphism and the identity of Trypanosoma brucei gambiense.

Thirty-two isolates from man in known areas of Gambian trypanosomiasis, in the Sudan, Kenya, Zaire, Nigeria, Ivory Coast, Burkina Faso, Liberia and Senegal, were examined by isoenzyme electrophoresis of 11 enzymes. Comparisons were also made with our previously published results on 23 other stocks of similar origins, which had been examined in the same manner. All those stocks of low initial virulence to laboratory rodents, which thus conform to the accepted view of the behaviour of Trypanosoma brucei gambiense can be identified by characteristic combinations of enzyme patterns, especially certain aminotransferase markers. A limited study of superoxide dismutase polymorphism suggested a further marker of value. The isolates of high initial virulence to rodents, which are thus behaviourally akin to T. b. rhodesiense, did not share these characteristics. We conclude that there exists a homogeneous group of trypanosomes of wide dispersion throughout tropical Africa, characterized by certain isoenzyme combinations and low initial virulence to rodents, which corresponds to the classical concept of T. b. gambiense. The features of limited antigenic repertoire, high resistance to normal human serum and restriction fragment length polymorphisms of the genes for certain variant surface glycoproteins also appear to be characteristic of this group.

The Testryp CATT (Card Agglutination Test for Trypanosomiasis): a field study on gambiense sleeping sickness in Liberia.

The inhabitants of three Liberian villages in a known gambiense sleeping sickness area with a low prevalence were examined comparatively with the haematocrit centrifugation technique (HCT), the miniature anion exchange centrifugation technique (m-AECT), the indirect fluorescent antibody test (IFAT) and the Testryp CATT (Card Agglutination Test for Trypanosomiasis). Among 418 persons examined was one individual from whom trypanosomes could be isolated with the HCT and m-AECT. This positive case and another 21 individuals had positive titres of 1:40 and more in the IFAT. The same number, but not the same individuals, showed positive reactions in the CATT in the 1:5 serum dilution. The 1:25 dilution of blood and serum was inferior to the corresponding 1:5 dilution and could therefore be omitted. The 'false positive' rate in the CATT was 5.0% in the serum, 10.1% in the blood, and 11.5% in blood or serum. The combination of HCT and m-AECT in the field did not achieve better results than the CATT or IFAT. Thus the CATT can be recommended as the preferable technique for screening of gambiense trypanosomiasis in the field.

Mycobacterium marinum infection in gnotobiotic nu/nu mice pretreated with cyclophosphamide and silica.

Germfree nude mice (NMRJ-nu/nu) and germfree nu/+ mice were infected with Mycobacterium marinum in the footpad test. After pretreatment with 400 mg/kg cyclophosphamide, significantly higher amounts of M. marinum were reached in the footpad and in the liver. On the other hand, the yield of M. marinum could not be elevated by in vivo inhibition of macrophages with silica.

Isoenzymes of South American Paragonimus peruvianus and P. ecuadoriensis.

The characterization of adult Paragonimus peruvianus by isoenzyme electrophoresis of four enzymes, a peptidase, alanine aminotransferase, glucosephosphate isomerase and glucose 6-phosphate dehydrogenase revealed a complete identity of all six individuals examined. Electrophoretic patterns of eight Paragonimus ecuadoriensis showed uniformity concerning the same enzymes. Both species compared were monomorphous with respect to three enzymes and could only be separated by the peptidase. The close relationship, already established by morphology, between adults of P. peruvianus is therefore supported.

West African dogs as a model for research on trypanotolerance.

Autochthonous dogs from Liberia/West Africa were reared trypanosome-free and brought to West Berlin/germany. Together with Beagle dogs they were infected cyclically by tsetseflies with Trypanosoma congolense. While the European dogs died soon of the trypanosomal infection, the African dogs developed milder parasitaemias and remained clinically unaffected. The authors' opinion is, that this dog model could make a contribution to research on trypanotolerance.

Morphological identification of simulium sanctipauli and S. yahense in Liberia and comparison of results with those of enzyme electrophoresis.

Simulium sanctipauli and S. yahense have been incriminated as the main vectors of Onchocerca volvulus in the rain-forest zone of Liberia. An accurate identification of the females of the two species which breed in different types of water-courses but are morphologically similar is essential for a proper study of their epidemiological importance and the planning of control measures. Various external characters were examined for their diagnostic value by the comparison of identifications using these characters and those based on enzyme electrophoresis (phosphoglucomutase, trehalase). It was shown that the females of the two species can be separated reliably by the use of external morphological characters. The occasional finding in the same fly of phosphoglucomutase variants typical for each species indicated the possible occurrence of natural hybridization between S. sanctipauli and S. yahense.

Identity of Trypanozoon stocks isolated from man and a domestic dog in Liberia.

The isoenzyme patterns (electrophoresis on thin-layer starch gel) and the human serum resistance (blood incubation infectivity test) of a Trypanozoon stock from a dog and 14 stocks from man in Liberia were examined. The complete conformity of criteria for man-infectivity of the dog stock with 5 human stocks and the very close similarity to the 9 remaining stocks from the same epidemiological locality indicates the dog as a reservoir host of gambiense sleeping sickness.