Drug Resistance in Natural Isolates of Leishmania donovani s.l. Promastigotes Is Dependent of Pgp170 Expression

Resistance of pathogens to drugs is a growing concern regarding many diseases. Parasites like Leishmania, Plasmodium and Entamoeba histolytica; and neoplastic cells, present the multidrug-resistant phenotype rendering chemotherapy ineffective. The acquired resistance of Leishmania to antimony has generated intense research on the mechanisms involved but the question has not yet been resolved. To test the hypothesis that drug efflux in Leishmania, as measured by flow cytometry using the fluorescent dye Rhodamine-123, is largely dependent on the number of efflux pumps an isolate can express, the amount of Pgp 170 molecules was assessed in ten field isolates (5 “resistant” and 5 “susceptible”) using: Western Blotting, Confocal and Transmission Electron Microscopy, and proteomics. Their survival after exposure to three antileishmanial drugs, in vitro, was evaluated and clinical data were compared to the in vitro results. All isolates were resistant to Glucantime but susceptible to Miltefosine, whilst Amphotericin B was more effective on the “susceptible” isolates. The MDR gene, expressing the transmembrane efflux pump Pgp 170, appears to play a key role in the phenomenon of drug resistance. When “susceptible” versus “resistant” parasites were compared, it was shown that the higher the number of Pgp 170 molecules the higher the Rhodamine-123 efflux from the parasite body and, when exposed to the drug, the number of efflux pumps increased. However, the rate of this increase was not linear and it is possible that there is a maximum number of Pgp 170 molecules an isolate can express. Nevertheless, the phenomenon is a complex one and other factors and proteins are involved in which the HSP-70 group proteins, detected in the “resistant” isolates, may play a significant role.     

Application and assessment of the Environmental Vulnerability Index in Greece

The Environmental Vulnerability Index (EVI) was developed by the Pacific Islands Applied Geoscience Commission as a global composite index that quantifies the vulnerability of an area's environment. Greece has been selected as reference area due to its current physical and anthropogenic conditions that may lead to environmental instabilities in the natural, social, and economic infrastructure environment. Hence, in the present approach, using data on Greece, an effort to define the range of information that the EVI may provide for the pertinent country is presented and a discussion on whether the index may be further developed is conveyed. Advantages as well as shortcomings of the Index are also delineated.     

Leishmania infantum and Toxoplasma gondii: Mixed infection of macrophages in vitro and in vivo

Although macrophages have a microbicidal role in the immune system they themselves can be infected by pathogens. Often a simultaneous infection by more than one microbe may occur in a single cell. This is the first report of coinfection of macrophages with Toxoplasma gondii and Leishmania infantum, in vitro and in vivo. L. infantum does not cause severe disease in mice but T. gondii, RH strain, is lethal. Cell culture studies using THP-1 macrophages dually infected in vitro revealed that 4.3% harbored both parasites 24 h after infection. When mice were infected with both parasites on the same day 7.3% of the infected cells carried both parasites 7 days later. Yet, if mice were first infected with L. infantum and then with Toxoplasma (5 days post-infection) 18.7% of the macrophages hosted either parasite but concomitant infection could not be found and mice, already harboring L. infantum, survived Toxoplasma’s lethal effect.     

Leishmaniadonovani s.l.: Evaluation of the proliferation potential of promastigotes using CFSE staining and flow cytometry

Leishmania infantum causes visceral leishmaniasis in all countries in the Mediterranean basin. It uses Phlebotomine sandflies as vectors where the promastigote stage develops, reproduces and becomes infective. Therefore the reproductive power of the promastigotes determines the inoculum size of the isolate. Ten Leishmania strains from Cyprus: two Leishmania donovani and eight L. infantum were used to study the proliferation capacity of the promastigotes. Population increase during a 6-day culture period was assessed quantitatively, by haematocytometer enumeration, and qualitatively by following the division history of each population during the same period by CFSE staining and flow cytometry. The strains exhibited different proliferation rates with L. infantum showing higher multiplication rates than L. donovani. These differences may represent their fitness capabilities and their ability to synchronize the multiplication activity of individual members in the population for the production of a sizeable inoculum in time for the vector’s blood meal.     

Impact of KRAS,BRAF, PIK3CA Mutations,PTEN, AREG,EREG Expression and Skin Rash in ≥2nd Line Cetuximab-Based Therapy of Colorectal Cancer Patients

Background

To investigate the predictive significance of KRAS, BRAF, PIK3CA mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in metastatic colorectal cancer (mCRC) patients treated with cetuximab containing salvage chemotherapy.

Methods

Primary tumors from 112 mCRC patients were analyzed. The worst skin toxicity during treatment was recorded.

Results

KRAS, BRAF and PIK3CA mutations were present in 37 (33%), 8 (7.2%) and 11 (9.8%) cases, respectively, PTEN was lost in 21 (19.8%) cases, AREG and EREG were overexpressed in 48 (45%) and 51 (49%) cases. In the whole study population, time to tumor progression (TTP) and overall survival (OS) was significantly lower in patients with KRAS (p = 0.001 and p = 0.026, respectively) or BRAF (p = 0.001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.018 and p = 0.013, respectively) or EREG (p = 0.002 and p = 0.004, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). In KRAS wt patients TTP and OS was significantly lower in patients with BRAF (p = 0.0001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.021 and p = 0.004, respectively) or EREG (p = 0.0001 and p<0.0001, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). TTP was significantly lower in patients with PIK3CA mutations (p = 0.01) or lost PTEN (p = 0.002). Multivariate analysis revealed KRAS (Hazard Ratio [HR] 4.3, p<0.0001), BRAF mutation (HR: 5.1, p<0.0001), EREG low expression (HR: 1.6, p = 0.021) and absence of severe/moderate skin rash (HR: 4.0, p<0.0001) as independent prognostic factors for decreased TTP. Similarly, KRAS (HR 2.9, p = 0.01), BRAF mutation (HR: 3.0, p = 0.001), EREG low expression (HR: 1.7, p = 0.021), absecence of severe/moderate skin rash (HR: 3.7, p<0.0001) and the presence of undifferantited tumours (HR: 2.2, p = 0.001) were revealed as independent prognostic factors for decreased OS.

Conclusions

These results underscore that KRAS-BRAF mutations and EREG expression can be used as biomarkers to further select patients undergoing anti-EGFR treatment.     

Ability of bone graft substitutes to support the osteoprogenitor cells: An in-vitro study

AIM: To compare seven commercially available bone graft substitutes(BGS) in terms of these properties and without using any additional biological growth factors.METHODS: Porcine osteoprogenitor cells were loaded on seven commercially available BGS and allowed to proliferate for one week followed by osteogenic induction. Staining for live/dead cells as well as scanning electron microscopy(SEM) was carried out to determine viability and cellular binding. Further outcome measures included alkaline phosphatase(ALP) assays with normalisation for DNA content to quantify osteogenic potential. Negative and positive control experiments were carried out in parallel to validate the results.RESULTS: Live/dead and SEM imaging showed higher viability and attachment with β-tricalcium phosphate(β-TCP) than with other BGS(P 0.05). The average ALP activity in nmol/mL(normalised value for DNA content in nmol/μg DNA) per sample was 657.58(132.03) for β-TCP, 36.22(unable to normalise) for calcium sulphate, 19.93(11.39) for the Hydroxyapatite/Tricalcium Phosphate composite, 14.79(18.53) for polygraft, 13.98(8.15) for the highly porous β-Tricalcium Phosphate, 5.56(10.0) for polymers, and 3.82(3.8) for Hydroxyapatite.CONCLUSION: Under the above experimental conditions, β-TCP was able to maintain better the viability of osteoprogenitor cells and allow proliferation and differentiation(P 0.05).     

NSAIDS inhibit in vitro MSC chondrogenesis but not osteogenesis: implications for mechanism of bone formation inhibition in man

The non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for analgesia but may inhibit bone formation. We investigated whether the reported NSAID effect on bone is related to inhibition of bone marrow mesenchymal stem cell (MSC) proliferation and osteogenic and chondrogenic differentiation and evaluated both cyclooxygenase (COX)-1 and COX-2 specific drugs. The effects of seven COX-1 and COX-2 inhibitors on MSC proliferation and osteogenic and chondrogenic differentiation were tested using Vybrant, sodium 3′-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT), functional and quantitative assays of MSC differentiation. The MSC expression of COX-1 and COX-2 and prostaglandin E2 (PGE-2) levels were evaluated serially during lineage differentiation by quantitative PCR and ELISA. None of the NSAIDs at broad range of concentration (range 10⁻³ to 100 μg/ml) significantly affected MSC proliferation. Surprisingly, MSC osteogenic differentiation inhibition was not evident. However, NSAIDs affected chondrogenic potential with a reduction in sulphated glycosaminoglycans (sGAG) content by 45% and 55% with diclofenac and ketorolac, respectively (P < 0.05 compared to controls). Parecoxib and meloxicam, more COX-2 specific reagents inhibited sGAG to a lesser degree, 22% and 27% respectively (P < 0.05 compared to controls). Cartilage pellet immunohistochemistry confirmed the above results. Pellet chondrogenesis was associated with increased COX-1 expression levels but not COX-2, and COX-1 specific drugs suppressed MSC PGE-2 more than COX-2 specific inhibitors. These findings suggest that NSAIDs may inhibit bone formation via blockage of MSC chondrogenic differentiation which is an important intermediate phase in normal endochondral bone formation.     

BRAFV600E Mutation Analysis in Patients with Metastatic Colorectal Cancer (mCRC) in Daily Clinical Practice: Correlations with Clinical Characteristics,and Its Impact on Patients’ Outcome

Background

To prospectively evaluate the usefulness of the BRAFV600E mutation detection in daily clinical practice in patients with metastatic Colorectal Cancer (mCRC).

Patients and Methods

504 mCRC patients treated with systemic chemotherapy ± biologics were analyzed.

Results

A statistically significant higher incidence of the BRAF mutation was observed in patients with ECOG-PS 2 (p=0.001), multiple metastatic sites (p=0.002),> 65 years old (p=0.004), primary tumors located in the colon (p<0.001), high-grade tumors (p=0.001) and in those with mucinous features (p=0.037). Patients with BRAFV600E mutated tumors had a statistically significantly reduced progression-free survival (PFS) compared to wild-type (wt) ones (4.1 and 11.6 months, respectively; p<0.001) and overall survival (OS) (14.0 vs. 34.6 months, respectively; p<0.001). In the multivariate analysis the BRAFV600E mutation emerged as an independent factor associated with reduced PFS (HR: 4.1, 95% CI 2.7–6.2; p<0.001) and OS (HR: 5.9, 95% CI 3.7–9.5; p<0.001). Among the 273 patients treated with salvage cetuximab or panitumumab, the BRAFV600E mutation was correlated with reduced PFS (2.2 vs. 6.0 months; p<0.0001) and OS (4.3 vs. 17.4 months; p<0.0001).

Conclusions

The presence of BRAFV600E-mutation in mCRC characterizes a subgroup of patients with distinct biologic, clinical and pathological features and is associated with very poor patients’ prognosis.     

Biological and molecular profile of fracture non‐union tissue: current insights

Delayed bone healing and non‐union occur in approximately 10% of long bone fractures. Despite intense investigations and progress in understanding the processes governing bone healing, the specific pathophysiological characteristics of the local microenvironment leading to non‐union remain obscure. The clinical findings and radiographic features remain the two important landmarks of diagnosing non‐unions and even when the diagnosis is established there is debate on the ideal timing and mode of intervention. In an attempt to understand better the pathophysiological processes involved in the development of fracture non‐union, a number of studies have endeavoured to investigate the biological profile of tissue obtained from the non‐union site and analyse any differences or similarities of tissue obtained from different types of non‐unions. In the herein study, we present the existing evidence of the biological and molecular profile of fracture non‐union tissue.