Heparin-binding epidermal growth factor-like growth factor protects rat intestine from ischemia/reperfusion injury

BACKGROUND: We have shown previously that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is cytoprotective for intestinal epithelial cells exposed to hypoxia in vitro. We now examine the effects of HB-EGF on the recovery of small intestine from ischemic injury in vivo. METHODS: Segmental intestinal ischemia of 60-min duration was produced in adult rats by occlusion of a first-order branch of the superior mesenteric artery. Recombinant HB-EGF (100 microg) was injected intraluminally into the proximal small bowel after 45 min of ischemia in experimental animals, and buffered saline was injected in control animals. Animals were sacrificed after 48 h, and the affected bowel was resected, processed, and examined microscopically, with histologic grading of the ischemic injury. Additional animals were allowed to recover for up to 1 month to evaluate mortality differences. RESULTS: Intraluminal administration of HB-EGF resulted in significantly decreased extent and severity of ischemia/reperfusion injury, with significantly decreased grade of injury in the HB-EGF-treated compared with nontreated animals (average injury grade 0.66 compared with 2.44, respectively). Moreover, the mortality rate was significantly lower in the HB-EGF-treated animals compared with nontreated animals (0% vs 25%, respectively). HB-EGF-treated animals had increased weight gain in the postischemia recovery period. CONCLUSIONS: We conclude that HB-EGF, given intraluminally, reduces both the amount and the severity of ischemia/reperfusion injury in the small bowel, reduces the mortality associated with intestinal ischemia, and may enhance intestinal recovery. The in vitro and in vivo cytoprotective effects of this growth factor suggest that it may, in the future, be clinically useful in treating patients with intestinal ischemia.     

Evaluation of vascular prostheses made of carbon fiber

The whole work was aimed at experimental estimation of the healing process in case of carbon fibre vascular prostheses implanted into the abdominal aorta of a dog. The tests applied to carbon fibre woven prostheses made in the USSR. All metrological and chemical tests were performed at the Knitting Industry Main Centre for Research and Development "Tricomed" in Lód?. Experimental examinations were performed on 12 dogs. Prostheses were implanted into dog's abdominal aorta "end to the end" with the help of threads type Mersilen 4-0 or Prolen 4-0. The dogs were observed for 53 to 121 days, i.e. their behaviour, condition of the postoperative wound and presence of pulse in the femoral arteries were taken into consideration. Upon the completion of the observation autopsy was performed. Thus obtained material was fixed in 9% formalin and next various dyeing methods were applied to it. During examination in autopsy no inflammatory reactions in the tissues adjacent to implanted prostheses were discovered. In each case there was a continuous and firm adhesion of prosthesis and neighbouring inferior caval vein. A newly developed so called "neoadventitia" in the lumen of the prostheses constituted a solid and uniform film covering the wall of the prosthesis. Haematoma in the nearabout of the prosthesis was found in two cases only. Close to anastomosis ordinary clusters of lymphocytes and numerous fibroblasts as well as collagen fibre between prosthesis plexus were discovered. On the basis of the examinations performed a conclusion could be drawn, i.e. the process of healing was followed by regular structure of new aorta walls together with well developed flexible and springy fibre and neglidgeble immunological reactions.     

Imatinib inhibits proliferation of Ewing tumor cells mediated by the stem cell factor/KIT receptor pathway, and sensitizes cells to vincristine and doxorubicin-induced apoptosis.

PURPOSE AND EXPERIMENTAL DESIGN: The stem cell factor/KIT receptor loop may represent a novel target for molecular-based therapies of Ewing tumor. We analyzed the in vitro impact of KIT blockade by imatinib in Ewing tumor cell lines. RESULTS: KIT expression was detected in 4 of 4 Ewing tumor cell lines and in 49 of 110 patient samples (44.5%) by immunohistochemistry and/or Western blot analysis. KIT expression was stronger in Ewing tumors showing EWS-FLI1 nontype 1 fusions. Despite absence of c-kit mutations, constitutive and ligand-inducible phosphorylation of KIT was found in all tumor cell lines, indicating an active receptor. Treatment with KIT tyrosine kinase inhibitor imatinib (0.5-20 micro M) induced down-regulation of KIT phosphorylation and dose response inhibition of cell proliferation (IC(50), 12-15 micro M). However, imatinib administered alone at doses close to IC(50) for growth inhibition (10 micro M) did not induce a significant increase in apoptosis. We then analyzed if blockade of KIT loop through imatinib (10 micro M) was able to increase the antitumor in vitro effect of doxorubicin (DXR) and vincristine (VCR), drugs usually used in Ewing tumor treatment. Addition of imatinib decreased in 15-20 and 15-36% of the proliferative rate of Ewing tumor cells exposed to DXR and VCR, respectively, and increased in 15 and 30% of the apoptotic rate of Ewing tumor cells exposed to the same drugs. CONCLUSIONS: Inhibition of Ewing tumor cell proliferation by imatinib is mediated through blockade of KIT receptor signaling. Inhibition of KIT increases sensitivity of these cells to DXR and VCR. This study supports a potential role for imatinib in the treatment of Ewing tumor.     

CD34+ cells from acute myeloid leukemia, myelodysplastic syndromes, and normal bone marrow display different apoptosis and drug resistance-associated phenotypes.

Myelodysplastic syndromes and acute myeloid leukemia (AML) are heterogeneous disorders in which conflicting results in apoptosis and multidrug resistance (MDR) have been reported. We have evaluated by multiparameter flow cytometry the expression of apoptosis- (APO2.7, bcl-2, and bax) and MDR-related proteins [P-glycoprotein (P-gp), multidrug resistance protein (MRP), and lung resistance protein (LRP)] specifically on bone marrow (BM) CD34+ cells, and their major CD32-/dim and CD32+ subsets, in de novo AML (n=90), high-risk myelodysplastic syndrome (n=9), and low-risk myelodysplastic syndrome (n=21) patients at diagnosis, and compared with normal BM CD34+ cells (n=6). CD34+ myeloid cells from AML and high-risk myelodysplastic syndrome patients displayed higher expression of bcl-2 (P <0.0001) and lower reactivity for APO2.7 (P=0.002) compared with low-risk myelodysplastic syndrome and normal controls. Similar results applied to the two predefined CD34+ myeloid cell subsets. No significant differences were found in the expression of P-gp, MRP, and LRP between low-risk myelodysplastic syndrome patients and normal BM, but decreased expression of MRP (P <0.03) in AML and high-risk myelodysplastic syndromes and P-gp (P=0.008) in high-risk myelodysplastic syndromes were detected. Hierarchical clustering analysis showed that low-risk myelodysplastic syndrome patients were clustered next to normal BM samples, whereas high-risk myelodysplastic syndromes were clustered together and mixed with the de novo AML patients. In summary, increased resistance to chemotherapy of CD34+ cells from both AML and high-risk myelodysplastic syndromes would be explained more appropriately in terms of an increased antiapoptotic phenotype rather than a MDR phenotype. In low-risk myelodysplastic syndromes abnormally high apoptotic rates would be restricted to the CD34- cell compartments.     

Plitidepsin has a cytostatic effect in human undifferentiated (anaplastic) thyroid carcinoma.

Undifferentiated (anaplastic) thyroid carcinoma is a highly aggressive human cancer with very poor prognosis. Although there have been a few studies of candidate treatments, the fact that it is an infrequent tumor makes it very difficult to design clinical trials. A strong association has been observed between undifferentiated thyroid carcinoma and TP53 mutations in numerous molecular genetic and expression studies. Plitidepsin (Aplidin, PharmaMar, Madrid, Spain) is a novel anticancer compound obtained from a sea tunicate. This compound has been reported to induce apoptosis independently of TP53 status. We investigated the actions of plitidepsin in human thyroid cancer cells. In initial experiments using primary cultured cells from a differentiated (papillary) carcinoma, we found that 100 nmol/L plitidepsin induced apoptosis, whereas lower doses were cytostatic. Because our aim was to study the effects of plitidepsin at clinically relevant concentrations, subsequent experiments were done with a dosage regimen reflecting plasma concentrations observed in previously reported clinical trials: 100 nmol/L for 4 hours, followed by 10 nmol/L for 20 hours (4(100)/20(10) plitidepsin). This plitidepsin dosage regimen blocked the proliferation of a primary undifferentiated/anaplastic thyroid carcinoma culture obtained in our laboratory and of a commercial cell line (8305C) obtained from an undifferentiated thyroid carcinoma; however, it did not induce apoptosis. The proportion of cells in the G(1) phase of the cell cycle was greatly increased and the proportion in the S/G(2)-M phases greatly reduced, suggesting that plitidepsin blocks G(1)-to-S transition. Levels of the cyclin D1/cyclin-dependent kinase 4/p21 complex proteins were decreased and, in line with this, the levels of unphosphorylated Rb1 increased. The decrease in cell cycle proteins correlated with hypoacetylation of histone H3. Finally, we did experiments to assess how rapidly tumor cells return to their initial pretreatment proliferative behavior after 4(100)/20(10) plitidepsin treatment. Cells from undifferentiated tumors needed more than 3 days to recover logarithmic growth, and after 7 days, cell number was still significantly lower than in control cultures. 4(100)/20(10) plitidepsin inhibited the growth in soft agar. Together, our data show that plitidepsin is able to block in vitro cell cycle progression at concentrations similar to serum concentrations observed in vivo, and that this effect is persistent for several days after plitidepsin removal. Whether plitidepsin will prove to be clinically useful in the treatment of undifferentiated thyroid cancers remains to be established. However, our results raise the possibility that plitidepsin might be effective alone or in combination with radiotherapy and/or other drug treatments.     

Successful treatment of pediatric plasmacytoid dendritic cell tumors with a contemporary regimen for acute lymphoblastic leukemia

Dendritic cell leukemia (DCL) or hematodermic tumor is an uncommon subtype of acute leukemia. In contrast to adult cases, children tend to have a less aggressive course. The diagnosis of DCL should be considered when its characteristic morphologic features are present and leukemic cells co‐express CD4 and CD56. Cases of DCL among pediatric patients have been reported to respond to therapeutic regimens for acute lymphoblastic leukemia, but details regarding the specifics of therapy are lacking. Pediatr Blood Cancer 2013; 60: E38–E41. © 2013 Wiley Periodicals, Inc.     

Optical aberrations in the mouse eye

PURPOSE: The mouse eye is a widely used model for retinal disease and has potential to become a model for myopia. Studies of retinal disease will benefit from imaging the fundus in vivo. Experimental models of myopia often rely on manipulation of the visual experience. In both cases, knowledge of the optical quality of the eye, and in particular, the retinal image quality degradation imposed by the ocular aberrations is essential. In this study, we measured the ocular aberrations in the wild type mouse. METHODS: Twelve eyes from six four-week old black C57BL/6 wild type mice were studied. Measurements were done on awake animals, one being also measured under anesthesia for comparative purposes. Ocular aberrations were measured using a custom-built Hartmann-Shack system (using 680-nm illumination). Wave aberrations are reported up to fourth order Zernike polynomials. Spherical equivalent and astigmatism were obtained from the 2nd order Zernike terms. Modulation Transfer Functions (MTF) were estimated for the best focus, and through-focus, to estimate depth-of-focus. All reported data were for 1.5-mm pupils. RESULTS: Hartmann-Shack refractions were consistently hyperopic (10.12+/-1.41 D, mean and standard deviation) and astigmatism was present in many of the eyes (3.64+/-3.70 D, on average). Spherical aberration was positive in all eyes (0.15+/-0.07 microm) and coma terms RMS were significantly high compared to other Zernike terms (0.10+/-0.03 microm). MTFs estimated from wave aberrations show a modulation of 0.4 at 2c/deg, for best focus (and 0.15 without cancelling the measured defocus). For that spatial frequency, depth-of-focus estimated from through-focus modulation data using the Rayleigh criterion was 6D. Aberrations in the eye of one anesthetized mouse were higher than in the same eye of the awake animal. CONCLUSIONS: Hyperopic refractions in the mouse eye are consistent with previous retinoscopic data. The optics of the mouse eye is far from being diffraction-limited at 1.5-mm pupil, with significant amounts of spherical aberration and coma. However, estimates of MTFs from wave aberrations are higher than previously reported using a double-pass technique, resulting in smaller depth-of-field predictions. Despite the large degradation imposed by the aberrations these are lower than the amount of aberrations typically corrected by available correction techniques (i.e., adaptive optics). On the other hand, aberrations do not seem to be the limiting factor in the mouse spatial resolution. While the mouse optics are much more degraded than in other experimental models of myopia, its tolerance to large amounts of defocus does not seem to be determined entirely by the ocular aberrations.     

Oxazoline scaffold in synthesis of benzosiloxaboroles and related ring-expanded heterocycles: diverse reactivity,structural peculiarities and antimicrobial activity

Two isomeric benzosiloxaborole derivatives 3a and 5a bearing fluorine and 4,4-dimethyl-2-oxazolin-2-yl substituents attached to the aromatic rings were obtained. Both compounds were prone to hydrolytic cleavage of the oxazoline ring after initial protonation or methylation of the nitrogen atom. The derivative 3c featuring N-methylammoniumalkyl ester functionality was successfully subjected to N-sulfonylation and N-acylation reactions to give respective derivatives which demonstrates its potential for modular synthesis of structurally extended benzosiloxaboroles. Compound 5c bearing N-ammoniumalkyl ester underwent conversion to a unique macrocyclic dimer due to siloxaborole ring opening. Furthermore, an unexpected 4-electron reduction of the oxazoline ring occurred during an attempted synthesis of 5a. The reaction gave rise to an unprecedented 7-membered heterocyclic system 4a comprising a relatively stable B–O–B–O–Si linkage and stabilized by an intramolecular N–B coordination. It could be cleaved to derivative 4c bearing BOH and SiMe₂OH groups which acts as a pseudo-diol as demonstrated by formation of an adduct with Tavaborole. Apart from the multinuclear NMR spectroscopy characterization, crystal structures of the obtained products were determined in many cases by X-ray diffraction. Investigation of biological activity of the obtained compounds revealed that derivatives 3e and 3f with pendant N-methyl arylsulfonamide groups exhibit high activity against Gram-positive cocci such as methicillin-sensitive Staphylococcus aureus ATCC 6538P, methicillin-resistant S. aureus (MRSA) ATCC 43300 as well as the MRSA clinical strains, with MIC values in the range of 3.12–6.25 mg L⁻¹. These two compounds also showed activity against Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 6057 (with MICs of 25–50 mg L⁻¹). The results of the antimicrobial activity and cytotoxicity studies indicate that 3e and 3f can be considered as potential antibacterial agents, especially against S. aureus MRSA.

Transformations of oxazoline–benzosiloxaborole conjugates gave rise to novel boracyclic systems as well as functionalized derivatives featuring antibacterial activity.