Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio).

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

Carp edema virus from three genogroups is present in common carp in Hungary.

Hungary is an important carp producer with intensive trading relationships with farms in other carp-producing areas in Europe. Carp in Europe were recently found infected with carp edema virus (CEV), a poxvirus which causes the koi sleepy disease (KSD) syndrome. Moribund carp were collected from 17 fish farms and angling ponds in different regions of Hungary. Histological analysis of gills from these carp revealed a proliferation of the interlamellar epithelium and an infiltration by eosinophilic cells. In 13 of 17 of these carp, CEV DNA was detected by qPCR and in seven fish more than 1 × 104 copies of virus-specific DNA sequences per 250 ng of DNA, which could be considered as clinically relevant and a cause of disease. A phylogenetic analysis of the sequences revealed that all three genogroups of CEV were present in Hungarian common carp with genogroup I being most abundant. These results support the hypothesis of a prolonged presence of CEV in European carp populations and suggest that previous outbreaks of KSD were not recorded or misdiagnosed. Hence, a testing of carp and koi for infection with CEV should be included into disease surveillance programmes to prevent further spreading of this disease.

Novel members of quinoline compound family enhance insulin secretion in RIN-5AH beta cells and in rat pancreatic islet microtissue.

According to clinical data, some tyrosine kinase inhibitors (TKIs) possess antidiabetic effects. Several proposed mechanisms were assigned to them, however their mode of action is not clear. Our hypothesis was that they directly stimulate insulin release in beta cells. In our screening approach we demonstrated that some commercially available TKIs and many novel synthesized analogues were able to induce insulin secretion in RIN-5AH beta cells. Our aim was to find efficient, more selective and less toxic compounds. Out of several hits, we chose members from a compound family with quinoline core structure for further investigation. Here we present the studies done with these novel compounds and reveal structure activity relationships and mechanism of action. One of the most potent compounds (compound 9) lost its affinity to kinases, but efficiently increased calcium influx. In the presence of calcium channel inhibitors, the insulinotropic effect was attenuated or completely abrogated. While the quinoline TKI, bosutinib substantially inhibited tyrosine phosphorylation, compound 9 had no such effect. Molecular docking studies further supported our data. We confirmed that some TKIs possess antidiabetic effects, moreover, we present a novel compound family developed from the TKI, bosutinib and optimized for the modulation of insulin secretion.

Pharmacophore and binding analysis of known and novel B-RAF kinase inhibitors.

The extensively investigated serine/threonine kinase, B-RAF, is a member of the RAS/RAF/MEK/ERK pathway. It plays important role in the regulation of cell growth, differentiation and survival. The mutation of B-RAF occurs frequently in melanomas and colon tumors; therefore, it is considered as an outstanding therapeutic target. In recent years a great number of B-RAF inhibitors have been reported and this number is expected to increase. The aim of our work was to compare the structures and binding mode of the published B-RAF inhibitors, and then to apply the correlations found for the explanation of our experimental results. In the first part of this paper we describe the main pharmacophore features of the co-crysallized B-RAF inhibitors published in the literature, focusing on the binding modes and common structural elements. In the second part we present and characterize our recently developed B-RAF inhibitor family by application of in silico methods and in vitro kinetic profiling. The inhibitory activity of these compounds was determined in biochemical kinase- and cell-based assays. The docking and assay results support our conclusion that the presented compound family belongs to the type I 1/2 subgroup, they inhibit B-RAF and B-RAF(V600E) mutant in a sub-micromolar range and most of them show selectivity towards B-RAF(V600E) mutant expressing cell lines with equal or even better IC50 values than sorafenib.

Developing FGFR4 inhibitors as potential anti-cancer agents via in silico design, supported by in vitro and cell-based testing.

Fibroblast growth factor receptor-4 (FGFR4) is a tyrosine kinase with a range of important physiological functions. However, it is also frequently mutated in various cancers and is now generating significant interest as a potential therapeutic target. Unfortunately, biochemical characterization of its role in disease, and further evaluation as a drug target is hampered by lack of a specific inhibitor. We aimed to discover new inhibitors for FGFR4 ab initio using a strategy combining in silico, in vitro and cell-based assays. We used the homologous FGFR1 to calculate docking scores of a chemically-diverse library of approximately 2000 potential kinase inhibitors. Nineteen potential inhibitors and ten randomly- selected negative controls were taken forward for in vitro FGFR4 kinase assays. All compounds with good docking scores significantly inhibited FGFR4 kinase activity, some with sub-micromolar (most potent being V4-015 with an IC(50) of 0.04 µM). Four of these compounds also demonstrated substantial activity in cellular assays using the FGFR4- overexpressing breast carcinoma cell line, MDA-MB453. Through immunoblot assays, these compounds were shown to block the phosphorylation of the FGFR4 adaptor protein, FGFR substrate protein-2α (FRS2α). The most potent compound to date, V4-015, suppressed proliferation of MDA-MB453 cells at sub-micromolar concentrations, activated the pro-apoptotic caspases 3/7 and inhibited cellular migration. While achieving complete selectivity of this compound for FGFR4 will require further lead optimization, this study has successfully identified new chemical scaffolds with unprecedented FGFR4 inhibition capacities that will support mechanism of action studies and future anti-cancer drug design.

Novel, selective CDK9 inhibitors for the treatment of HIV infection.

Cyclin Dependent Kinases (CDKs) are important regulators of cell cycle and gene expression. Since an up-to-date review about the pharmacological inhibitors of CDK family (CDK1-10) is not available; therefore in the present paper we briefly summarize the most relevant inhibitors and point out the low number of selective inhibitors. Among CDKs, CDK9 is a validated pathological target in HIV infection, inflammation and cardiac hypertrophy; however selective CDK9 inhibitors are still not available. We present a selective inhibitor family of CDK9 based on the 4-phenylamino-6- phenylpyrimidine nucleus. We show a convenient synthetic method to prepare a useful intermediate and its derivatisation resulting in novel compounds. The CDK9 inhibitory activity of the derivatives was measured in specific kinase assay and the CDK inhibitory profile of the best ones (IC(50) < 100 nM) was determined. The most selective compounds had high selectivity over CDK1, 2, 3, 5, 6, 7 and showed at least one order of magnitude higher inhibitory activity over CDK4 inhibition. The most selective molecules were examined in cytotoxicity assays and their ability to inhibit HIV-1 replication was determined in cellular assays.

The testis histology of artificially maturated European eel (Anguilla anguilla L.) at the end of sexual maturation, and spermatozoa ultrastructure in freshwater rearing. Short communication.

The artificial induction of sexual maturation of European eel males was carried out by using weekly hCG administrations. Histological pictures showed that the testis tissues developed and regressed naturally and no pathological changes took place under the conditions of artificial rearing in freshwater. According to light and electron microscopic investigations the morphology and motility of the spermatozoa of males kept in freshwater proved to be similar to those in seawater. The authors suppose that freshwater rearing of males is not a barrier factor in the artificial propagation of European eels.

The intestinal parasite Pomphorhynchus laevis as a sensitive accumulation indicator for the platinum group metals Pt, Pd, and Rh.

Concentrations of the platinum group elements Pt, Pd, and Rh were analyzed by adsorptive cathodic stripping voltammetry (Pt, Rh) and total-reflection X-ray fluorescence (Pd) in the intestinal helminth Pomphorhynchus laevis and its host Barbus barbus. The fish were caught in the Danube river south of the city of Budapest (Hungary) and were exposed to ground catalytic material for 28 days. Following exposure all three precious metals were taken up and accumulated in host organs and the parasites. Interestingly, in all tissues of the unexposed controls Pt was found, whereas Pd was not present in the muscle of the controls and Rh was not detectable in muscle and intestine of unexposed barbel. All metals were found at significantly higher concentrations in the acanthocephalan than in the tissues of barbel. These results are discussed with respect to the application of P. laevis as an accumulation indicator for metals.

The intestinal parasite Pomphorhynchus laevis (Acanthocephala) from barbel as a bioindicator for metal pollution in the Danube River near Budapest, Hungary.

Concentrations of As, Al, Ag, Ba, Bi, Cd, Co, Cr, Cu, Fe, Ga, Mg, Mn, Ni, Pb, Sb, Sn, Sr, Tl, V and Zn were analyzed by inductively coupled mass spectrometry (ICP-MS) in the intestinal helminth Pomphorhynchus laevis and its host Barbus barbus. The fish were caught in the Danube river downstream of the city of Budapest (Hungary). Ten out of twenty one elements analyzed were found at higher concentrations in the acanthocephalan than in different tissues (muscle, intestine, liver and kidney) of barbel. Considering the fish tissues, most of the elements were present at highest concentrations in liver, followed by kidney, intestine and muscle. Spearman correlation analyses indicate that there is competition for metals between the parasites and the host. The negative relationships between parasite number and metal levels in organs of the barbel support this hypothesis. The bioconcentration factors for Ag, As, Ba, Bi, Cu, Ga, Mn, Pb, Sr, Tl, and Zn showed that the parasites concentrated metals to a higher degree than the fish tissues. They accumulated the metals As, Cd, Cu, Fe, Ni, Pb, Sr and Zn even better than established bioindicators such as the mussel Dreissena polymorpha as revealed by data from the literature. The results presented here emphasize that acanthocephalans of fish are very useful as sentinels for metal pollution in aquatic ecosystems. Ratio of metal concentrations in the parasites and the host tissues provide additional information. Not including acanthocephalans in accumulation bioindication studies with fishes (as still customarily done) may lead to false results.

Redescription of Myxobolus hungaricus Jaczó, 1940 (Myxosporea: Myxobolidae), a parasite of bream (Abramis brama L.).

Myxobolus hungaricus Jaczó, 1940 forms relatively large plasmodia on the gills of bream (Abramis brama). The authors give a redescription of this deficiently described parasite on the basis of spores collected from the original habitat, which spores are indicated as a neotype. The plasmodia of the parasite can be found on the gills typically in the spring months, and are located in the central part of the gill filaments. They start to develop in the lumen of capillaries of the secondary lamellae and remain in intralamellar location also after they have reached the mature stage. The spores have a very characteristic shape, as on the surface of the spore valves a distinctly emerging ridge runs parallel to the sutural line. In addition to breams from the typical habitat, Lake Balaton, the plasmodia of M. hungaricus could be detected also on breams originating from the river Danube.

[Amyloid goiter]. / Az amyloid golyváról.

Amyloid goitre is at an extremely rare occurrence. Authors review the origin of disease and its symptoms, diagnostic and therapeutic tools. The disease may be due to either primary or secondary systemic or local amyloidosis. Diagnosis may be made even before surgery on anamnestic data, on very rapid growth of thyroid glands, on diffuse appearance, on other symptoms of systemic amyloidosis, on findings of iconographic procedures and on detection of amyloid in aspirates. Final diagnosis is based on histology. Surgical therapy is aiming at avoidance of the existing and the threatening consequences of expanding mass. The outcome is independent from thyroid surgery, it is related to other manifestations of amyloidosis. Concerning with the present case the chronic superior vena cava syndrome and chylous pleural effusion as first described symptoms and asymptomatic hyperthyroxinaemia is emphasised. Neither other organ involvement, nor primary amyloidogenous molecula was found during the 18 months follow up, so patient has secondary and localised amyloidosis.

Experimental detection of the actinospores of Myxobolus pseudodispar (Myxosporea: Myxobolidae) in oligochaete alternate hosts.

The development of Myxobolus pseudodispar Gorbunova, 1936, an intracellular myxosporean muscle parasite of the roach Rutilus rutilus L., was studied in experimentally infected oligochaetes. In one experiment, uninfected Tubifex tubifex Müller and Limnodrilus hoffmeisteri (Claparéde) were exposed to mature spores of M. pseudodispar. Triactinomyxon spores developed both in T. tubifex and L. hoffmeisteri specimens. Triactinospores were first released from the oligochaetes 76 d after initial exposure. At that time, pansporocysts containing 8 triactinospores were located in the gut epithelium of experimentally infected oligochaetes, but free actinosporean stages were also found in their gut lumen. Each triactinospore had 3 pyriform polar capsules and an elongated cylindrical sporoplasm with 8 secondary cells. The spore body joined the 3 caudal projections with a relatively long style. One of the 3 caudal projections was shorter than the other two. The total length of the triactinospore was on average 206.5 microns.

Light and electron microscopic studies on the development of Sphaerospora colomani Baska, 1990 and Chloromyxum inexpectatum Baska, 1990.

During a parasitological survey conducted on sterlets (Acipenser ruthenus L.), the developmental stages of two new coelozoic species of Myxosporea were demonstrated in the kidney and in the urinary passages. Sphaerospora colomani develops vegetative stages in the interstitial capillaries of the kidney in winter. The ultrastructure of a large primary cell containing secondary, tertiary and quaternary cells was examined. The vegetative developmental forms were located beside the tubule in the cytoplasm of the endothelial cell. The primary cell developed in the cytoplasm of the endothelial cell, making the cell greatly enlarged. Young, developing and fully vacuolated secondary cells were seen in the cytoplasm. Two tertiary cells developed in each vacuole that contained free nuclei and quaternary cells. Each developmental stage of the parasite bore processes to increase the surface. The pseudoplasmodia of Chloromyxum inexpectatum were found mainly in the lower passages and, less often, in the renal tubules of all fish specimens examined. The vegetative stages were located among the capillaries of the Bowman's capsules. The structure of the Bowman's capsule had changed due to the xenoma developing inside. A thin, shaded area represented the demarcation zone between the xenoma and the host cells. The cytoplasm of the xenoma formed by host and parasitic cells contained nuclei and young, differentiated secondary cells. In the more developed secondary cells, generally two tertiary cells developed and, later on, a quaternary cell appeared in each.

Sphaerospora infection of American catfishes (Ictalurus punctatus and I. nebulosus) in Europe.

The occurrence in Europe of a Sphaerospora species described in North America is reported. Based upon its morphological characteristics, the parasite could be identified with the species S. hankai described from brown bullhead in Canada. This parasite was found to infect channel catfish (Ictalurus punctatus) cultured in farm ponds in Italy and brown bullhead (Ictalurus nebulosus) living in the supply channels of fish ponds in Hungary. The spores and sporogonic developmental stages were situated in the lumen of the renal tubules. In the authors' opinion, S. ictaluri described from channel catfish can be considered synonymous with S. hankai.

Ultrastructural observations on different developmental stages of Goussia sinensis (Chen, 1955), a parasite of the silver carp (Hypophthalmichthys molitrix Valenciennes, 1844).

The development of Goussia sinensis, a coccidium parasitizing the intestine of the silver carp (Hypophthalmichthys molitrix) was studied by electron microscopy. All stages developed in the epithelial cells, less frequently in the goblet cells, and were located within a parasitophorous vacuole. In some cases one cell was invaded by several merozoites. Eight to sixteen merozoites were formed within the meront by ectomerogony. The ultrastructural processes characteristic of gamogony were the same as those found for Goussia spp. parasitizing other species of fish. A hitherto unknown mechanism of oocyst wall formation was observed. The oocyst membrane developing within the zygote surrounded only part of the zygote material. Thus, a small part of the zygote material left the oocyst proper. It is suggested that this zygote residue and the necrotic host cell constitute the so-called "yellow bodies" which include the excreted oocyst. The oocyst wall was 40 to 60 nm thick. Oocyst sporulation took place within the fish. The sporocysts consisted of two hemispheres connected by sutures and had a 100 to 120 nm thick double wall. They were surrounded by sporocyst veils fixed to the oocyst wall by membranes.

Light and electron microscopic studies on various developmental stages of Hoferellus cyprini (Doflein, 1898).

The development of Hoferellus cyprini (Myxosporea) in common carp was studied by light and electron microscopy. It has been established that in the autumn the early developmental stages are located in a cell syncytium formed from the epithelium of the renal tubules. The authors share the opinion of Lom and Dyková [13] that each intracellular trophozoite corresponds to a primary parasitic cell which contains secondary, tertiary and quaternary cells having formed by internal cleavage. From December the trophozoites are released into the tubular lumen, and the secondary cells are transformed into coelozoic plasmodia. Spores are formed inside the Plasmodia in the ureter or renal tubules in April. The sporoblast cells correspond to the quaternary cells of the trophozoites. On the surface of each of the two spore valves 10 striations run longitudionally and continue in bristles at the caudal end of the spores. Morphologically, the spores are consistent with the description of Mitraspora cyprini Fujita, 1912. Therefore, the authors regard M. cyprini as a synonym of Hoferellus cyprini (Doflein, 1898).