Antitumor effect of the tyrosine kinase inhibitor nilotinib on gastrointestinal stromal tumor (GIST) and imatinib-resistant GIST cells

<p>Antitumor effect of the tyrosine kinase inhibitor nilotinib on gastrointestinal stromal tumor (GIST) and imatinib-resistant GIST cells</p

apoptosis assay

<p>apoptosis assay date</p

in vivo drug assay

<p>in vivo drug assay date</p

Figure5

<p>Figure 5</p> <p>Antitumor activity of nilotinib on GIST and imatinib-resistant GIST cells. Cells were maintained in supplemented medium for 12 h, and then incubated with nilotinib (0~100μM) for 72 h. Cell viability was determined by comparing treated cells with the untreated control. Data are means of triplicates from a representative experiment.</p

Figure1

<p>Nilotinib antitumor activity in GIST xenograft models. (A) Immunohistochemistry staining for KIT in xenograft lines established from human GISTs: GK1X, GK2X and GK3X. (B) Tumor tissue fragments (~5 mm3) were transplanted s.c. into the backs of BALB/cSlc-nu/nu mice that were randomized into 3 groups (n = 6-8). Doses of 40 mg/kg/day of imatinib, nilotinib or pure water (control) were administered by oral gavage daily for 28 days. Tumor size was measured every two to three days. (C) Tumor growth inhibition (TGI) on the day of evaluation was calculated as the ratio of tumor volume on the evaluation day to that on day 1.</p> <p> </p

Figure4

<p>Figure 4</p> <p>Quantitative phosphorylation analysis. Parental (GK1C and GK3C; red histograms) or imatinib-resistant GIST cell lines (GK1C-IR and GK3C-IR, blue histograms) were fixed and stained with anti phospho-KIT (Tyr719), anti phospho-PDGFRA (Tyr754), anti phospho-SRC (Tyr416), anti phospho-AKT (Ser473) and anti phospho-ERK1/2 (Thr202/Tyr204). Finally, cells were detected with Alexa Fluor 488 donkey anti-rabbit IgG antibody (Isotype control was reacted only with the secondary antibody). The MFI (mean of fluorescence intensity) values were calculated by FlowJo. GK1C: p-KIT=3.21, p-PDGFRA=10.3, p-SRC=7.19, p-AKT=20.3, p-ERK1/2=37.8. GK1C-IR: p-KIT=3.30, p-PDGFRA=12.8, p-SRC=9.35, p-AKT=20.5, p-ERK1/2=94.4. GK3C: p-KIT=2.65, p-PDGFRA=7.29, p-SRC=5.35, p-AKT=19.5, p-ERK1/2=32.2. GK3C-IR: p-KIT=3.89, p-PDGFRA=9.82, p-SRC=8.31, p-AKT=21.3, p-ERK1/2=115.</p

in vitro drug assay

<p>in vitro drug assay date</p

Bacteriological Assessment of Healthcare-Associated Pneumonia Using a Clone Library Analysis

<div><p>Background</p><p>The causative pathogens of healthcare-associated pneumonia (HCAP) remain controversial, and the use of conventional cultivation of sputum samples is occasionally inappropriate due to the potential for oral bacterial contamination. It is also sometimes difficult to determine whether methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a true causative pathogen of HCAP.</p><p>Methods</p><p>We evaluated the bacterial diversity in bronchoalveolar lavage fluid (BALF) using molecular and cultivation methods in 82 HCAP patients. BALF specimens were obtained from the lesions of pneumonia using bronchoscopy. The bacterial flora was analyzed according to the clone library method using amplified fragments of the 16S ribosomal RNA gene with universal primers. In addition, sputum cultures and the above specimens were assessed.</p><p>Results</p><p>Eighty (97.6%) of the 82 BALF samples obtained from the patients with HCAP showed positive polymerase chain reaction results. The predominant phylotypes detected in the BALF in this study included bacteria common in cases of community- and hospital-acquired pneumonia. In addition, the phylotypes of streptococci and anaerobes were detected in 19 (23.2%) and 8 (9.8%) cases, respectively. In particular, phylotypes of streptococci were highly detected among the patients 75 of age or older. <i>Staphylococcus aureus</i> was cultured in 23 (28.0%) cases using conventional cultivation methods and detected in only 6 (7.3%) cases as predominant phylotypes according to the clone library method.</p><p>Conclusions</p><p>The clone library analysis of BALF in the HCAP patients detected heterogeneous bacteria and a high incidence of streptococci compared with that observed using cultivation methods. In addition, the results of our study may indicate a lower incidence of MRSA than previously expected in HCAP patients.</p></div

An Unclassified Microorganism: Novel Pathogen Candidate Lurking in Human Airways

<div><p>During the assessments of the correlation of the diseases and the microbiota of various clinical specimens, unique 16S ribosomal RNA (rRNA) gene sequences (less than 80% similarity to known bacterial type strains) were predominantly detected in a bronchoalveolar lavage fluid (BALF) specimen from a patient with chronic lower respiratory tract infection. The origin of this unique sequence is suspected to be the causative agent of the infection. We temporarily named the owner organism of this sequence “IOLA” (Infectious Organism Lurking in Airways). In order to evaluate the significance of IOLA in human lung disorders, we performed several experiments. IOLA-16S rRNA genes were detected in 6 of 386 clone libraries constructed from clinical specimens of patients with respiratory diseases (in our study series). The gene sequences (1,427 bp) are identical, and no significantly similar sequence was found in public databases (using NCBI blastn) except for the 8 shorter sequences detected from patients with respiratory diseases in other studies from 2 other countries. Phylogenetic analyses revealed that the 16S rRNA gene of IOLA is more closely related to eukaryotic mitochondria than bacteria. However, the size and shape of IOLA seen by fluorescent <i>in-situ</i> hybridization are similar to small bacteria (approximately 1 µm with a spherical shape). Furthermore, features of both bacteria and mitochondria were observed in the genomic fragment (about 19 kb) of IOLA, and the GC ratio of the sequence was extremely low (20.5%). Two main conclusions were reached: (1) IOLA is a novel bacteria-like microorganism that, interestingly, possesses features of eukaryotic mitochondria. (2) IOLA is a novel pathogen candidate, and it may be the causative agent of human lung or airway disease. IOLA exists in BALF specimens from patients with remarkable symptoms; this information is an important piece for helping solve the elusive etiology of chronic respiratory disorders.</p></div

Clinical and laboratory features of the 82 patients with HCAP.

<p>Data are presented as n (%) or mean ± SD unless otherwise stated.</p><p><i>Definition of abbreviations</i>: HCAP, healthcare-associated pneumonia; BMI, body mass index; BP, blood pressure; SpO₂, pulse oximetric saturation; PaO₂, partial pressure of arterial oxygen; BUN, blood urea nitrogen; PSI, pneumonia severity index; SD, standard deviation</p><p>^§1BMI was evaluated in 59 patients</p><p>^§2Respiratory rate was evaluated in 68 patients</p><p>^§3Arterial blood gas analysis was performed in 51 patients</p><p>^§4 0, can be active without any problems or limitations, daily life the same as before the onset; 1, intense activity limited, but can walk and perform light work or work while sitting; 2, can walk and perform all personal care, but cannot work; more than 50% of daytime hours out of bed; 3, can only do limited personal care; more than 50% of daytime hours spent in bed or chair; 4, cannot move at all or perform personal care, all day spent in bed or chair</p><p>Clinical and laboratory features of the 82 patients with HCAP.</p