Novel engineered TRAIL-based chimeric protein strongly inhibits tumor growth and bypasses TRAIL resistance.

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.

AD-O53.2--a novel recombinant fusion protein combining the activities of TRAIL/Apo2L and Smac/Diablo, overcomes resistance of human cancer cells to TRAIL/Apo2L.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors became promising molecules for selective targeting of tumor cells without affecting normal tissue. Unfortunately, cancer cells have developed a number of mechanisms that confer resistance to TRAIL\Apo2L-induced apoptosis, which substantiates the need for development of alternative therapeutic strategies. Here we present a recombinant variant of TRAIL\Apo2L peptide, named AD-O53.2, fused to the peptide-derived from Smac/Diablo protein-the natural inhibitor of the apoptotic X-linked IAP (XIAP) protein considered as a pro-apoptotic agent. The proposed mechanism of action for this construct involves specific targeting of the tumor by TRAIL\Apo2L followed by activation and internalization of pro-apoptotic peptide into the cancer cells. While in the cytoplasm , the Smac\Diablo peptide inhibits activity of X-linked IAP (XIAP) proteins and promotes caspase-mediated apoptosis. AD-O53.2 construct was expressed in E.coli and purified by Ion Exchange Chromatography (IEC). Derived protein was initially characterized by circular dichroism spectroscopy (CD), HPLC-SEC chromatography, surface plasmon resonance, protease activation and cell proliferation assays. Our Smac/Diablo-TRAIL fusion variant was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect with the IC50 values in femtomolar range for the most sensitive cell lines, while it remained ineffective against non-transformed HUVEC cells as well as isolated normal human and rat hepatocytes. Importantly, the construct was well tolerated by animals and significantly reduced the rate of the tumor growth in colon and lung adenocarcinoma animal models.

[Frequency of drug resistant tuberculosis in Poland in 2000 as compared to 1997]. / Czestosc wystepowania gruzlicy lekoopornej w Polsce w 2000 R. w porównaniu z 1997 r.

Prevalence of drug resistance to one drug and multidrug resistance--MDR in different categories of tuberculosis patients is an important information about the susceptibility pattern of Mycobacterium tuberculosis isolates against antimycobacterial drugs. Poland joined WHO/IUATLD global project on TB drug resistance surveillance, and carried out in 1996/1997 the first prospective survey, simultaneously on primary and acquired drug resistance. This study is repeated in 2000 according to the WHO/IUATLD protocol. The programme covered the whole country. A total of 16 regional centers participated in the co-operative study. 3705 questionnaires and cultures were obtained from patients who excreted TB bacilli during the 12-months from 1 st. January to 31st December 2000. Drug resistance tests to INH, RMP, SM, EMB were performed on Lowenstein-Jensen medium according to the proportion method or/and Bactec 460 TB system. 3705 TB patients (3037 new and 668 treated cases) bacteriologically confirmed by culture were included in one-year study. Primary resistance to any drug was found in 6.12% (CI 5.27-6.56) of new cases. 35 patients (1.15%, CI 0.77-1.35) were infected with MDR strains. Acquired resistance to any drug was found in 16.6% (CI 5.27-6.56), 8.53% (CL 6.41-9.6) of the patients who excreted MDR strains. We have found increased resistance from 3.6% in 1997 to 6.12% (p < 0.001) in 2000 and MDR from 0.6% in 1997 to 1.15% (p < 0.001) in 2000 in untreated tuberculosis patients in Poland. The rate of resistance in the group of treated TB patients was very similar in 1997 (17.0%) and in 2000 (16.6%); except 20% increase of MDR cases--(7.0% in 1997, and 8.53% in 2000). We observed an increase in drug resistant tuberculosis first time during 40 years long period of its monitoring. Regular monitoring of drug resistance in TB patients in Poland is recommended.

[Sensitivity of microscopy for detection of mycobacterium tuberculosis and MOTT (mycobacteria other than tuberculosis) on the basis of analysis 22.218 clinical materials submitted in 1998-2001 to the Department of Microbiology in National Tuberculosis and Lung Diseases Research Institute in Warsaw, Poland]. / Czulosc metody bakterioskopowej w wykrywaniu pratków gruzlicy i MOTT na podstawie analizy 22.218 badan diagnostycznych przeprowadzonych w Zakladzie Mikrobiologii IGiCHP w okresie 1998-2001.

The results of 22.218 respiratory specimens sent to our laboratory were studied to determine the sensitivity of the Ziehl-Neelsen (Z-N) stain and microscopy-fluorescence method for detection of M.tbc and MOTT. There were no AIDS patients among analyzed cases. Smears were positive for acid fast bacilli (AFB) 60.0% (480 of 800) of specimens growing M. tuberculosis and 25.1% (219 of 872) of specimens growing the six common species of MOTT. Smear positivity by species was 28.1% (141 of 502) for M. kansasii, 29.4% (43 of 146) for MAIC, 28.7% (35 of 122) for M. xenopi. No smear was positive for M. gordone (43 cultures), M. fortuitum (33 cultures), M. scrofulaceum (26 cultures). The rate of nonculturable mycobacteria on L-J medium was 0.2%; mean rate of contamination was 4.6%. We also analyzed the relation between the number of AFB seen on the smear and time of the growth of M. tuberculosis and relation between abundance of the culture growth and AFB seen on smears. These study suggest that the sensitivity of microscopy for Mycobacterium tuberculosis is comparable with the data of others authors. Sensitivity of microscopy is lower in MOTT detection than for M.tbc (p < 0.001). Time of growth and abundance of the M. tuberculosis cultures were adequate to AFB seen in microscopy.

[Evaluation of Bactec MGIT 960 fluorescent method in diagnosis of tuberculosis]. / Przydatnosc metody fluorescencyjnej Bactec MGIT 960 w mikrobiologicznym diagnozowaniu gruzlicy.

We evaluated the fluorescent Bactec MGIT 960 the new system in Poland, which is a fully automated, non-invasive, system for growth and detection of Mycobacterium with a capacity to incubate and continuously monitor 960 of 7-ml culture tubes. This system is equipped with special oxygen-quenching fluorescent sensor, which permits to continuously monitoring of microbial growth. Processed specimens were inoculated into Bactec MGIT 960, Bactec 460 Tb and MB/BacT as well as on to Lowenstein-Jensen slants. The greatest number of isolates of M. tuberculosis complex was recovered by using Bactec MGIT 960 system (49/19.5%). Other systems detected M. tuberculosis complex as follows: Bactec 460 Tb (47/18.7%), MB/BacT (43/17/1%), L-J (38/15.1%). Detection mean time of mycobacterial growth in smear-positive specimens were 15.4 days for Bactec MGIT 960, 16.2 days for Bactec 460 Tb, 15.1 days for MB/BacT and 28.2 days for L-J medium The rates of contamination for each of the system were: 3.8% for Bactec MGIT 960 and Bactec 460 Tb, 3.6% for MB/BacT and 2.9% for L-J. In conclusion Bactec MGIT 960 system is a valuable alternative of radiometric, semi automated Bactec 460 Tb system.