An orthotopic model of ductal adenocarcinoma of the pancreas in severe combined immunodeficient mice representing all steps of the metastatic cascade.

INTRODUCTION: Clinically relevant animal models are needed to evaluate new therapeutic strategies against pancreatic adenocarcinoma, which is almost incurable by established treatment. AIMS: To establish and characterize a metastatic orthotopic transplant model for pancreatic ductal adenocarcinoma in severe combined immunodeficient (SCID) mice. METHODOLOGY: Human pancreatic ductal carcinoma cells, PancTu 1, were implanted either subcutaneously or orthotopically into the pancreas. RESULTS: After 4 weeks, orthotopic transplantation resulted in an extensive local tumor growth of an undifferentiated ductal adenocarcinoma with slight to moderate desmoplastic reaction. The tumor growth and spread resembled the situation in humans, including invasion into adjacent organs causing biliary and stomach obstruction. In addition, tumor metastases to regional lymph nodes of the pancreas, lung, liver, mesentery, and diaphragm, and attached to the kidneys, spleen, and reproductive organs were observed. In contrast, no invasion or metastases could be demonstrated by subcutaneous implanted PancTu I cells. Using immunohistochemical analysis, even single human tumor cells could be detected in blood vessels and metastatic organs, providing evidence that the orthotopic transplant model appropriately reflects the entire process of the metastatic cascade. CONCLUSION: This cancer model in SCID mice appears to be a powerful tool to investigate the identity of metastasis-associated genes and to evaluate preclinically the potency of novel antimetastatic agents in ductal adenocarcinoma of the pancreas.

Inhibitory effect of a matrix metalloproteinase inhibitor on growth and spread of human pancreatic ductal adenocarcinoma evaluated in an orthotopic severe combined immunodeficient (SCID) mouse model.

The present study was aimed at evaluating the effect of the matrix metalloproteinase (MMP) inhibitor prinomastat (AG3340) on tumor progression using an orthotopic pancreatic carcinoma model in severe combined immunodeficient mice. In controls, receiving vehicle only, the poorly differentiated ductal adenocarcinoma invaded into adjacent organs and metastasized to different sites in the abdomen and to the lungs. Treatment with prinomastat, intraperitoneally twice daily for 21 days, reduced primary tumor volume significantly to 19.0 (+/-7.7)% of control, with induction of necrosis, differentiation, and fibrotic tissue in the pancreatic tumors. Invasion was not observed in 63% of prinomastat-treated mice, and metastases were reduced markedly. Surprisingly, prinomastat-treated tumors had on average higher microvessel densities as a consequence of an increased angiogenesis in perinecrotic tumor areas. We conclude that prinomastat is highly effective in inhibiting pancreatic carcinoma growth and progression in an orthotopic cancer model. This model appears to be a valuable tool to investigate the potency of novel antimetastatic strategies in pancreatic ductal adenocarcinoma by specifically targeting certain MMPs.

Further studies on thermal resistance of bovine parvovirus against moist and dry heat.

To supplement the results of thermal resistance of bovine parvovirus (Haden strain, BPV) published previously, we carried out assays at 60 degrees C (moist heat) to compare the thermal resistance of BPV with that of hepatitis B-virus (HBV). What we know about the resistance of HBV at a temperature of 60 degrees C is mainly based on data collected within the context of blood product pasteurization. The results suggest that at a temperature of 60 degrees C, BPV shows thermal resistance comparable to HBV. Thus, BPV--which is easier to handle--can be considered a good test virus to verify the efficacy of thermal disinfection techniques against HBV. BPV is very resistant against dry heat of 100 degrees C, the inactivation largely depending upon the residual moisture of the lyophilisate. Reducing the residual moisture from 2% to less than 1%, the exposure time has to be prolonged by ca. 2.5 times to achieve the same virucidal effect.

Microbiological efficacy of superheated steam. I. Communication: results with spores of Bacillus subtilis and Bacillus stearothermophilus and with spore earth.

For the spores of Bacillus subtilis and Bacillus stearothermophilus as well as for spore earth (acc. DIN 58,946 Part 4 of August 1982), the dependence of resistance on the superheating of the steam used to kill germs was determined. A material (glass fibre fleece) was used as the germ carrier which does not superheat on contact with steam. The temperature of the saturated steam was 100 degrees C (B. subtilis) and 120 degrees C (B. stearothermophilus and spore earth). The yardstick for the resistance of the spores or bioindicators was the exposure period of the saturated or superheated steam at which 50% of the treated test objects no longer showed any viable test germs. The spores of Bacillus subtilis were far more sensitive to superheating of steam and reacted far more than the spores of Bacillus stearothermophilus and the germs in the spore earth. When superheating by 4 Kelvin the spores of Bacillus subtilis were approximately 2.5 times more resistant than they were to saturated steam. The resistance of Bacillus stearothermophilus and spore earth was only slightly higher up to superheating by 10 Kelvin. The spores of Bacillus subtilis had the highest resistance during superheating by 29 Kelvin; they were 119 times more resistant than they were to saturated steam. The resistance maximum of the spores of Bacillus stearothermophilus was at an superheating by around 22 Kelvin. However, the spores were only 4.1 times more resistant than they were to saturated steam. When using steam to kill germs, we must expect superheated steam. This raises the question whether the spores of Bacillus stearothermophilus, with their weaker reaction to the superheating of steam, are suitable as test germs for sterilisation with steam in all cases.

[Further studies on the falsification of the steam resistance of bioindicators by superheating]. / Weitere Untersuchungen über die Verfälschung der Dampf-Resistenz von Bioindikatoren durch Uberhitzung.

As a supplement to a preceding paper (Zbl. Hyg., 194 (1993). 369-279) the resistance of bioindicators has been investigated which differed only in the material of which the carriers are made (filter paper and glass fibre fleece, respectively). The conditions of the tests were such that further evidence could be expected for the fact that the characteristic values of bioindicators can be falsified by superheating of the carrier and its envelope. As a test organism Enterococcus faecium has been used. To avoid side effects, the bacteria have been dried on to the carriers from a suspension in water distillators. The exposition basket of the resistometer has been loaded with 3 rows of 15 indicators (disc shaped; diameter: 6 mm), arranged parallel. At a temperature of 68 degrees C the dependency of (relative) frequency of indicators having surviving test organisms capable of multiplying on exposure time to saturated steam has been determined. Free exposed indicators made of filter paper required considerably longer exposure periods than indicators made of glass fibre fleece to change from "(nearly) all the indicators have test organisms capable of multiplying" to "(nearly) all the indicators are free from test organisms capable of multiplying". The exposure time to free 50% of carriers made of filter paper from test organisms capable of multiplying (t50%) amounted to 44.1 minutes. The frequency of indicators free from test organisms was higher at the ends than in the middle of the exposed rows. When such indicators had been wetted before exposure, they showed a t50% value of 2.3 minutes, and the frequency of indicators free from surviving test organisms was distributed over the exposed indicators evenly. Free exposed indicators made of glass fibre fleece showed a t50% value of 4.4 minutes. The frequency of indicators free from test organisms was evenly distributed. Wetting of the indicators before exposure changed t50% value only slightly. When indicators made of glass fibre fleece had been exposed between two layers of filter paper they showed a very high t50% value (47.5 minutes), and the frequency of indicators free from test organisms was unevenly distributed. Indicators being sterile could be found mainly at the ends of the exposed rows. In an envelope of parchment paper bioindicators made of glass fibre fleece showed considerably higher characteristic values than when free exposed. Superheating of the carriers amounted to about 3 Kelvin.(ABSTRACT TRUNCATED AT 400 WORDS)

[Superheating of germ carriers falsifies the steam resistance of bioindicators]. / Uberhitzung der Keimträger verfälscht die Dampf-Resistenz von Bioindikatoren.

In the course of experiments on the resistance of bioindicators in saturated steam the authors noticed that carriers made of filter paper showed superheating by hygroscopic condensation. The differences in temperature between the bioindicators and the saturated steam amounted to about 5 Kelvin. Even after 20 minutes temperature equilibrium has not been reached. As a result of overheating and reduced water activity the frequency of bioindicators with surviving test organisms was increased. Beyond that, the microbiologic results depended on storage conditions of the bioindicators before putting them into the resistometer. The lower (higher) the-relative humidity, the higher (lower) the superheating, and the higher (smaller) the frequency of indicators with test organisms having survived. Carriers made of glass fibre fleece did not show any superheating and related effects. But carriers onto which small amounts of a suspension of test organisms in blood have been dried on, gave a superheating of about 1.7 Kelvin. Under identical conditions t50% values of bioindicators made of filter paper and bioindicators made of glass fibre fleece differed considerably. Experiments with Enterococcus faecium as test organism at 75 degrees C resulted in 14.4 and 1.7 minutes, respectively. Therefore, the high resistance of test organisms dried onto filter paper is an artefact caused by superheating. The experimental results should have consequences on the manufacture and use of bioindicators as well as chemoindicators and on the evaluation of results got with them.

[A simple apparatus for the determination of the resistance of bioindicators to saturated steam at temperatures less than 100 degrees C., tested with Enterococcus faecium as test microbe]. / Eine einfache Apparatur zur Bestimmung der Resistenz von Bioindikatoren gegen gesättigten Wasserdampf bei Temperaturen von weniger als 100 degrees C, erprobt mit Enterococcus faecium als Testkeim.

An apparatus is described by means of which the resistance of microbiological indicators to water vapor at temperatures below 100 degrees C can be determined. The apparatus can be assembled from parts generally available in laboratories. The principle of the apparatus consists in the production of water vapor of the desired temperature under conditions of reduced pressure and its recondensation to water after having passed a special chamber. Accordingly, the device consists of a heated round-bottom flask serving as steam generator, an exposure chamber (B), and a condenser (D) attached to a receiver (E). The bioindicators are exposed to the water vapor in the exposure chamber. A bypass located between the steam generator and the condenser allows for continuous operation even when the exposure chamber is opened. The reduced pressure was achieved by means of a waterjet pump and adjusted by two tandem-joined pressure-regulating valves as needed. The apparatus was tested using water vapor of 73, 75 and 77 degrees C, respectively, and bioindicators containing Enterococcus faecium as test organism. In the range of exposure periods in which bioindicators change from the status "all indicators having surviving test organisms" to the status "all indicators free from surviving test organisms" the bioindicators showed D values of 5.7, 4.4 and 2.9 min, respectively. For the temperature dependence of resistance a z value of 12.5 Kelvin resulted.

[The significance of heat activation for the testing of bioindicators on surviving microorganisms exposed to formaldehyde]. / Bedeutung der Hitzeaktivierung für die Prüfung von Bioindikatoren auf überlebende Keime, die Formaldehyd ausgesetzt waren.

Heat activation is a special phenomenon: After an additional heat treatment, a larger share of the bacterial spores which had been exposed to formaldehyde proves to be viable than without such heat activation. Model studies have been performed to test the effects of heat activation on the examination of bioindicators and test objects for surviving organisms. Test objects (cotton threads of 1 cm length) contaminated with spores of Bacillus stearothermophilus were used for these trials. The test objects were exposed to a 2% formaldehyde solution at 60 degrees C. After periods of action of 30, 45, 60 ... and 105 min, formaldehyde adhering to the test objects was neutralized. For testing these objects for surviving organisms, they were placed into a nutrient medium and incubated for 40 days at 56 degrees C. The investigation consisted of 2 parallel test series which only differed in one single point. In one series, the test objects were incubated at 56 degrees C as soon as they had been placed into the nutrient solution. In the other series, the test objects were exposed to a temperature of 95 degrees C for 1 h (heat activation) before starting incubation. The culture tubes were checked daily to see whether signs of growth (turbidity and deposits) could be observed. The frequencies of test objects with surviving organisms depending on the period of action of formaldehyde and the period of incubation determined in this way are based on the examination of 72 test objects each. Without heat activation, the share of test objects on which surviving test organisms could be detected, increased slowly with the period of incubation. Only after 30 days the counts did not increase any more when continuing the incubation (cf. Fig. 1). In the test series in which the spores had been subjected to heat activation before the incubation period, useful results were obtained already after 3 days. They only changed slightly when incubation was continued. Moreover, the frequency of test objects on which surviving organisms could be detected was always considerably higher than without heat activation. When the frequency of test objects with surviving organisms was plotted against the period of action of formaldehyde (cf. Fig. 2A), S-shaped curves resulted.(ABSTRACT TRUNCATED AT 400 WORDS)

[Dependence of microbiologic test results of formaldehyde gas sterilization methods on the nature of the test material]. / Abhängigkeit der mikrobiologischen Prüfungsergebnisse von Formaldehyd-Gassterilisationsverfahren von der Materialbeschaffenheit des Testkörpers.

The efficiency of a formaldehyde gas sterilization procedure was evaluated with the aid of test pieces consisting of various materials. Both rigid and flexible tubes served as test pieces. The tubes were 75 cm long with an inner diameter of 1 mm and were sealed at one end. The bioindicators were placed inside the tubes close to the sealed end. Dried spores of Bacillus stearothermophilus adhering to linen threads served as test organisms. The test results varied according to the material of the test pieces and the thickness of their walls (see Table 1). In flexible tubes made of silicon rubber, all bioindicators became sterile, in tubes of stainless steel, all bioindicators exhibited test organisms that had survived. The findings for materials such as polyvinyl chloride, polyethylene, polyamide and polytetrafluorethylene ranged between these two extremes; the frequencies of bioindicators containing viable germs were 10, 55, 68 and 85%, respectively. Rigid and flexible tubes which had been sealed at both ends served to demonstrate that silicon rubber and polyvinyl chloride were highly permeable for formaldehyde and water vapour. Also the other plastic materials tested were permeable for formaldehyde and water vapour but longer exposure periods were needed to create conditions in the interior of the tubes that would result in a killing of the test organisms (see Fig 2). In this respect, polyamide exhibited a peculiar behaviour. The number of viable spores remained at the initial level for a long period before a decline took place. From the results of testing, it is concluded that test pieces must conform to the objects to be sterilized not only in their dimensions (length, inner diameter) but also in the characteristics of their material. The walls of the test pieces should not have a higher permeability for formaldehyde and water vapour than the material to be sterilized. The highest demands on the efficiency of formaldehyde gas sterilization procedures are those created by mental tubes and thick-walled flexible polytetrafluorethylene. Instruments and devices to be sterilized by a formaldehyde gas procedure should be preferentially made of materials which are sufficiently permeable for formaldehyde and water vapour as e.g. silicon rubber. Such gas-permeable components may considerably facilitate the sterilization of cavities which have a small lumen and are difficult to reach.

[An expedient semi-automatic procedure for the preparation of large quantities of bioindicators especially for use in gas sterilization processes]. / Ein rationelles halbautomatisches Verfahren zur Herstellung grosser Mengen Bioindikatoren, insbesondere für Gas-Sterilisationsverfahren.

Bioindicators serve to test the efficacy of disinfection and sterilization procedures. Such indicators mostly consist of a support (filter paper, as a rule) to which micro-organisms have been fixed by drying. The authors have used a thread as support and a special apparatus for semi-automatic preparation of the bioindicators. The components of the device are either commercially available or may be prepared from commercially available material without difficulty. The principle of the method is as follows: The thread serving as the support is drawn slowly, at constant speed, through the suspension of test organisms and dried in an air stream immediately afterwards. The apparatus consists of a cylindrical glass tube of a few centimeters in diameter, an electric motor slowly rotating the cylinder, a fan, a magnetic stirrer, and an ice-water bath. A small vial containing the germ suspension is immersed in the ice-water bath. The vial is sealed by a screw cap with two glass tubes of about 3 mm inner diameter passing through it. One of the glass tubes being bent in its upper part reaches far down into the vial to leave just enough play for free rotation of a magnetic stirring rod. This tube serves to introduce the thread into the germ suspension. The second straight tube does not reach as far down as the first one. Its lower opening should not be immersed in the germ suspension. This tube serves as a guide for the returning thread. Preparation begins by winding the thread to be soaked with the suspension around the cylinder.(ABSTRACT TRUNCATED AT 250 WORDS)

[What should be the length and inner diameter of the testing device for microbiological efficacy testing of formaldehyde gas sterilization methods?]. / Welche Länge und lichte Weite soll der Testkörper für die mikrobiologische Wirksamkeitsprüfung von Formaldehyd-Gassterilisationsverfahren besitzen?

The series of tests described in a preceding publication (Spicher and Borchers, 1983) has been continued in a modified way. This time, the dependency of the microbiological test results of a formaldehyde gas sterilization procedure on length and inner diameter of the tubes serving as test pieces was examined. The tubes were 1 or 2 m in length with an inner diameter of 1 or 2 mm. The tests were performed with four different preparations of bioindicators. Spores of Bac. stearothermophilus served as test germs. The preparations differed in the type of suspension used for the preparation of the bioindicators: distilled water, diluted blood (10%), undiluted blood, 10% albumin solution. The spore suspensions had been dried on linen thread. During the test procedure, the bioindicators were located near the sealed end of the tube. After completion of the sterilization procedure, the bioindicators were examined for viable germs. In tubes of identical length, the frequency of indicators carrying viable germs was always higher in those of 1 mm than in those of 2 mm inner diameter. In tubes of identical inner diameter, the frequency of indicators carrying viable germs in those of 2 m length was always higher than in those of 1 m length. This regularity was independent of the type of bioindicators used. The bioindicators for the preparation of which a 10% albumin solution had been employed showed the highest resistance. A somewhat lower resistance was found for the bioindicators prepared with undiluted blood. The bioindicators for which the spores had been suspended in diluted blood proved to have the lowest resistance. If the spores had been suspended in distilled water, the resistance of the bioindicators was a little lower than that of those suspended in undiluted blood, but was higher than that of the dried spores with diluted blood. The test results confirm the effectiveness of the method proposed earlier, i.e. to deposit the bioindicators in special test pieces (e.g. tubes or sounds) for the microbiological testing of formaldehyde gas sterilization procedures. These test pieces must be at least as long and as narrow as the longest and narrowest cavity of the object to be sterilized (tubes, catheters). In order to standardize the microbiological testing of formaldehyde gas sterilization procedures and to guarantee a certain minimum efficiency, the bioindicator as well as the test piece and its size (length and inner diameter) should be standardized.(ABSTRACT TRUNCATED AT 400 WORDS)

[Dependency of a microbiological test of a formaldehyde gas sterilization procedure on the shape of objects to be sterilized]. / Abhängigkeit der mikrobiologischen Prüfungsergebnisse eines Formaldehyd-Gassterilisationsverfahrens von der Form der zu sterilisierenden Objekte.

During the last decade, a number of procedures have been developed by different firms for the sterilization of heat-sensitive instruments using a mixture of formaldehyde and water vapor at a temperature of approximately 60 degrees C as means of sterilization. Instruments to be sterilized by this technique as e.g. sounds and catheters normally have long narrow cavities. Therefore, the formaldehyde gas sterilization procedures have to be tested primarily for their capability of achieving a sufficient microbicidal effect within those cavities. For this purpose, the bioindicators are placed into special test pieces. The test pieces commonly in use differ widely in their construction, shape, and size. They mostly consist of some hollow cylinder with an attached capillary or a tube (see Table 1). The authors demonstrated by means of models that the variety of test pieces in use meant that the sterilization procedures had to meet quite different requirements. The models consisted of flexible tubes differing in diameter and length and were connected to short glass tubes. These glass tubes having identical or wider inner diameters than the flexible tubes served as receptacles containing the bioindicators. Spores of Bacillus stearothermophilus served as test organisms. The spores were suspended in defibrinated sheep blood and dried on filter paper. The efficiency of the sterilization technique was measured in terms of the relative number of indicator strips with surviving germs (i.e. non-sterilized indicators) after treatment of the test pieces with the formaldehyde gas. At first, the test results were examined as to their dependency on the length of the flexible tubes. These tubes were 3 mm wide and 5 to 100 cm long, each being sealed at one end and with the bioindicators placed near the sealed end. The percentage of indicators with surviving germs increased with the length of the tubes. After the sterilization process, nearly all indicators (92%) contained in the 1 m tubes proved to be non-sterile (see Table 2). The same results were obtained with tubes open at both ends, with the bioindicators located in the middle section of the tubes (see Table 3). Using tubes of 1 m length, the dependency of the test results on the inner diameter of the test pieces was demonstrated. While all indicators placed into tubes of 3 mm inner diameter still contained surviving germs, those in the tubes of 9 mm inner diameter were all sterile (see Table 4).(ABSTRACT TRUNCATED AT 400 WORDS)