Realistic multi-cellular dosimetry for 177Lu-labelled antibodies: model and application.

Current preclinical dosimetric models often fail to take account of the complex nature of absorbed dose distribution typical of in vitro clonogenic experiments in targeted radionuclide therapy. For this reason, clonogenic survival is often expressed as a function of added activity rather than the absorbed dose delivered to cells/cell nuclei. We designed a multi-cellular dosimetry model that takes into account the realistic distributions of cells in the Petri dish, for the establishment of survival curves as a function of the absorbed dose. General-purpose software tools were used for the generation of realistic, randomised 3D cell culture geometries based on experimentally determined parameters (cell size, cell density, cluster density, average cluster size, cell cumulated activity). A mixture of Monte Carlo and analytical approaches was implemented in order to achieve as accurate as possible results while reducing calculation time. The model was here applied to clonogenic survival experiments carried out to compare the efficacy of Betalutin®, a novel 177Lu-labelled antibody radionuclide conjugate for the treatment of non-Hodgkin lymphoma, to that of 177Lu-labelled CD20-specific (rituximab) and non-specific antibodies (Erbitux) on lymphocyte B cells. The 3D cellular model developed allowed a better understanding of the radiative and non-radiative processes associated with cellular death. Our approach is generic and can also be applied to other radiopharmaceuticals and cell distributions.

Bystander effects in cell death induced by photodynamic treatment UVA radiation and inhibitors of ATP synthesis.

Confluent layers of MDCK II cells were treated with four different photosensitizers (a purified version of hematoporphyrin derivative [Photofrin], tetra(3-hydroxyphenyl)porphine [3-THPP], meso-tetra(4-sulphonatophenyl)porphine [TPPS4] and ALA-induced Protoporphyrin IX) and irradiated with blue light, with UVA without exogenous photosensitizers, or incubated with the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone and 2-deoxy-D-glucose. Necrotic and apoptotic cells were detected about 4 h later by fluorescence microscopy. Dead cells appeared in distinct clusters in the confluent layers. The number of dead cells in these clusters was determined by manual counting and image analysis. Forty-one of the 43 experimental distributions of dead cells in clusters were found to be significantly different from a Monte Carlo simulation of the distribution of independently inactivated cells. However, a Monte Carlo simulation model, assuming that each dead cell increased the probability of inactivation of adjacent cells, fitted 34 of the 43 observed distributions of dead cells in clusters, indicating a significant bystander effect for all the investigated treatments. The bystander-effect model parameter, defined as a cell's increase in probability of dying when it has dead neighbors, was significantly lower for 3-THPP-PDT and TPPS4-PDT than for Photofrin-PDT, ALA-PDT and treatment with metabolic inhibitors.

Gap junctional intercellular communication is not a major mediator in the bystander effect in photodynamic treatment of MDCK II cells.

Photodynamic treatment (PDT) of confluent MDCK II cells resulted in a noticeable clustering of dead cells, consistent with a significant bystander effect. Likewise, PDT of cells in microcolonies resulted in an overabundance of microcolonies that had responded to the treatment as a single unit, that is, in which either all or no cells were dead. Confluent MDCK II cells appeared to communicate via gap junction channels, while cells in microcolonies did not. Monte Carlo simulation models were fitted to the distributions of dead cells in confluent monolayers and in microcolonies. The simulations showed that the degree of the bystander effect was higher in microcolonies than in confluent cells, suggesting that gap junction communication may be involved in the bystander effect. However, when the gap junction hypothesis was tested by treatment of microcolonies with 30 microM dieldrin, an inhibitor of gap junctional intercellular communication, there was no reduction of the bystander effect, indicating that this effect was not mediated by gap junctional intercellular communication. PDT influenced phosphorylation of tyrosine residues in several proteins in the cells. Protein phosphorylation is important in cellular signaling pathways and may be involved in the bystander effect, for example by influencing the mode of cell death.

The bystander effect in photodynamic inactivation of cells.

Treatment of MDCK II cells with the lipophilic photosensitizer tetra(3-hydroxyphenyl)porphyrin and light was found to induce a rapid apoptotic response in a large fraction of the cells. Furthermore, the distribution of apoptotic cells in microcolonies of eight cells was found to be different from the binomial distribution, indicating that the cells are not inactivated independently, but that a bystander effect is involved in cell killing by photodynamic treatment. The observation of a bystander effect disagrees with the common view that cells are inactivated only by direct damage and indicates that communication between cells in a colony plays a role in photosensitized induction of apoptosis. The degree of bystander effect was higher for cells dying by necrosis than for cell dying by apoptosis.

The mode of cell death induced by photodynamic treatment depends on cell density.

Madison Darby canine kidney II (MDCK II) cells were seeded out at two different densities and incubated with 125 micrograms/mL of the photosensitizer meso-tetra(4-sulfonatophenyl)porphine (TPPS4) for 18 h, washed and irradiated with blue light. Four hours later the cells were studied by fluorescence microscopy. Apoptotic cells were detected by virtue of the distinct condensation and fragmentation of chromatin, and necrotic cells were detected by uptake of propidium iodide. In addition apoptosis was measured by the TdT assay. The fraction of apoptotic cells and the fraction of necrotic cells were determined for both cell densities at various levels of survival. With < 55% total cell death the apoptotic fraction was significantly higher for cells in confluent monolayers than for cells growing in microcolonies at equitoxic doses. Confluent cells were 2.9 times more sensitive than cells in microcolonies partly due to a 1.5 times higher uptake of TPPS4 in monolayer cells. The difference in mode of cell death for the different cell densities was not related to any observable difference in subcellular localization pattern of TPPS4 at equitoxic doses of photodynamic treatment.

Cooperative inactivation of cells in microcolonies treated with UVA radiation.

Microcolonies of Madison-Darby canine kidney cells (MDCK II) were exposed to UVA radiation, and the number of cells with membrane damage was determined by staining with propidium iodide and fluorescence microscopy. The cells were clearly damaged in a nonrandom manner: The distribution of damaged cells per microcolony was incompatible with the assumption that the cells were damaged independently. The data were accurately described by a so-called propagated damage model in which a damaged cell can influence its neighbors in a propagating manner. These findings do not agree with the common view that optical radiation interacts with cells in a way in which damage manifested in a cell is the result of absorption of light in the same cell.

Cooperative effects of photodynamic treatment of cells in microcolonies.

Microcolonies of 2-8 Madison-Darby canine kidney cells (MDCK II) and Chinese hamster lung fibroblasts (V79) cells were incubated with the photosensitizer Photofrin and exposed to light, and the resulting number of dead cells per colony was determined. The distribution of this number was found to be incompatible with the assumption that cells are inactivated independently. The experimental distributions were significantly different from the binomial distribution expected from this assumption, but in accordance with a model in which an inactivated cell can inactivate adjacent cells with a certain probability. These findings are contrary to the common view that damage caused by radiation is limited to the cell in which the primary damage takes place. Our findings clearly indicate some kind of cooperativity between cells treated with Photofrin and light.

Assessment of safety and protective value of a cell culture modified strain "C" vaccine of hog cholera/classical swine fever virus.

The protective value of a commercial strain "C" vaccine of classical swine fever (CSF) was tested in weaner pigs. Vaccinated animals were challenged intranasally with the virulent hog cholera virus (HCV) strain ALFORT/187 in groups of four pigs each at one to four weeks post vaccination, respectively. Non-vaccinated control animals were challenged in the same manner. Some vaccinated pigs seroconverted as early as one week post vaccination with all pigs yielding neutralizing antibodies (nAb) against the vaccine virus at two weeks post vaccination. After challenge no clinical signs were observed in any of the vaccinated animals whereas non-vaccinated control animals developed fever starting in general on the fourth day post challenge. In vaccinated pigs no challenge virus could be isolated from leucocyte samples taken on days 3 to 7 post challenge while HCV was isolated from buffy coat leucocytes of all non-vaccinated animals. Six out of eight control animals were sacrificed and viral antigen was detected in tonsils, mandibular lymph node and spleen in four animals, exclusively in tonsils in one animal and none in another animal. Two non-vaccinated animals that survived the experiment seroconverted after challenge and developed nAb against the HCV strain ALFORT/187.

[Field infection with BVD virus in swine: epidemiology and diagnosis]. / Feldinfektion mit BVD-Virus beim Schwein: Epidemiologie und Diagnostik.

In a pig breeding herd in Lower Saxony infertility of breeding sows had been repeatedly observed. Growth retardation and post mortem findings in two piglets gave clinical indication to swine fever/hog cholera. A virus was isolated and typed by monoclonal antibodies as pestivirus not identical with hog cholera virus (HCV). In neutralization tests applying the field isolate, HCV and bovine viral diarrhea (BVD) virus the sera breeding sows and weaner pigs yielded high neutralizing antibody titres against the pestivirus field isolate but low titres against HCV. Specific antibodies against HCV were ruled out by a complex trapping blocking (CTB) ELISA. Intranasal inoculation of a weaner pig with spleen homogenate led to a short-termed viraemia without clinical signs but seroconversion with high antibody titres against the homologous pestivirus. In an in-contact pig no virus was detected and no antibody demonstrated within a period of 18 weeks.

[Prevalence of antibodies against the viruses of European swine fever, Aujeszky's disease and "porcine reproductive and respiratory syndrome" in wild boars in the federal states Sachsen-Anhalt and Brandenburg]. / Prävalenz von Antikörpern gegen die Viren der Europäischen Schweinepest, der Aujeszky'schen Krankheit und des "Porcine reproductive and respiratory syndrome" (PRRS) bei Wildschweinen in den Bundesländern Sachsen-Anhalt und Brandenburg.

During the hunting season from 1991/1992 blood samples were collected from wild boar shot in the Federal States of Sachsen-Anhalt (482 samples) and Brandenburg (177 samples) which corresponds to 2.1 and 0.4% of the total hunting bag. All sera were screened in a complex trapping blocking (CTB) ELISA for antibodies against hog cholera virus (HCV) and in an indirect ELISA for antibodies against Aujeszky's disease virus (ADV). Additionally the sera were tested for neutralizing antibodies against HCV strain ALFORT/187, bovine viral diarrhoea virus (BVDV) strains NADL and 1138/69, and against an ADV field isolate. In case of questionable results sera were tested against HCV strain "BERGEN", HCV vaccine strain "RIEMS" and three HCV field isolates from wild boar. The serological testing for antibodies against "porcine reproductive and respiratory syndrome virus" (PRRSV) was carried out in indirect immunoperoxidase monolayer assay (IPMA). Four sera (Sachsen-Anhalt) reacted positive in CTB-ELISA. Seven sera yielded neutralizing antibodies against HCV but only one of the "non-negative" samples scored positive in both techniques, ELISA and VNT. Two sera (Brandenburg) had low neutralizing antibody titres against Alfort/197 but scored negative in CTB-ELISA. Screening for antibodies against ADV of 640 sera led to 13 positive sera including 5 positive findings in both ELISA and VNT. Antibodies against PRRSV were detected in two sera which were collected Sachsen-Anhalt. Estimations resulted in a prevalence of about 5% for antibodies against HCV.

Antibody prevalence of hog cholera, bovine viral diarrhoea and Aujeszky's disease virus in wild boars in northern Germany.

During the hunting season 1990/1991 a total of 841 blood samples was collected from shot wild boar corresponding to about 2.11% of the total hunting bag in Lower Saxony. All the sera were screened for neutralizing antibodies (nAb) to hog cholera virus (HCV) and bovine viral diarrhoea virus (BVDV) by direct neutralizing peroxidase linked antibody (NPLA) assay. For the detection of antibodies (Ab) against HCV a Complex Trapping Blocking (CTB) ELISA was used. Cytotoxic sera were retested using an indirect immunoperoxidase technique. Additionally all sera were tested for antibodies to Aujeszky's disease virus (ADV) in a commercial indirect ELISA and 722 sera for ADV nAb in a virus neutralization test (VNT) with and without the addition of guinea pig complement. Screening of 841 sera yielded six sera neutralizing HCV strain ALFORT/187 and two sera with a positive CTB-ELISA reaction. A total of seven sera not identical with the HCV seropositive sera yielded BVDV neutralizing antibodies. The majority of HCV nAb positive samples were found in a region close to the former border to the German Democratic Republic (GDR) west of the State of Sachsen-Anhalt. Screening for ADV detected five sera positive in ELISA amongst 12 sera yielding nAb against ADV. Positive samples were obtained in the regions of Brunswick, Lüneburg and Hannover.

Clinical, virological and serological findings after intranasal inoculation of pigs with bovine viral diarrhoea virus and subsequent intranasal challenge with hog cholera virus.

Five groups of weaner pigs were intranasally inoculated with constant doses of bovine viral diarrhoea virus (BVDV) strain OSLOSS/2482. Four weeks post primary inoculation (p.p.i.) the animals were intranasally challenged with decreasing doses of hog cholera virus (HCV) strain Alfort/187. Clinical signs were not observed apart from a short febrile period (2 days, > 40 degrees C) in one animal. Another animal died intercurrently without showing any pathological signs. Virus isolation from leucocyte samples taken regularly during one week post challenge detected HC viraemia in most animals that had received HCV doses > 100 TCID50 per animal. Using monoclonal antibody (mab) analysis all isolates obtained were proven to be HCV. Serological investigations using the virus neutralization test (VNT) yielded HC neutralizing antibodies in all groups with higher titres in those animals having received HCV doses > 100 TCID50. However, HCV specific neutralizing antibodies never exceeded the BVDV antibody titre. A complex trapping blocking (CTB) ELISA applying a HCV specific mab detected HCV specific antibodies in animals that had gone through HC viraemia while discriminating BVDV specific antibodies.

A review on classical swine fever infections in pigs: epizootiology, clinical disease and pathology.

A review is given on classical swine fever (CSF) including epizootiology, clinical disease and pathology. Under the item of epizootiology the history of CSF is briefly summarized. Ways of transmission are described with special reference to CSF in wild boars. The chapter about clinical disease includes the description of different courses of CSF such as peracute, acute, subacute form and chronic disease with reference to the course of transplacental infection and fate of the progeny associated with the "carrier sow syndrome". The most typical lesions in CSF are summarized in the chapter of pathology.

Clinical, post mortem and virological findings after simultaneous inoculation of pigs with hog cholera and bovine viral diarrhoea virus.

The clinical course, post mortem lesions as well as virological and serological results after simultaneous intranasal inoculation of pigs with bovine viral diarrhoea virus (BVDV) and hog cholera virus (HCV) are described. Five groups of four weaners received constant doses of BVDV strain OSLOSS/2482 and tenfold decreasing doses of HCV strain ALFORT/187. Doses of 1,000 and 100 TCID50 of HCV in groups A and B of pigs led to fever and severe clinical signs in all animals of two groups, whereas at higher dilution of inoculum two, three or four animals survived without any clinical signs in the respective groups (C-E). Leucocyte samples taken from febrile animals and from normal pigs on five consecutive days were inoculated into both fetal calf kidney (FCK) and PK (15) cell cultures. Virus isolates were differentiated with BVDV and HCV specific monoclonal antibodies. HCV viraemia was detected in febrile animals exclusively, and BVDV viraemia occurred in not affected animals on days 3 to 7 post inoculation. Neutralizing antibodies (nab) against BVDV appeared before HCV nab in surviving animals of groups C and D after receiving low doses of HCV (10 or 1 TCID50). No BVDV nab were detected in group E that had received such a high dilution of HCV in addition to BVDV that theoretically no HCV was applied.

[Use of monoclonal antibodies for the differential diagnosis of pestivirus infections in swine]. / Anwendung monoklonaler antikörper zur Differentialdiagnose von Pestivirusinfektionen beim Schwein.

Monoclonal antibodies (mab) specific for hog cholera virus (HCV), bovine viral diarrhoea virus (BVDV) or pestivirus were applied for the differential diagnosis of pestivirus infections in pigs. Field virus isolated from 8 confirmed classical swine fever outbreaks and one suspect case was propagated in PK(15) cell cultures and identified by direct immunofluorescence (IFA) and peroxidase linked antibody (PLA) assays. Peroxidase-linked HCV, BVDV and pestivirus specific mab were applied in direct PLA for differentiation. Nine isolates were classified as members of the genus pestivirus. Eight isolates showed a positive reaction with an HCV mab. One isolates reacted with BVDV specific mab only. For further characterization an indirect PLA was performed using a collection of different HCV and BVDV specific mabs. Some of the HCV isolates also showed a weak reaction with BVDV specific mab.

A new approach for the diagnosis of hog cholera.

Pestiviruses were isolated from seven cases of suspect hog cholera. Using peroxidase conjugates of monoclonal antibodies (Mabs) six isolates were identified as hog cholera viruses (HCV), while one isolate was of ruminant origin, possibly bovine viral diarrhea virus. In parallel attempts were made to develop an ELISA for the detection of HCV-specific antibodies in pig sera. The Mab HCTC26 coated to polystyrol plates efficiently captured the major viral glycoprotein gp53 from crude antigen suspensions prepared from infected cells. The immobilized gp53 served as diagnostic antigen. Five pigs experimentally infected with the HCV strain Glentorf were sequentially bled and the development of antibodies was monitored by neutralization tests and the ELISA. Results showed that both tests detected antibodies simultaneously after infection. Titres measured by ELISA were slightly higher than those registered by neutralization.

Identification of conserved epitopes on a hog cholera virus protein.

Eight monoclonal antibodies directed against the hog cholera virus (HCV) strain Alfort/187 and displaying broad cross-reactivity with other HCV strains were characterized. An enzyme immunoassay on fixed monolayers of porcine or bovine cells infected with 14 different strains and isolates of HCV and 12 bovine viral diarrhea viruses (BVDV), respectively, showed that all antibodies reacted with HCV only. Seven antibodies recognized all HCV tested, thus indicating that they were directed against conserved epitopes. All antibodies neutralized the homologous strain and different patterns of the other HCV tested. Radioimmunoprecipitation analysis showed that the monoclonal antibodies were directed against a doublet of 56-60 kDa, presumably representing the major envelope glycoprotein of HCV. The results of reciprocal antibody blocking assays allowed the mapping of two distinct conserved antigenic domains on this protein.

Differentiation of hog cholera and bovine virus diarrhoea viruses in pigs using monoclonal antibodies.

Monoclonal antibodies against hog cholera and bovine viral diarrhoea viruses were assayed on organ tissue sections of experimentally infected animals. The animals had been infected simultaneously with both viruses. The antibodies were tested using an indirect immunofluorescence test and an indirect enzyme immunoassay with a biotin/streptavidin/peroxidase detection system. A polyclonal hyperimmune serum was used as a control in direct immunofluorescence tests. Both techniques based on monoclonal antibodies were more sensitive and more specific than the conventional test, the enzyme immunoassay being more sensitive than the immunofluorescence test. Small amounts of BVD viral antigen were demonstrable with monoclonal antibodies in most organ tissues.