Ilse Schwendenwein

Establishment and characterization of a novel canine B-cell line derived from a spontaneously occurring diffuse large cell lymphoma

Cell lines derived from spontaneous tumors serve as a research tool for cancer cell biology and new anti-cancer drug development. Isolation and propagation of canine lymphoma cell lines is difficult, thus only a few are available. Now we have established a new B-cell lymphoma cell line CLBL-1 from a dog with confirmed stage IV diffuse large cell lymphoma. Immunophenotyping of these CLBL-1 cells showed positive staining for CD11a, CD79alphacy, CD45, CD45RA, MHC II and cells were negative for CD3, CD4, CD5, CD8, CD11d, CD14, CD21, CD34, CD56 and T-cell receptor-gammadelta (TCR-gammadelta). PCR analysis for TCR-gamma and immunoglobulin heavy chain (IgH) gene rearrangements yielded a monoclonal result for the IgH gene. Furthermore, the clonality of IgH gene rearrangement was confirmed by sequencing of 16 positive bacterial clones. As canine lymphoma resembles non-Hodgkins lymphoma (NHL) in humans in many respects, this new cell line, will promote translational and comparative lymphoma research in humans and dogs.

Cell therapy using microencapsulated 293 cells transfected with a gene construct expressing CYP2B1, an ifosfamide converting enzyme, instilled intra-arterially in patients with advanced-stage pancreatic carcinoma: a phase I/II study.

Matthias Lohr (principal investigator) · Zoltan Tibor Bago · Helga Bergmeister · Manfred Ceijna · Mathias Freund · Wolfgang Gelbmann · Walter H. Gunzburg · Ralf Jesnowski · Johannes Hain · Karlheinz Hauenstein Wolfgang Henninger · Anne Hoffmeyer · Peter Karle · Jens-Christian Kroger · Gunther Kundt · Stefan Liebe Udo Losert · Petra Muller · Alexander Probst · Katrin Puschel · Matthias Renner · Renate Renz · Robert Saller Brian Salmons · Maximilian Schuh · Ilse Schwendenwein · Kerstin von Rombs · Thomas Wagner · Ingrid Walter (coinvestigators)

Biocompatibility of polyethylene terephthalate (Trevira® hochfest) augmentation device in repair of the anterior cruciate ligament

The biocompatibility of a 3 mm band made of polyethylene terephthalate (Trevira hochfest) has been tested in an experimental study within right knee joints of 60 sheep. After transsecting the anterior cruciate ligament (ACL), two randomized groups were formed. In group I, the ACL was repaired according to the Marshall technique whilst in group II an additional 3 mm polyethylene terephthalate (PET) augmentation band was implanted using the through-the-condyle (TTC) procedure. To assess the biocompatibility of the augmentation device the knee joints of both groups were punctured and the synovial fluids were analyzed before, as well as 2, 6, 16, 26, and 52 weeks after the operation. In addition, the histologic appearance of excised suprapatellar pouches and ipsilateral inguinal and popliteal lymphatic noduli were examined. Comparing both groups no significant differences were found neither before nor after the augmented and non-augmented ACL repair. No pathological increase in the total protein concentration occurred after operation and no significant differences versus the preoperative analysis were found. No synovitis signalling a decrease in the glucose concentration was observed. The cytological examination revealed no increase of the leukocyte cell count results. Within the synovial specimen neither free nor phagocytosed PET wearparticles could be detected. In groups I and II the histological appearance of excised popliteal and inguinal lymphatic noduli showed a normal result. In 25% of the PET augmented ACL repairs, a slight concentration of PET wearparticles and solitary, multinuclear giant foreign body cells could be seen in the histological preparations of suprapatellar pouches.

Progression of burn wound depth by systemical application of a vasoconstrictor: an experimental study with a new rabbit model

The final depth of a necrosis resulting from burn trauma is determined within 3 days. The zone of stasis has the potential for complete regeneration or there may be ischemic influences that lead to necrosis. In our model, we examined the dermal influence of vasoconstrictors with reference to the development of burn necrosis. On the backs of New Zealand white rabbits (4.0-4.5 kg) standardized lesions were made with a heated aluminum stamp at 80 degrees C, 14 s in duration. The lesions were intradermal, whereby the border zone of the coagulated tissue was found in the middle two quarters of the dermis in 100% of untreated animals after 72 h. For dermal vasoconstriction epinephrine in a dose of 0.5 microg/kg/min was used. There were two groups of seven animals each. One group received epinephrine and the dosage was dependent on the clinical state of the animal. Several cycles were administered within a 3-day period. The reduction of skin perfusion was documented by Laser-Doppler-flowmetry. After 3 days, the skin with the lesions was excised and using a hematoxylin dye, a histological examination followed. The parameter used to determine the efficacy was the thickness of the uncoagulated part of the excised dermis. Over a period of 48 h, an average of 2.3 epinephrine cycles of average of 88 min per animal in duration resulted in an average reduction of skin diffusion of 41%. The uncoagulated part of the dermis in the epinephrine group was 28.6% average; in the control group, this was 43.5%. The statistical analysis revealed significant differences with a p-value of 0.0312 (significant, when value is less than 0.05). The test results indicate that temporary reduction of skin perfusion through external administration of vasocontrictors may lead to progression of burn necrosis in our animal model. Clinically, this result indicates that for patients with burn injuries and systemic inflammatory response syndrome who have insufficient volume therapy, the administration of vasocontrictors may produce similar results in the injured area.

Controlled partial skin thickness burns: an animal model for studies of burnwound progression.

A practicable, reliable and reproducible model for infliction of partial skin thickness burn lesions in rabbits is presented. The model is dedicated to experimental studies investigating the influence of drugs on burn wounds. A round aluminium stamp with a contact area of 4 cm2, weight 85 g, was heated up to 80 degrees C and applied for 14 s without additional pressure on the depilated dorsal skin of rabbits. This procedure produced the desired partial skin thickness burn injury. The depth of the burn lesions was investigated by HE-stained paraffin sections. The border of the necrotic zone was found in the central third of the dermis in 80% of cases, and in the central two quarters in 100%. These results are achieved when the rabbits hair at the site of infliction is in the anagen phase of the hair growth cycle. For obtaining reproducible results we recommend using rabbits of the same strain and weight, anagen hair growth phase, the described procedure of infliction, an identical stamp and the specified temperature and infliction time.

Expression of vascular endothelial growth factor and its receptors in canine lymphoma.

Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and has a pivotal role in tumour angiogenesis. The expression of VEGF and its receptors VEGFR-1 and VEGFR-2 was examined immunohistochemically in 43 specimens of canine lymphoma and in six normal lymph nodes. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect VEGF protein and mRNA, respectively. VEGF protein was expressed by 60% of the tumours with diffuse cytoplasmic labelling of the neoplastic cells. Endothelial cells, macrophages and plasma cells were also immunolabelled. VEGFR-1 was expressed by variable numbers of neoplastic cells in 54% of lymphoma specimens. VEGFR-1 was also expressed by macrophages, plasma cells, reticulum cells, and vascular endothelial cells. Macrophages and lymphocytes in germinal centres of normal lymph nodes were also immunoreactive with anti-VEGF and VEGFR-1. Most tumours did not express VEGFR-2 but in 7% of sections there was focal labelling of neoplastic and endothelial cells, with a cytoplasmic and perinuclear pattern. The observed variability in expression of VEGF and its receptors probably relates to the fact that lymphoma is a heterogeneous lymphoproliferative tumour. Individual differences in VEGF and VEGFR expression must be taken into account when VEGF and VEGFR-targeted approaches for anti-angiogenic therapy are considered in dogs.

Detection of Encephalitozoon cuniculi in the feline cataractous lens

PURPOSE Identification of Encephalitozoon cuniculi (E. cuniculi) as a possible causative agent for cataracts and uveitis in cats. METHODS Within a 12-month study period, cats that were presented with focal anterior cortical or mature cataract and secondary uveitis underwent a complete ophthalmic examination, complete blood count, serum biochemistry, serologic tests for E. cuniculi and tests for feline immunodeficiency virus (FIV), feline infectious peritonitis (FIP), feline leukemia virus (FeLV) and Toxoplasma gondii (T. gondii). PCR for DNA detection of E. cuniculi and T. gondii as well as cytologic examination of aqueous humor after paracentesis and phacoemulsified lens material were also performed. In addition histopathologic examination of the resected anterior lens capsule and attached lens epithelial cells was performed. Serologic testing for antibodies against E. cuniculi was also performed in 100 ophthalmologically healthy cats. RESULTS Eleven (19 eyes) European shorthair cats with a median age of 3.5 years were included. Nine of 11 cats had bilateral cataracts, with 12/19 eyes having focal anterior cortical cataracts and 7/19 eyes having mature cataracts. In 14/19 eyes anterior uveitis was present. All cats had a positive antibody titer (1:80-1:10,000) for E. cuniculi. Encephalitozoon cuniculi DNA was detected by PCR and sequencing in 18/19 lenses and in 10/19 aqueous samples. Five tentative positive results were detected by cytologic examination. Spores were detected in 15/19 samples of lens material with histopathologic staining. Only 2/100 ophthalmologically healthy cats showed a positive antibody titer for E. cuniculi. CONCLUSION Encephalitozoon cuniculi is a cause of focal anterior cortical cataract and anterior uveitis in cats.

Evaluation of mitoxantrone-loaded albumin microspheres following intraperitoneal administration to rats.

Mitoxantrone has demonstrated therapeutic efficacy in the regional treatment of intraperitoneal malignancies. However, severe local toxicity was dose limiting. Consequently, a new injectable sustained delivery formulation of mitoxantrone has been developed: the drug was incorporated (8.2 w/w%) in highly hydrophilic albumin microspheres. In vitro drug release profile was modified by matrix crosslinking extent. The extractable amount of residual crosslinking agent (glutaraldehyde) in the microspheres was lower than 6 ppm. Mitoxantrone concentration in peritoneal fluid and plasma was determined up to 72 h after intraperitoneal administration of 30, 60 and 120 mg mitoxantrone per m2 body surface area as solution and in the form of a dispersion containing mitoxantrone-loaded microspheres to rats. Data analysis revealed sustained release of mitoxantrone from microspheres into peritoneal fluid in all dosage groups. The initial high drug levels in peritoneal fluid and plasma observed after application of mitoxantrone in the solution form were prevented by administration of the drug incorporated in microspheres. However, tumoricidal drug levels in peritoneal fluid were maintained over a comparable time span. In addition, preliminary toxicity data suggest a superior local tolerability of mitoxantrone-loaded microspheres. The dose of intraperitoneally administered mitoxantrone might be increased from 30 to 60 mg per m2 body surface area using the slow release formulation. In conclusion, the described microsphere drug delivery system for mitoxantrone might overcome dose-limiting drug toxicity.

Intraarterial instillation of microencapsulated cells in the pancreatic arteries in pig.

JENS C. KROGER,a,f HELGA BERGMEISTER,b ANNE HOFFMEYER,c,d MANFRED CEIJNA,b PETER KARLE,d,e ROBERT SALLER,d ILSE SCHWENDENWEIN,b KERSTIN VON ROMBS,d STEFAN LIEBE,c WALTER H. GUNZBURG,e BRIAN SALMONS,d KARLHEINZ HAUENSTEIN,a UDO LOSERT,b AND MATTHIAS LOHRc aDepartment of Diagnostic and Interventional Radiology, University of Rostock, Germany bCenter for Biomedical Research, General Hospital, University of Vienna, Austria cDepartment of Medicine, University of Rostock, Germany dBavarian Nordic Research Institute, Munich, Germany eInstitute of Virology, University of Veterinary Sciences, Vienna, Austria

Diagnostic value of the neutrophil myeloperoxidase index in horses with systemic inflammation.

The myeloperoxidase index (MPXI) was investigated as a diagnostic indicator of systemic inflammation in a retrospective study using data from 859 hospitalised horses. A reference interval of 8.5-10.4 for the MPXI was established. In horses with systemic inflammatory response syndrome (SIRS), the MPXI was significantly lower than in healthy horses, those with localised inflammation and those with sepsis. The MPXI in horses with sepsis was also significantly lower than in healthy animals and those with localised inflammation. Horses in the SIRS group with leucopenia, white blood cell (WBC) count within the reference interval (WRI) or leucocytosis had significantly lower MPXIs than healthy horses, those with localised inflammation and those with sepsis in the same WBC count subgroups. In horses with sepsis and WBC count WRI, the MPXI was significantly lower than in healthy horses or those with localised inflammation. MPXI is a useful complementary tool to identify horses with systemic inflammation, especially if they have WBC counts WRI.