Clinical-parasitological Screening for Respiratory Capillariosis in Cats in Urban Environments.

Respiratory capillariosis is a widely distributed zoonotic parasitic disease caused by the nematode Capillaria aerophila (Trichocephalida, Trichuridae) that commonly infects wild carnivores but also cats and dogs. This retrospective study aims to describe cases of respiratory capillariosis in cats from the city of Belgrade, Serbia. Between 2015 and 2019, a total of 155 pet cats with or without respiratory symptoms were submitted to physical examination and parasitological examination of the feces. All cats lived indoor but had free access to outdoor. In suburban settlements, wild carnivores commonly share their living environments with owned cats and dogs. It can be assumed that more intense urbanization spreading into the natural habitats of will carnivores creates the opportunity for closer and more frequent contacts between the population of cats and feral carnivores which might increase the risk of feline contamination. The findings confirm the existence of capillaries in cats in urban areas of the city of Belgrade, contribute to a better understanding of the epidemiology of this nematode and warn that, because of close contacts between cats of pets and humans, capillaries can cause human infection.

Soil and Sand Contamination with Canine Intestinal Parasite Eggs as a Risk Factor for Human Health in Public Parks in Nis (Serbia).

Regarding geographical distribution and clinical relevance, the most common canine geohelminths are Toxocara canis, ancylostomatids, and Trichuris vulpis. Canine intestinal parasites from the soil and sand present an important potential serious human health hazard, especially for the children preschool and school - age. This paper aimed to establish the degree of contamination of soil and sand with zoonotic parasites from the canine feces and the degree of risk they could pose for human health in public places and playgrounds in the city of Nis. Our parasitological study involved 200 soil samples and 50 sand samples from the public parks in the city of Nis in southeastern Serbia (43°19'15″N, 21°53'45″ E). From several locations, about 100 g of soil and sand was collected based on the bioclimatic indices. Parasitological diagnosis was performed using conventional qualitative and quantitative coprological methods, abiding by the recommendations about the diagnosis of parasitic diseases. In 38 - 46 % of soil samples and 40 % of sand samples seven species of endoparasites were diagnosed. In the samples of soil, a medium and high degree of contamination with the ascarid T. canis (14 - 22 %) was detected, as well as a low and medium degree of contamination with ancylostomatids (4 - 12 %), and in the samples of sand, a variable degree of contamination with the helminths T. canis (26 %) and A. alata (16 %) was found. A statistically significant difference was found in the contamination with A. alata eggs between the samples of sand and samples of soil. The studied public surfaces represent the reservoir of zoonotic parasites, which is a public health problem requiring a synergistic action of several factors to be successfully resolved, i.e. the implementation of prevention, surveillance, and control measures.

Improving agricultural production of domestic rabbits in Serbia by follow-up study of their parasitic infections.

A study was made on the prevalence of some parasitic infections appearing in domestic rabbits obtained from individual breeders in Serbia in order to improve the agricultural production of these animals. Aside from economic reasons (meat production and reproduction) rabbits are bred for the research purposes, and races are kept as household pets. For these reasons, among others, it is important to gain knowledge of medical culprits including causes of parasitic diseases that compromise their health, well-being and cause economic losses. This parasitological research was conducted in the period from 2010 to 2015 in 8 epidemiological regions of Serbia, on 433 rabbits as representative samples of different races (154 individuals up to 1 years of age and 279 individuals older than 5). Out of the total number of examined rabbits parasitic infections were established in 82.68% of animals. We detected 3 species of endoparasites (Eimeria spp., Trichostrongylus spp., and Passalurus ambiguus) and 3 species of ectoparasites (Scabies from genera Sarcoptes, Psoroptes and Notoedres). In "kits" (small rabbits) coccidiosis was the most prevalent disease (50.65%), while in older animals trichostrongilidosis was common (39.07%). The most represented scabies infection was with the species Psoroptes cuniculi (12.01%). Aiming at better control on the health of rabbits, there is a growing need for continual monitoring of parasitic infections including appropriate diagnosis, application efficient therapeutic protocols and control measures.

A phase Ib dose-escalation study of everolimus combined with cisplatin and etoposide as first-line therapy in patients with extensive-stage small-cell lung cancer.

BACKGROUND: This phase Ib study aimed to establish the feasible everolimus dose given with standard-dose etoposide plus cisplatin (EP) for extensive-stage small-cell lung cancer (SCLC). PATIENTS AND METHODS: An adaptive Bayesian dose-escalation model and investigator opinion were used to identify feasible daily or weekly everolimus doses given with EP in adults with treatment-naive extensive-stage SCLC. A protocol amendment mandated prophylactic granulocyte colony-stimulating factor (G-CSF). Primary end point was cycle 1 dose-limiting toxicity (DLT) rate. Secondary end points included safety, relative EP dose intensity, pharmacokinetics, and tumor response. RESULTS: Patients received everolimus 2.5 or 5 mg/day without G-CSF (n=10; cohort A), 20 or 30 mg/week without G-CSF (n=18; cohort B), or 2.5 or 5 mg/day with G-CSF (n=12; cohort C); all received EP. Cycle 1 DLT rates were 50.0%, 22.2%, and 16.7% in cohorts A, B, and C, respectively. Cycle 1 DLTs were neutropenia (cohorts A and B), febrile neutropenia (all cohorts), and thrombocytopenia (cohorts A and C). The most common grade 3/4 adverse events were hematologic. Best overall response was partial response (40.0%, 61.1%, and 58.3% in cohorts A, B, and C, respectively). CONCLUSIONS: Everolimus 2.5 mg/day plus G-CSF was the only feasible dose given with standard-dose EP in untreated extensive-stage SCLC.

Influence of size scale and morphology on antibacterial properties of ZnO powders hydrothemally synthesized using different surface stabilizing agents.

Metal oxide nanoparticles represent a new class of important materials that are increasingly being developed for use in research and health-related applications. Although the antibacterial activity and efficiency of bulk zinc oxide were investigated in vitro, the knowledge about the antibacterial activity of ZnO nanoparticles remains deficient. In this study, we have synthesized ZnO particles of different sizes and morphologies with the assistance of different types of surface stabilizing agents - polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA) and poly (α,γ, l-glutamic acid) (PGA) - through a low-temperature hydrothermal procedure. The characterization of the prepared powders was preformed using X-ray diffraction (XRD) method and field emission scanning electron microscopy (FE SEM), as well as Malvern's Mastersizer instrument for particle size distribution. The specific surface area (SSA) of the ZnO powders was measured by standard Brunauer-Emmett-Teller (BET) technique. The antibacterial behavior of the synthesized ZnO particles was tested against gram-negative and gram-positive bacterial cultures, namely Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively. We compared the results of the antibacterial properties of the synthesized ZnO samples with those of the commercial ZnO powder. According to the obtained results, the highest microbial cell reduction rate was recorded for the synthesized ZnO powder consisting of nanospherical particles. In all of the examined samples, ZnO particles demonstrated a significant bacteriostatic activity.

Efficacy of everolimus (RAD001) in patients with advanced NSCLC previously treated with chemotherapy alone or with chemotherapy and EGFR inhibitors.

BACKGROUND: Treatment options are scarce in pretreated advanced non-small-cell lung cancer (NSCLC) patients. RAD001, an oral inhibitor of the mammalian target of rapamycin (mTOR), has shown phase I efficacy in NSCLC. METHODS: Stage IIIb or IV NSCLC patients, with two or fewer prior chemotherapy regimens, one platinum based (stratum 1) or both chemotherapy and epidermal growth factor receptor tyrosine kinase inhibitors (stratum 2), received RAD001 10 mg/day until progression or unacceptable toxicity. Primary objective was overall response rate (ORR). Analyses of markers associated with the mTOR pathway were carried out on archival tumor from a subgroup using immunohistochemistry (IHC) and direct mutation sequencing. RESULTS: Eighty-five patients were enrolled, 42 in stratum 1 and 43 in stratum. ORR was 4.7% (7.1% stratum 1; 2.3% stratum 2). Overall disease control rate was 47.1%. Median progression-free survivals (PFSs) were 2.6 (stratum 1) and 2.7 months (stratum 2). Common > or =grade 3 events were fatigue, dyspnea, stomatitis, anemia, and thrombocytopenia. Pneumonitis, probably or possibly related, mainly grade 1/2, occurred in 25%. Cox regression analysis of IHC scores found that only phospho AKT (pAKT) was a significant independent predictor of worse PFS. CONCLUSIONS: RAD001 10 mg/day was well tolerated, showing modest clinical activity in pretreated NSCLC. Evaluation of RAD001 plus standard therapy for metastatic NSCLC continues.

Use of c-KIT/PDGFRA mutational analysis to predict the clinical response to imatinib in patients with advanced gastrointestinal stromal tumours entered on phase I and II studies of the EORTC Soft Tissue and Bone Sarcoma Group.

Previous studies have shown that activating mutations of c-KIT/PDGFRA, potential therapeutic targets for imatinib mesylate, are implicated in the pathophysiology of gastrointestinal stromal tumours (GISTs). In this study, GISTs from 37 patients enrolled in an European Organisation for Research and Treatment of Cancer (EORTC) phase I/II clinical study of imatinib were examined for mutations of c-KIT/PDGFRA in order to explore whether the mutational status of the tumour predicts the clinical response to therapy. Mutations were screened by denaturing high-pressure liquid chromatography (DHPLC) and characterised by bi-directional DNA sequencing. Activating mutations of c-KIT or PDGFRA were found in 29 (78%) and 2 (6%) GISTs, respectively. Most c-KIT mutations involved exon 11 (n=24; 83%), all but one being an in-frame deletion; no isolated point mutations were found. The other c-KIT mutations included exon 9 AY 502-503 duplication (n=4; 14%) and exon 13 Lys-->Glu(642) missense mutation (n=1; 3%). Two tumours with no detectable c-KIT mutations demonstrated PDGFRA Asp-->Glu(842) amino acid substitutions. Patients with GISTs harbouring exon 11 mutations were more likely to achieve a partial response (PR) on imatinib therapy (83%) than all of the others (23%). The overall survival and progression-free survival rates for the entire group at 106 weeks were 78.3% and 46.9%, respectively. Based on a Kaplan-Meier analysis, patients with GISTs harbouring c-KIT mutations had longer median survival times and were less likely to progress than the other patients. These findings indicate that the mutational status of the c-KIT/PDGFRA oncoproteins could be useful to predict the clinical response of patients imatinib therapy.

18FDG-Positron emission tomography for the early prediction of response in advanced soft tissue sarcoma treated with imatinib mesylate (Glivec).

Imatinib mesylate (Glivec, formerly STI571) is the first effective systemic treatment for gastrointestinal stromal tumours (GISTs). Major changes in tumour volume, however, tend to occur late after the start of treatment. The aim of this study was to evaluate if [18F]-fluorodeoxyglucose-positron emission tomography (FDG-PET) can be used for the early evaluation of response to imatinib mesylate treatment in soft-tissue sarcomas (STS). 21 patients (17 GIST, 4 other STS) underwent FDG-PET imaging prior to and 8 days after the start of treatment. PET response (European Organization for Research and Treatment (EORTC) guidelines) was observed in 13 GISTs (11 Complete Responders, 2 partial responders. Subsequent computerised tomography (CT) response Response Evaluation Criteria in Solid Tumours (RECIST) was observed in 10 of these patients after a median follow up of 8 weeks. Stable or progressive disease was observed on PET in 8 patients and none of them achieved a response on CT. PET response was also associated with a longer progression-free survival (PFS) (92% versus 12% at 1 year, P=0.00107). We conclude that FDG-PET is an early and sensitive method to evaluate an early response to imatinib treatment.

Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal stromal tumours: a phase I study.

BACKGROUND: Gastrointestinal stromal tumours (GISTs) are rare tumours of the gastrointestinal tract characterised by cell-surface expression of the tyrosine kinase KIT (CD117). No effective systemic treatment is available. Imatinib (STI571) inhibits a similar tyrosine kinase, BCR-ABL, leading to responses in chronic myeloid leukaemia, and has also been shown to inhibit KIT. We did a phase I study to identify the dose-limiting toxic effects of imatinib in patients with advanced soft tissue sarcomas including GISTs. METHODS: 40 patients (of whom 36 had GISTs) received imatinib at doses of 400 mg once daily, 300 mg twice daily, 400 mg twice daily, or 500 mg twice daily. Toxic effects and haematological, biochemical, and radiological measurements were assessed during 8 weeks of follow-up. 18Fluorodeoxy-glucose positron-emission tomography (PET) was used for response assessment in one centre. FINDINGS: Five patients on 500 mg imatinib twice daily had dose-limiting toxic effects (severe nausea, vomiting, oedema, or rash). Inhibition of tumour growth was seen in all but four patients with GISTs, resulting in 19 confirmed partial responses and six as yet unconfirmed partial responses or more than 20% regressions. 24 of 27 clinically symptomatic patients showed improvement, and 29 of 36 were still on treatment after more than 9 months. PET scan responses predicted subsequent computed tomography responses. INTERPRETATION: Imatinib at a dose of 400 mg twice daily is well tolerated during the first 8 weeks, side-effects diminish with continuing treatment, and it has significant activity in patients with advanced GISTs. Our results provide evidence of a role for KIT in GISTs, and show the potential for the development of anticancer drugs based on specific molecular abnormalities present in cancers.

Tyrosine kinase inhibitor imatinib (STI571) as an anticancer agent for solid tumours.

Imatinib mesylate, also known as STI571 or CGP57148, is a competitive inhibitor of a few tyrosine kinases, including BCR-ABL, ABL, KIT, and the platelet-derived growth factor receptors (PDGF-R). It binds to the ATP-binding site of the target kinase and prevents the transfer of phosphate from ATP to the tyrosine residues of various substrates. At oral doses of 300 mg or greater, the vast majority of patients with chronic myeloid leukaemia achieve a haematological response and this is usually associated with limited toxicity. Imatinib also has substantial activity in Philadelphia chromosome-positive acute lymphoblastic leukaemia expressing the BCR-ABL fusion protein. Gastrointestinal stromal tumours (GISTs) have also been evaluated for clinical activity of imatinib. About 90% of malignant GISTs harbour a mutation in c-kit leading to KIT receptor autophosphorylation and ligand-independent activation. According to initial clinical studies, more than 50% of GISTs respond to therapy within a few months, and only about 10-15% progress. The potential for cure and the optimal length of treatment are currently not known. Several other human cancers may over-express KIT or PDGF-R, and clinical trials to evaluate the role of imatinib in the treatment of such cancers are currently ongoing. Imatinib is an example of a specifically designed, highly targeted cancer therapy, which poses novel requirements for both pathology laboratories and clinicians in terms of identifying the major molecular mechanisms involved in tumour growth.

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation.

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.

Analysis and purification of phorbol esters using normal phase HPLC and photodiode-array detection.

For the first time a normal-phase HPLC method using photodiode-array detection is described for the analysis and purification of phorbol esters. The use of the method is demonstrated with examples of 10 different tigliane and daphnane esters (TPA, DOPP, DOPPA, Sap A, Sap B, Sap C, Sap D, Thy A, Ro and Rx). Both analytical and semipreparative techniques were developed. The method has been used in the final purification of DOPP and Rx from plant extracts. The method can be employed in the areas of phytochemistry, biochemistry and pharmacology/toxicology, where small samples of the toxic materials are required for research.

Characterization of phorbol ester binding to protein kinase C isotypes.

A mixed micellar assay was used to study the in vitro binding of [3H]phorbol-12, 13-dibutyrate ([3H]PDBu) to pure recombinant protein kinase C (PKC)-alpha, -beta 1, -beta 2, -gamma, -delta, -epsilon, and -zeta isotypes expressed in the baculovirus/insect cell system. Scatchard analysis revealed that all isotypes except PKC-zeta were able to specifically bind PDBu, with Kd values ranging from 1.6 to 18 nM in the presence of calcium. In the absence of calcium PKC-alpha, -beta 1, -beta 2, and -delta were observed to have a 2-3-fold drop in affinity, although Bmax values remained unchanged, at a stoichiometry of 1.4-2.8 mol of PDBu/mol of enzyme. Competition with specific [3H]PDBu binding was assessed for the phorbol esters PDBu, 12-tetradecanoylphorbol-13-O-acetate, 12-deoxyphorbol-13-O-phenylacetate, 12-deoxyphorbol-13-O-phenylacetate-20-acetate, thymeleatoxin, resiniferatoxin, and sapintoxin A. Resiniferatoxin and 12-deoxyphorbol-13-O-phenylacetate-20-acetate were found to compete effectively only with PDBu bound to the PKC-beta 1 and -beta 2 isotypes and were the least potent of the phorbol esters tested (IC50, > 5 microM). The phorbol esters sapintoxin A, 12-deoxyphorbol-13-O-phenylacetate, 12-tetradecanoylphorbol-13-O-acetate, and PDBu (in order of potency) competed for binding to all isotypes (IC50 values ranging from 2 to 70 nM), with unchanged or slightly decreased potency when calcium was replaced by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thymeleatoxin, which was similar in other respects to these potent phorbol esters, was found to be less able to compete with binding to PKC-alpha and -epsilon isotypes (IC50, 3-5 microM). It appears that, whereas the binding of phorbol esters to PKC depends primarily on the C20 substituent, other areas of the molecule have an influence on this interaction and the PKC isotypes themselves display heterogeneity in their phorbol ester-binding characteristics.

HL-60 cell differentiation induced by phorbol- and 12-deoxyphorbol-esters.

The human promyelocytic leukaemia cell (HL-60) undergoes differentiation into a macrophage-like form when exposed to both tumour promoting- and non-promoting phorbol esters. We have investigated the effect of the two non-promoting phorbol esters, 12-deoxyphorbol-13-O-phenylacetate (Dopp) and 12-deoxyphorbol-13-O-phenylacetate-20-acetate (Doppa) on HL-60 cultures, and compared them with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All phorbol esters tested were found to be able to stop HL-60 proliferation and induce cell adherence and morphological changes characteristic of differentiation. TPA, fully differentiating at 1 nM, was more potent than Dopp and Doppa, which required 100 nM for full differentiation effects within the 4 day study. Doppa initially appeared weaker than Dopp at inhibiting incorporation of thymidine, the earliest effect studied, but we were able to detect rapid C-20 deacylation of Doppa, converting it to Dopp, using an HPLC protocol presented here. A detailed study of this thymidine incorporation inhibition showed that both TPA (10 nM or greater) and Dopp (500 nM or greater) have very similar time courses, with 50% inhibition occurring at approximately 12 h, in contrast to Doppa which had a significantly delayed time course at all doses tested. Exposure tests indicated that Dopp and Doppa could be washed from the cells much more easily than TPA. The data presented here strongly support the notion that the metabolic conversion of Doppa to Dopp by HL-60 cells was necessary to mediate its differentiating effects. Since protein kinase C (PKC)-beta 1, present in HL-60 cells, has been found to be the only PKC isotype activated so far in vitro by Doppa, our results suggest that activation of this isotype is not sufficient to drive HL-60 differentiation in vivo.

Cadmium-induced changes of antioxidant and metabolic status in red blood cells of rats: in vivo effects.

Chronic exposure of adult rats to dietary intake of cadmium (15 mg CdCl2/day/kg for 30 days) leads to development of anemia and thrombocytosis. Anemia is characterized by significant reticulocytosis (13.1 +/- 1.0%), anysocytosis, poikilocytosis, iron deficiency and marked alterations of antioxidant and metabolic status of red blood cells. Activities of SOD, catalase, GPx and GR were significantly increased in red blood cells of cadmium-treated rats. In treated animals cadmium induced an increase of red cell reduced and oxidized glutathione with no changes of GSSG/GSH ratio. However, significant reduction of lipid peroxidation was found. Plasma levels of tocopherol and ascorbate, as well as activity of glutathione-S-transferase, were all significantly increased in cadmium-treated rats. The energy metabolism of red blood cells was deeply altered in cadmium-treated rats. The levels of ATP, ADP, AMP and TAN were significantly increased while ATP/ADP ratio and adenylate energy charge (AEC) were significantly reduced. The level of 2,3-BPG was somewhat lower, but 2,3-BPG/Hb ratio was considerably higher, in red blood cells of cadmium-treated rats.