Long-term and efficient expression of human ß-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.

Targeted integration of a therapeutic gene at specific loci in safe genomic regions by a non-viral vector can restore the function of the damaged gene. This approach also minimizes the potential genotoxic effects of transferred DNA. In this study, we have developed a non-viral vector that functions according to site-specific recombination (SSR). The vector contained a bacterial backbone and puromycin resistance gene (pur(r)), a ß-globin expressing cassette and an attB recombination site. We used phiC31 integrase to insert a copy of the vector into specific genomic locations of a human hematopoietic cell line. Site-specific integration of the vector with one or two copies in the transcriptionally active regions of the genome was confirmed. After genomic integration, we used Cre recombinase to remove the bacterial backbone and pur(r). This removal was verified by negative selection and genomic PCR screening. Following deletion of these sequences, the stable ß-chain expression was continued for several months in the absence of selective pressure. Consequently, this vector may potentially be a powerful tool for ex vivo correction of ß-globinopathies such as ß-thalassemia through successful genomic integration of a functional copy of the globin gene into the patient's target cells.

Spontaneous intrauterine "ping-pong" fracture: review and case illustration.

We report a case of a closed outer-table parietal "ping-pong" skull fracture occurring in a 4 190-gram female infant born at 39 weeks and 5 days gestation after an uneventful Cesarean section (Apgar scores of 9 and 9 at one and five minutes). There was no maternal history of abdominal trauma during pregnancy and there were no complications or difficulties with Cesarean section delivery. Neurological examination was normal. Computed tomography with three-dimensional reconstruction images showed a 4 x 5 cm depression in the right parietal bone with a medial lucency consistent with a fracture of the superior margin of the skull and leftward deviation of the sagittal suture and sinus. Spontaneous resolution did not occur by one month of age and the skull fracture was repaired with excellent cosmetic results. Rarely has a case of spontaneous intrauterine skull fracture been reported in an atraumatic Cesarean delivery. We believe this fracture resulted from a chronic in utero process without associated trauma as evidenced by deviation of the sagittal suture and sinus.

Compact retarding-potential Mott polarimeter.

A simple compact retarding-potential Mott polarimeter is described that operates at an electron accelerating voltage of 25 kV. With a thorium target the instrument provides efficiencies eta [=S2eff(I/I0), where Seff is the effective asymmetry (Sherman) function and I/I0 is the scattering efficiency] of approximately 1.3 x 10(-4) which are similar to the best values obtained using earlier Mott polarimeters. The present instrument, however, occupies a much smaller volume and is suitable for a wide range of applications involving angle- and/or energy-resolved polarization measurements.

Cytotoxicity and antiangiogenic effects of Rhus coriaria, Pistacia vera and Pistacia khinjuk oleoresin methanol extracts.

Angiogenesis, formation of new blood vessels, play an important role in some diseases such as cancer and its metastasis. Using angiogenesis inhibitors, therefore, is one of the ways for cancer treatment and prevention of metastasis. Medicinal plants have been shown to play a major role in the treatment of a variety of cancers. In this direction, cytotoxic and angiogenic effects of oleo gum resin extracts of Rhus coriaria, Pistacia vera and Pistacia khinjuk from Anacardiaceae family were studied. For IC50 values, cytotoxic effects of the plant extracts were evaluated at different concentrations (1, 10, 20, 40, 80,100 µg/ml) against human umbilical vein endothelial normal cell (HUVEC) and Y79 cell lines using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In vitro tube formation on matrigel base was used to evaluate angiogenic effects in the presence of increasing concentrations (50, 100, 250 µg/ml) of the extracts. Vascular endothelium growth factor was used as angiogenesis stimulator. Gas chromatography results showed that α-pinene and ß-pinene were the major essential oils constituents of all plant extracts. According to the MTT assay results, the R. coriaria resin extract was more cytotoxic than those of P. vera and P. khinjuk extracts (IC50, 9.1 ± 1.6 vs 9.8 ± 2.1 and 12.0 ± 1.9, respectively; P<0.05). Cytotoxic effects of all extracts against Y79 cell line was significantly higher than those of HUVEC used as a normal cell line (P<0.05). Tube formation assay also showed that extract of R. coriaria resin inhibited angiogenesis more significantly than other tested extracts (P<0.05). It could be concluded that R. coriaria resin extract possess cytotoxic effect and antiangiogenesis against cancer cells and as an anticancer natural product has a good potential for future studies.

Effect of human Wharton's jelly mesenchymal stem cell secretome on proliferation, apoptosis and drug resistance of lung cancer cells.

Multipotent mesenchymal stem cells (MSCs) are recently found to alter the tumor condition. However their exact role in tumor development is not yet fully unraveled. MSCs were established to perform many of their actions through paracrine effect. Thus investigation of MSC secretome interaction with tumor cells may provide important information for scientists who are attempting to apply stem cells in the treatment of the disease. In this study we investigated the effect of human Wharton's jelly derived MSC (WJ-MSCs) secretome on proliferation, apoptotic potential of A549 lung cancer cells, and their response to the chemotherapeutic agent doxorubicin. WJ-MSCs were isolated from human umbilical cord and then characterized according to the International Society for Cellular Therapy criteria and WJ-MSC secretome was collected. BrdU cell proliferation assay and Annexin V-PI staining were used for the evaluation of cytotoxic and proapoptotic effects of WJ-MSC secretome on A549 cells. WJ-MSC secretome neither induced proliferation of lung cancer cells nor affected the apoptotic potential of the tumor cells. We also studied the combinatorial effect of WJ-MSC secretome and the anticancer drug doxorubicinwhich showed no induction of drug resistance when A549 cells was treated with combination of WJ-MSC secretome and doxorubicin. Although MSCs did not show antitumor properties, our in vitro results showed that MSC secretome was not tumorigenic and also did not make lung cancer cells resistant to doxorubicin. Thus MSC secretome could be considered safe for other medical purposes such as cardiovascular, neurodegenerative, and autoimmune diseases which may exist or occur in cancer patients.

Nontraumatic rupture of varicocele.

A case of a nontraumatic scrotal hematoma thought to result from a ruptured varicocele is reported. It is postulated that a high venous pressure generated during a Valsalva maneuver while defecating may have precipitated the variceal hemorrhage.

Utricular papilloma.

A benign prostatic utricular papilloma was found in a twenty-seven-year-old man complaining of urethral bleeding and intermittent hematuria after sexual intercourse. Local endoscopic resection was performed. Since the natural history of this lesion is not known, follow-up cystourethroscopy will be performed.

Self-inflicted foreign bodies involving lower urinary tract and male genitals.

A great variety of self-inflicted foreign bodies have been removed from the lower urinary tract and male external genitalia. These foreign bodies were inserted or applied for autoerotic, psychiatric, therapeutic, or no definite reasons by the patient. Most patients were too ashamed to admit they had inserted or applied any object and usually presented when a complication had occurred from the foreign body such as difficulty voiding, hematuria, pain or swelling, extravasation, or abscess formation. Inspection and palpation diagnosed foreign bodies involving the male external genitalia in 3 patients and anterior urethra in 6. Radiographic studies with and without contrast medium and endoscopy were required to diagnose foreign bodies in the posterior urethra and bladder in 9 patients, and the exact location, shape, and orientation of the foreign bodies in all sites, radiolucent objects, and the complications of mucosal laceration and extravasation. All 16 foreign bodies in the anterior urethra (below urogenital diaphragm) were removed by endoscopic manipulation in the 6 patients. Nine foreign bodies in the posterior urethra or bladder (above the urogenital diaphragm) were removed by endoscopic manipulation in 2 females and 2 males, while suprapubic cystostomy was required in 1 female and 4 males. The 3 foreign bodies applied to the external genitalia of 3 males were removed carefully to avoid injury to the underlying skin. Foreign bodies lying below the urogenital diaphragm were palpable and readily removed endoscopically while foreign bodies above the urogenital diaphragm required greater endoscopic manipulation or open surgical procedures.

Spontaneous rupture of hydronephrotic renal pelvis with massive hemorrhage.

Spontaneous rupture of a hydronephrotic renal pelvis with massive hemorrhage presents as a perplexing diagnostic challenge to the urologist. The preoperative distinction between this condition and spontaneous rupture of the kidney (Wunderlich disease) may prove uncertain or equivocal despite the application of exhaustive diagnostic modalities. Surgical intervention should follow initial stabilization, and nephrectomy has been the procedure of choice.

Enterocystoplasty in the management and reconstruction of the pediatric neurogenic bladder.

Eighteen children and young adults with neurogenic bladder underwent enterocystoplasty as part of urinary undiversion or for treatment of incontinence associated with reduced bladder compliance or detrusor sphincter dyssynergia. In 12, tubular sigmoid enterocystoplasty with transureteroureterostomy was performed with the smaller diameter ureter implanted into the bowel tenia. In two patients the ileocecal segment was used to augment the bladder, and the ureters were anastomosed to the ileum. In four patients the cecum or a patch of sigmoid colon was used to augment the bladder. Young-Dees bladder neck reconstruction was performed on eight patients at the time of surgery; one later required bladder neck reconstruction, and two later required an artificial sphincter. After a mean follow-up of 20 months, 16 of the 17 available for follow-up are continent with clean intermittent catheterization every 3 to 4 hours. Nine patients require anticholinergic or smooth muscle relaxing medication to increase functional bladder capacity. Most of the patients need chronic antimicrobial treatment to control bacteriuria.

Congenital ureteral valves in children: case report and review of the literature.

Congenital ureteral valves are a rare cause of ureteral obstruction. While only 17 pediatric cases have been reported in the literature, this condition should be considered in the differential diagnosis of the child with upper urinary tract obstruction. The pediatric cases are reviewed, and we present an additional case in which unilateral hydronephrosis caused by a ureteral valve was first detected by ultrasound in utero.

The influence of HLA matching on renal allograft survival at a single pediatric center.

Between January 1, 1969, and February 2, 1988, 331 pediatric renal transplants were performed at a single pediatric center. Of these 225 were first cadaveric allografts. Graft allocation was independent of the outcome of human lymphocyte antigen (HLA) matching. The results of class I antigen matching were analyzed in 224 transplants, and actuarial graft survivals were calculated. Class II antigen matching was analyzed in 80 patients from January 1, 1982, and submitted to the same analysis. HLA matching could not be demonstrated to be beneficial. It is proposed that any matching benefit is small and obscured in our series by the dominance of other factors.

Synthesis and characterization of folate-targeted dextran/retinoic acid micelles for doxorubicin delivery in acute leukemia.

Folate and retinoic acid grafted/dextran (FA-RA/DEX) copolymers with different molecular weight of DEX were synthesized using carbonyldiimidazole and dimethylaminopyridine for targeted delivery of doxorubicin (DOX) in acute myelogenous leukemia (AML). The copolymers structure was confirmed by (1)H NMR and FTIR. Critical micelle concentration (CMC) of each copolymer was determined using pyrene as a fluorescent probe. DOX was loaded in micelles by the direct dissolution method. Physical properties of micelles, including particle size, zeta potential, drug loading efficiency, and drug release profiles, were examined. The orientation of the folate ligand on the surface of the micelles was studied by X-ray photoelectron spectroscopy (XPS) technique. The cytotoxicity of micelles loaded with DOX at different concentrations was studied in KG1 cells using MTT assay and their cellular uptake by flow cytometry technique. FTIR and (1)H NMR spectra confirmed successful production of the targeted micelles and XPS spectra showed the surface orientation of folate. R15D10F7 copolymer produced micelles with particle size of 82.86 nm, polydispersity index of 0.3, zeta potential of -4.68 mV, drug loading efficiency of 96%, and release efficiency of 63%. DOX loaded in folate-targeted micelles of RA/DEX was more toxic than that in nontargeted micelles and free drug and seems promising in reducing drug resistance in AML.

Polymeric delivery of siRNA for dual silencing of Mcl-1 and P-glycoprotein and apoptosis induction in drug-resistant breast cancer cells.

Enhanced survival mechanisms of malignant cells in combination with elevated levels of drug transporters can sustain an undesirable resistance against drug therapy. Short interfering RNA (siRNA) delivery against targets involved in aberrant mechanisms is a promising approach and we hypothesize that simultaneous silencing of multiple targets could prove more advantageous than common approach to silence individual targets. To explore this approach, we targeted anti-apoptotic proteins myeloid cell leukemia 1 (Mcl-1) and survivin along with the efflux pump P-glycoprotein (P-gp) in drug-resistant breast cancer cells. Polymeric siRNA delivery was employed for this purpose by using small polyethylenimine (PEI) substituted with lipids. While silencing Mcl-1 caused ∼90% cell death in wild-type cells, this effect was less significant in P-gp over-expressing cells. An additive effect for Mcl-1 and P-gp silencing was evident in the latter cells, where simultaneous silencing of these targets created a significantly higher effect compared with silencing each individual target. Prolonged exposure of wild-type cells to doxorubicin (DOX) resulted in upregulation of P-gp, breast cancer resistance protein, survivin and Mcl-1. Dual silencing of P-gp and Mcl-1 again resulted in an additive effect in resistance-induced cells, which displayed an increased dependency on Mcl-1 for survival. Cytotoxic effect of DOX was also enhanced in resistance-induced cells after silencing Mcl-1. We conclude that polymer-mediated siRNA delivery can silence multiple targets simultaneously and reverse drug resistance.

Synthesis, analysis and cytotoxic evaluation of some hydroxypyridinone derivatives on HeLa and K562 cell lines.

A range of iron bidentae ligands containing the chelating moiety 3- hydroxypyridin-4-ones (HPOs) have been synthesized via a single or a three-step synthetic pathway. In the single-step reaction, maltol was directly reacted by suitable primary amine and in the second synthetic method; benzylated maltol was reacted with related amines to give 1-substuted-2-methyl-3-benzyloxypyridin-4-one derivatives. Finally, removal of the benzyl group under acidic conditions was performed by catalytic hydrogenation to yield the favored bidentate chelators as HCl salt. The partition coefficient of the free ligands and their iron (III) complexes between an aqueous phase buffered at pH 7.4 and 1-octanol were also determined. The cytotoxic effects of these iron chelators against HeLa and K562 cell lines were evaluated using MTT assay and the results showed that cytotoxicity was closely related to the lipophilicity of compounds so that the most lipophilic compound (4g) revealed the highest activity and compound 4e as a more hydrophilic agent (Kpart; 0.05) showed the lowest cytotoxic effect.

Use of magnetic folate-dextran-retinoic acid micelles for dual targeting of doxorubicin in breast cancer.

Amphiphilic copolymer of folate-conjugated dextran/retinoic acid (FA/DEX-RA) was self-assembled into micelles by direct dissolution method. Magnetic iron oxide nanoparticles (MNPs) coated with oleic acid (OA) were prepared by hydrothermal method and encapsulated within the micelles. Doxorubicin HCl was loaded in the magnetic micelles. The characteristics of the magnetic micelles were determined by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), transmission electron microscopy (TEM), and vibrating sample magnetometer (VSM). The crystalline state of OA-coated MNPs and their heat capacity were analyzed by X-ray diffraction (XRD) and differential scanning calorimetry (DSC) methods, respectively. The iron content of magnetic micelles was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Bovine serum albumin (BSA) was used to test the protein binding of magnetic micelles. The cytotoxicity of doxorubicin loaded magnetic micelles was studied on MCF-7 and MDA-MB-468 cells using MTT assay and their quantitative cellular uptake by fluorimetry method. TEM results showed the MNPs in the hydrophobic core of the micelles. TGA results confirmed the presence of OA and FA/DEX-RA copolymer on the surface of MNPs and micelles, respectively. The magnetic micelles showed no significant protein bonding and reduced the IC50 of the drug to about 10 times lower than the free drug.

In vitro cytotoxic evaluation of some synthesized COX-2 inhibitor derivatives against a panel of human cancer cell lines.

Celecoxib is a non-steroidal anti-inflammatory drug (NSAID) developed as a selective inhibitor of cyclooxygenase-2 (COX-2) for the treatment of rheumatoid arthritis disease. Recently some other mechanisms have been identified for anti cancer activity of these agents including induction of apoptosis, inhibition of tumor vascularization, stimulation of antitumor immune responses and inhibition of cellular protein synthesis. The cytotoxic effects of four synthesisized analogues of celecoxib (coded as D, E, F and G) were evaluated against Hela, MDA-MB-231, A-2780-s and HT-29 cancer cells, using MTT assay; Also their induction of apoptosis using DNA fragmentation analysis were studied. MTT assay showed that cell survival percent of COX-2 positive cell lines (HT-29, MDA-MB-231 and Hela; p≤0.05) were decreased significantly after exposure to the tested COX-2 inhibitors while little effect was observed on the COX-2 negative cell line (A-2780-s). Results also showed that A-2780-s and Hela were the most resistant and the most sensitive cell lines to these compounds, respectively. Moreover, in DNA fragmentation assay, induction of apoptosis was confirmed by electrophoretic pattern of separated DNA fragments in Hela cell line. Compounds E and G in comparison with D and F exerted more cytotoxic effect on COX-2 positive cell lines (Hela, HT-29 and MDA-MB-231). This could be due to the hydrophobic substituent (Cl, CH3) located at the para position of phenyl ring leading to more lipophilicity and cell uptake. In addition, these COX-2 inhibitors induced apoptosis on Hela cell-line, which could be considered as one of the cytotoxic mechanisms of these compounds as potential anti cancer agents.

Synthesis and biological evaluation of bidentate 3-hydroxypyridin-4-ones iron chelating agents.

A series of 3-hydroxypyridin-4-one derivatives (HPOs) were synthesized and their partition coefficient values (K(part)) were determined. The cytotoxic effects of these iron chelators against Hela cancer cells were also evaluated. The IC(50) of HPOs was determined using MTT assay. Among these ligands, compound 4e (K(part)=5.02) with an IC(50) of 30 µM and 4f (K(part)=0.1) with an IC(50) of 700 µM showed the lowest and highest IC(50)s, respectively. In conclusion, the introduction of a more hydrophobic functional group (such as butyl in compound 4e) on the nitrogen of pyridinone ring resulted in higher cytotoxic activity of ligands.

Effect of cerium lanthanide on Hela and MCF-7 cancer cell growth in the presence of transferring.

The anti-cancer activity of metal ions in the lanthanide group is being considered recently. It has been reported that cerium salts might stimulate the metabolism and therefore, produce anti-cancer effects. However, little is known about the effects of protein-cerium complex in controlling cancer cell growth. The aim of the present study was to elucidate the possible pathways for the cytotoxic effect of cerium in the presence of apo-transferrin on two cancer cell lines (Hela and MCF-7), that express transferrin receptors 3-4 fold higher than normal cells. The effect of different concentrations of cerium (0.1, 1, 10, 100 µM) in the presence and absence of transferrin for 48 h and 72 h incubation periods (37°C, 5% CO2 and 95% humidity) was studied using the MTT assay. The results showed that cerium has a cell-proliferation inhibitory activity which is significantly increased by transferrin protein. Compared with the direct treatment of cancer cells with cerium, the presence of transferrin assisted inhibition of cell proliferation by 20% and 40% in Hela and MCF-7 cells, respectively. Though apo-transferrin could lightly induce cell growth particularly in MCF-7 cells by itself, this phenomenon could not overcome the cerium-protein cell-proliferation inhibition activity. In conclusion, our results indicate that at a certain concentration, the cerium compounds could be possibly involved in the control of cell proliferation and inhibiting the growth of cancer cells.

Cytotoxic evaluation of doxorubicin in combination with simvastatin against human cancer cells.

Doxorubicin is a broad spectrum antibiotic used in the treatment of cancers. Its dose dependent cardiotoxicity is the most serious side effect causing withdrawal of drug from hard chemotherapeutic regimen. Statins are shown to be cytotoxic in concentrations higher than the effective doses for the treatment of hypercholesterolemia (40 mg/day). Co-administration of statins and chemotherapeutic agents suppose to be synergic although there are some controversies in the literature. In this study, cytotoxic effects of doxorubicin alone and in combination with simvastatin on Hela tumor cell line were evaluated. Different concentration of doxorubicin and simvastatin were added to the cultured cells and incubated for 72 h. Cell survival was evaluated using MTT and trypan blue exclusion assays. The results indicated that simvastatin in low concentration (0.25 µM) seems to be growth stimulator although cell viability was reduced in concentrations of ≥2 µM. Doxorubicin alone at all tested concentrations (0.1, 1 and 2 µM) was a cell growth inhibitor. It was also shown that percent cell viability was reduced in a decreasing manner with the following protocols: 1) co-administration of doxorubicin and simvastatin in different concentrations; 2) addition of simvastatin after incubation of cells with doxorubicin and 3) addition of doxorubicin after incubation of cells with simvastatin. It could be concluded that between 3 tested protocols combination of doxorubicin and simvastatin after 72 h incubation, showed the highest cytotoxicity against Hela cells.