Csaba Jakab

Strong synergy of heat and modulated electromagnetic field in tumor cell killing

Background and Purpose:Hyperthermia is an emerging complementary method in radiooncology. Despite many positive studies and comprehensive reviews, the method is not widely accepted as a combination to radiotherapy. Modulated electrohyperthermia (mEHT; capacitive, electric field modulated, 13.56 MHz) has been used in clinical practice for almost 2 decades in Germany, Austria and Hungary. This in vivo study in nude mice xenograft tumors compares mEHT with “classic” radiative hyperthermia (radHT).Material and Methods:Nude mice were xenografted with HT29 human colorectal carcinoma cells. 28 mice in four groups with seven animals each and two tumors per animal (totally 56 tumors) were included in the present study: group 1 as untreated control; group 2 treated with radHT at 42 °C; group 3 treated with mEHT at identical 42 °C; group 4 treated with mEHT at 38 °C (by intensively cooling down the tumor). 24 h after treatment, animals were sacrificed and the tumor cross sections studied by precise morphological methods for the respective relative amount of “dead” tumor cells.Results:The effect of mEHT established a double effect as a synergy between the purely thermal (temperature-dependent) and nonthermal (not directly temperature-dependent) effects. The solely thermal enhancement ratio (TER) of cell killing was shown to be 2.9. The field enhancement ratio (FER) at a constant temperature of 42 °C was measured as 3.2. Their complex application significantly increased the therapeutic enhancement to 9.4.Conclusion:mEHT had a remarkable cancer cell-killing effect in a nude mice xenograft model.Hintergrund und Ziel:Die Hyperthermie ist eine aufstrebende ergänzende Therapie in der Radioonkologie. Trotz zahlreicher positiver Studien und umfassender Reviews ist diese Methode immer noch nicht als Kombination zur Radiotherapie anerkannt. Die modulierte Elektrohyperthermie (mEHT; kapazitiv mit moduliertem elektrischem Feld, 13,56 MHz) wird seit fast 2 Jahrzehnten in Deutschland, Österreich und Ungarn klinisch angewandt. Die vorliegende In-vivo-Studie vergleicht in einem Xenograft-Nacktmaus-Tumormodell die mEHT mit der „klassischen“ radiativen Hyperthermie (radHT).Material und Methodik:Nacktmäuse wurden mit humanen kolorektalen HT29-Tumorzellen xenotransplantiert. 28 Mäuse in vier Gruppen zu je sieben Tieren mit zwei Tumoren pro Tier (gesamt 56 Tumoren) wurden in diese Studie einbezogen: Gruppe 1 als unbehandelte Kontrollgruppe; Gruppe 2 behandelt mit radHT bei 42 °C; Gruppe 3 behandelt mit mEHT ebenfalls bei 42 °C; Gruppe 4 behandelt mit mEHT bei 38 °C (durch intensive Kühlung des Tumors). 24 h nach der Behandlung wurden die Tiere getötet und die Tumorquerschnitte morphologisch auf den jeweiligen Anteil „toter“ Tumorzellen untersucht.Ergebnisse:Die Behandlung mit mEHT zeigte eine doppelte Wirkung als Synergie zwischen dem ausschließlich thermalen (temperaturabhängigen) und dem nichtthermalen (nicht direkt temperaturabhängigen) Effekt. Folgende Faktoren wurden gemessen: die durch alleinige Hyperthermie bedingte Verstärkung der Zellzerstörung („thermal enhancement ratio“ [TER]) mit dem Faktor 2,9; der alleinige Feldverstärkungseffekt („field enhancement ratio“ [FER]) bei konstanter Temperatur von 42 °C mit dem Faktor 3,2; die Kombination beider Effekte mit einem signifikant erhöhten Faktor von 9,4.Schlussfolgerung:Die durch ein moduliertes elektrisches Feld (13,56 MHz) erzeugte mEHT hatte in einem Nacktmaus-Xenograft-Tumormodell einen ausgeprägten tumorzellabtötenden Effekt.

Efficacy of protected sodium butyrate, a protected blend of essential oils, their combination, and Bacillus amyloliquefaciens spore suspension against artificially induced necrotic enteritis in broilers

Necrotic enteritis caused by Clostridium perfringens leads to serious economical losses to the poultry industry. There is a growing need to find effective, nontoxic, antibiotic alternatives to prevent and cure the disease. In our study, the efficacy of protected sodium butyrate at 1.5 g/kg (BP70), a Bacillus amyloliquefaciens spore suspension with 10(9) cfu/g (BAL; Ecobiol), a protected blend of essential oils (1%) at 1.5 g/kg (EO), and a combination of sodium butyrate with essential oils (1%) protected with vegetable fat at 1.5 g/kg (BP70+EO; Natesse) was investigated in an artifical C. perfringens-infection model. Body weight gain, gross pathological and histopathological lesion scores, villus lengths, and villus length:crypt depth ratio was determined and compared with the control group. Broilers infected with C. perfringens and treated with essential oils or the combination of sodium butyrate and essential oils showed significantly better BW gain (P < 0.05), increased villus length and villus length:crypt depth ratio (P < 0.001), and decreased gross pathological and histopathological lesion scores (P < 0.05) compared with the control. Sodium butyrate alone and B. amyloliquefaciens spore suspension had no beneficial effects on the course of the disease in this study. According to our results, the protected combination of sodium butyrate and essential oils, as well as the protected essential oils, can be potential candidates for the prevention and treatment of necrotic enteritis in broiler chickens.

Naturally occurring parvoviral infection in Hungarian broiler flocks

The major enteric disease (ED) complex in broiler chickens is runting–stunting syndrome and in turkey broilers is poult enteritis mortality syndrome. Viruses from numerous families have been identified in the intestinal tracts of poultry with ED, such as Astroviridae, Coronaviridae, Reoviridae, Rotaviridae, and Parvoviridae. The objective of the present study was to directly demonstrate the presence of the scarcely known chicken parvovirus (ChPV) and turkey parvovirus (TuPV) in Hungarian flocks experiencing clinical signs of ED. ChPV and TuPV infection were demonstrated in 15 chicken flocks and two turkey flocks, in intestinal samples collected between 2008 and 2010. The histopathological investigation revealed enteritis in the duodenum and jejunum, and atrophy of the lymphoid organs. Indirect immunohistochemistry (IHC) suggested the intestinal epithelium of chickens and turkeys as a potential replication site of the virus, similarly to other parvoviruses, while in case of the turkey samples IHC positivity was also observed in the bursa of Fabricius, liver and pancreas. However, no direct connection could be established between the presence of the pathogen in the above-mentioned tissues and the histopathological changes observed in the investigated flocks. The phylogenetic analysis performed on the partial nucleic acid sequence of the NS1 gene revealed an evident clustering tendency of the ChPV and TuPV strains, but also highlighted the potential reciprocal role of these two species in the epidemiology of these viruses. The role of the ChPV and TuPV in the ED is far from understood, but the results of the present study emphasize the fact that in certain, still not fully elucidated conditions, ChPV and TuPV may participate in the emergence of ED in chicken flocks, as suggested by previous experimental infections.

Acute Oxidative Stress Affects IL-8 and TNF-α Expression in IPEC-J2 Porcine Epithelial Cells

Reactive oxygen species are implicated in cell and tissue damage in a number of diseases including acute and chronic inflammation of the gut. Effects of H2O2 exposure on non-carcinogenic porcine epithelial cell line, IPEC-J2 cells cultured on collagen-coated membrane inserts were monitored based on transepithelial electrical resistance (TER) change, extent of necrotic cell damage, gene expression of inflammatory cytokines IL-8 and TNF-α. Furthermore, the junction proteins claudin-1 and E-cadherin were also investigated by immunohistochemistry. Peroxide (1mM) increased IL-8 and TNF-α gene expression levels significantly allowing 1 h recovery time without affecting the cellular distribution of junction proteins, TER and cell survival rate. In conclusion, the IPEC-J2 cell line on membrane insert was introduced as a fast and reliable investigation tool for oxidative stimuli-triggered intestinal inflammation and in the future as a screening method for antioxidant and probiotic candidates.

Evaluation of microvessel density (MVD) in canine mammary tumours by quantitative immunohistochemistry of the claudin-5 molecule

In our recent investigation, angiogenesis was evaluated and quantified by immunohistochemical evaluation of microvessel density (MVD) using claudin-5 (CLDN-5) as a marker for vascular endothelium in 67 canine mammary gland tumours. Computer image analysis was used to measure the intratumoural MVD. Higher intratumoural MVD was detected in malignant simple neoplasms compared with benign tumours. Furthermore, the results of MVD were correlated with histological grade, higher grades being accompanied by higher MVD. In simple adenomas and grade I tubular-tubulopapillary simple carcinomas the intratumoural microvessels were wide and regular in shape with evident erythrocytes in their lumen. In grade III solid carcinomas the microvessels were smaller, less regular and had irregular shape, often without a distinct lumen, and isolated endothelial cells were frequently present. In the complex carcinomas MVD was low and the intratumoural microvessels were mostly irregular in shape without a distinct lumen. The evaluation of MVD by CLDN-5 immunohistochemistry may give useful additional information on the angiogenic potential of breast cancers in dogs.

Expression and localisation of claudin-1, -2, -3, -4, -5, -7 and -10 proteins in the normal canine mammary gland.

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumour development. The aim of the present study was to analyse the expression of claudin-1, -2, -3, -4, -5, -7 and -10 in normal canine mammary glands. Samples from the inguinal mammary regions of 20 non-castrated, 1-13 years old female dogs were studied. Immunohistochemical analysis was performed on conventional specimens and tissue microarrays. The results of the immunohistochemical reactions detecting claudins in tissue sections were photodocumented. The immunoreactivity of claudins was quantitatively analysed on digital images using Leica QWin morphometry software. Intense membranous immunolabelling was found for claudin-1, -3 and -7, intense membranous with non-granular cytoplasmic immunolabelling for claudin-2, moderate membranous immunolabelling for claudin-4 and -5, and weak membranous immunolabelling for claudin-10. The occurrence of tight junctions was confirmed by ultrathin section electron microscopy. The available data suggested that claudins might be proteins preserved throughout the evolution of mammals. The results of our study support the concept that they are indeed preserved, since the same type of claudins, in identical distribution, could be detected in our canine mammary tissue samples as could be found in human mammary tissue.

Misleading results of the MboII-based identification of type 2a canine parvovirus strains from Hungary reacting as type 2c strains

Type 2 canine parvovirus (CPV2) infection is one of the most frequent causes of death in the young, susceptible canine populations worldwide. Since its emergence in the 1970s, several variants have been described. In the present study the authors describe the genetic analysis of 24 Hungarian CPV2 strains collected from 2004 to 2008. Surprisingly, the genetic and phylogenetic investigations of all these strains revealed that all of them were type 2a CPVs. On the other hand, the genetic analysis provided substantial evidence to demonstrate that due to a seemingly constant point mutation present in most of the Hungarian CPV2a strains, a previously described MboII-based rapid identification of CPV2c strains unfortunately cannot be reliably used any more.

Evaluation of EnSeal®, an adaptive bipolar electrosurgical tissue-sealing device

Relatively few, and inconsistent, data are available in the literature about the properties of EnSeal®, an electrosurgical tissue-sealing device. For this reason, we conducted control safety tests on experimental pigs. The mean burst pressure of sealed vessels (2-7 mm in diameter) proved to be 873.89 ± 120.57 mmHg (n = 60). Surface temperature increased to 69.25 ± 0.98 °C in average (n = 22). The mean diameter of the collateral microscopic thermal injury zone was found to be 0.28 ± 0.04 mm, and it did not show significant differences among the groups of tissues studied (n = 183). During our studies, the device worked reliably and met the relevant requirements in all cases. It can be established that EnSeal® enables high-safety clinical interventions at high blood pressure values, in different tissues and even at sites adjacent to heat-sensitive tissues, and thus it paves the way for new operative solutions in both human and veterinary surgery. In our opinion, the discrepancies between data reported in the literature arise from differences in the design of studies and in the designated limit values. To ensure standardisation, we recommend the use of the nitroblue-tetrazolium chloride/lactate dehydrogenase (NBTC/LDH) enzyme histochemical technique for studying thermal injury induced by the different performance levels and application times of devices operating with electromagnetic energy.

Skin and skeletal system lesions of european pond turtles (Emys orbicularis) from natural habitats

Water pollution is known to play an important role in the pathogenesis of plastron, carapace and skin diseases of turtles. In this study, a total of 150 European pond turtles (Emys orbicularis) of different age and both sexes, originating from natural habitats in Serbia, were examined for morphological changes of the skin, plastron, carapace and skeletal system. The turtles were taken out from their natural habitats in Lake Ludas, Lake Palic and Lake Tresetiste. After artificial hibernation, they were subjected to detailed examination, sampled and treated, and finally returned into their natural habitat. Biopsies from the skin and shell were subjected to histopathological examination and microbiological analysis. X-ray scanning was also performed to detect changes in the skeletal system. Macroscopic changes of the skin, most frequently degenerative, inflammatory or neoplastic diseases, were diagnosed in 49.33% of the turtles examined. Dermatitis of different origin and form was the most prominent histopathological finding (28.00%). In the plastron, inflammatory and degenerative processes were frequently found. Osteopathy and mechanical injuries were the dominant findings. Macroscopic changes of the plastron, carapace and skeletal system were diagnosed in 67.33% of the turtles examined. Using X-ray scanning, generalised osteopathy, anomalies and malformations of different aetiology were also diagnosed on the tail and legs. Microbiological examinations showed the presence of a variety of bacterial and fungal agents, either primary pathogens or potential polluters, which invaded the skin and shell, or were present in cloacal swab samples. Bacterial infection was diagnosed in 76.66% of the turtles, first of all in those with skin and shell necrosis. Mycoses were diagnosed in 33.33% of the animals.

Postmortem small babesia-like morphology of Babesia canis — Short communication

Here we report a case of canine babesiosis with unusual morphology of the causative agent. A male, seven-week-old Labrador retriever puppy, exhibiting severe anaemia and haemoglobinuria, was presented at the Clinic of Internal Medicine in February 2011. The puppy was euthanised. The most relevant pathological changes were icterus, severe splenomegaly, generalised lymphadenopathy and haemoglobin nephrosis. Samples were collected from various organs for histology within one hour post mortem. Impression smears were also prepared from the spleen after overnight storage at 4 °C. Tissue sections and smears showed the presence of multiple, coccoid intraerythrocytic bodies that measured 1-2 μm and resembled small babesiae. No large piroplasms were seen. DNA was extracted from the spleen, and a conventional PCR was performed for the amplification of a 450-bp region of the 18S rRNA gene of piroplasms. The causative agent was identified as Babesia canis canis, with 99% sequence identity to other European isolates. Sequence identity to B. gibsoni was only 91%. This is the first account to verify that the morphology of the large canine piroplasm, B. canis, can be uniformly small babesia-like post mortem or following the storage of tissue samples.