Transurethral autologous myoblast injection for treatment of urinary incontinence in children with classic bladder exstrophy.

PURPOSE: The application of autologous myoblasts is an area of active research that may represent an improved alternative for the treatment of urinary incontinence. In this study we investigated the effectiveness of autologous myoblast injection for the treatment of urinary incontinence in children with classic bladder exstrophy. MATERIALS AND METHODS: Seven boys and 1 girl with persistent urinary incontinence were entered in the study. All children had undergone staged bladder repair and bladder neck reconstruction, and 5 patients had received 1 to 3 transurethral injections of bulking agent. Autologous myoblasts were isolated from abdominal muscle biopsy and cultured before endourethral injection. After the procedure patients underwent pelvic floor electrical stimulation and continued pelvic floor exercises that had been started at least 1 year before injection. The clinical outcomes (based on a 24-hour voiding diary), and cystometric and urodynamic studies were evaluated. Followup ranged from 12 to 18 months (average 15.3). RESULTS: There was a significant, time dependent improvement in urinary continence. At final followup all 7 boys (88% of patients) were socially dry (daytime dryness more than 3 hours), including 3 (38%) who were completely dry. Urodynamic studies revealed an increase in mean bladder capacity (p <0.001), detrusor leak point pressure (p <0.001) and average maximum urinary flow (p <0.01). All 7 boys (vs only 2 patients preoperatively) achieved normal voiding with demonstrable voiding detrusor contraction in the presence of a compliant stable bladder (p <0.05). CONCLUSIONS: Our results suggest that transurethral autologous myoblast injection is a valid option for the treatment of structural urinary incontinence in children with classic bladder exstrophy. However, favorable preoperative urodynamic profiles and postoperative pelvic floor electrical stimulation may have contributed to the outcome in this series.

The interaction of cytochrome P450 17alpha with NADPH-cytochrome P450 reductase, investigated using chemical modification and MALDI-TOF mass spectrometry.

The lysine residues of guinea pig P450 17alpha were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17alpha prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17alpha was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17alpha in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17alpha with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.

Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.

A Simple, Rapid, and Efficient Method for Isolating Mesenchymal Stem Cells from the Entire Umbilical Cord.

Several reports have been published on the isolation, culture, and identification of mesenchymal stem cells (MSCs) from different anatomical regions of the umbilical cord (UC). UC is suitable for standardizing methods of MSC isolation because it is a uniform source with high MSC numbers. Although the UC is considered a medical waste after childbirth, ethical issues for its use must be considered. An increased demand for MSCs in regenerative medicine has made scientists prioritize the development of MSC isolation methods. Several research groups are attempting to provide a large number of high-quality MSCs. In this study, we present a modulated explant/enzyme method (MEEM) to isolate the maximum number of MSCs from the entire UC. This method was established for the isolation of MSCs from different anatomical regions of the UC altogether. We could retrieve 6 to 10 million MSCs during 8 to 10 days of primary culture. After three passages, we could obtain 8-10 × 108 cells in 28-30 days. MSCs isolated by this method express CD73, CD90, CD105, and CD44, but they do not express hematopoietic markers CD34 and CD45 or the endothelial marker CD31. The genes SOX2, OCT4, and NANOG are expressed in isolated MSCs. The capacity of these MSCs to differentiate into adipocytes and osteocytes highlights their application in regenerative medicine. This method is simple, reproducible, and cost efficient. Moreover, this method is suitable for the production of a large number of high-quality MSCs from an UC in less than a month, to be used for cell therapy in an 80-kg person.

A Simple, Rapid, and Efficient Method for Isolating Mesenchymal Stem Cells From the Entire Umbilical Cord.

Several reports have been published on the isolation, culture, and identification of mesenchymal stem cells (MSCs) from different anatomical regions of the umbilical cord (UC). UC is suitable for standardizing methods of MSC isolation because it is a uniform source with high MSC numbers. Although the UC is considered a medical waste after childbirth, ethical issues for its use must be considered. An increased demand for MSCs in regenerative medicine has made scientists prioritize the development of MSC isolation methods. Several research groups are attempting to provide a large number of high-quality MSCs. In this study, we present a modulated explant/enzyme method (MEEM) to isolate the maximum number of MSCs from the entire UC. This method was established for the isolation of MSCs from different anatomical regions of the UC altogether. We could retrieve 6 to 10 million MSCs during 8 to 10 days of primary culture. After three passages, we could obtain 8-10 × 10(8) cells in 28-30 days. MSCs isolated by this method express CD73, CD90, CD105, and CD44, but they do not express hematopoietic markers CD34 and CD45 or the endothelial marker CD31. The genes SOX2, OCT4, and NANOG are expressed in isolated MSCs. The capacity of these MSCs to differentiate into adipocytes and osteocytes highlights their application in regenerative medicine. This method is simple, reproducible, and cost efficient. Moreover, this method is suitable for the production of a large number of high-quality MSCs from an UC in less than a month, to be used for cell therapy in an 80-kg person.

Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling.

OBJECTIVE: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison's disease. DESIGN: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH-21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. METHODS: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. RESULTS: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% +/- 24% (n = 6), 86.7% +/- 7.6% (n = 6) and 86.5% +/- 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 +/- 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. CONCLUSIONS: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

Prevention and detection of Mycoplasma contamination in cell culture.

One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture.

Antibody formation following botulinum toxin type A (Dysport) injection in children with intractable bladder hyper-reflexia.

OBJECTIVES: To investigate the pattern of anti-Dysport antibody (ADA) formation after Dysport injection in patients with neuropathic bladder. Antibody formation may lead to failure or allergic reactions in patients undergoing Dysport injection. METHODS: Forty-four children with neuropathic bladder were enrolled and classified into 3 groups: group I, without history of previous injection (n = 8); group II, with history of one or more injections (n = 7); and group III, who had been injected 3-36 months before this study (n = 29). Groups I and II were subjected to Dysport injection. Fifty-five age-matched healthy children were selected as controls. Urinary incontinence score was assessed before and 6 months after injection. Under cystoscopic guidance, Dysport (10 IU/kg) was injected into the detrusor muscle, sparing the trigone and ureteral orifices. ADA level was measured by enzyme-linked immunosorbent assay technique before injection and then monthly for at least 4 months in groups I and II, and for just once in group III and control subjects. RESULTS: ADA level was increased 1-2 months after the last injection in 3 (38%) of group I and 5 (71%) of group II. However, ADA level in group III was not higher than controls. All patients had complete or partial improvement in urinary incontinence score except for 1 patient in group I. No resistance to treatment was detected. CONCLUSIONS: Increment of ADA titer in patients is not permanent. Repeated injections will not boost the immune system to produce higher levels of antibody. Increased levels of ADA may not be associated with treatment failure at follow-up visit.

Proliferation and differentiation of mesenchymal stem cell on collagen sponge reinforced with polypropylene/polyethylene terephthalate blend fibers.

Although tissue-engineered scaffolds made from collagen sponge are suitable for cell infiltrating, easily supplying oxygen and nutrients to cells, and removing the waste products, their mechanical properties are not satisfactory to be used as scaffold materials for tissue engineering applications. To improve mechanical properties of collagen, a novel porous scaffold for bone tissue engineering was prepared with collagen sponge reinforced by polypropylene/polyethylene terephthalate (PP/PET) fibers. Collagen solution (6.33 mg/mL) with PP/PET fibers (collagen/fiber ratio [w/w]: 1.27, 0.63, 0.42, 0.25) was freeze-dried, followed by cross-linking of combined dehydrothermal and glutaraldehyde. A scanning electron microscopy-based analysis of surface of the sponges demonstrated that the sponge with collagen/fiber <0.25 exhibited homogenous and interconnected pore structure with an average pore size of 200 µm. Incorporation of PP/PET fibers significantly enhanced the compressive strength of the collagen sponge. Proliferation and osteogenic differentiation of mesenchymal stem cell in collagen sponges reinforced with PP/PET fibers incorporation were significantly enhanced compared with collagen sponge without PP/PET incorporation. We conclude that incorporation of PP/PET fibers not only improves the mechanical properties of collagen sponge, but also enables mesenchymal stem cells to positively improve their proliferation and differentiation.

Comparison of two proliferation assays MTT and ltt in immunodeficient patients.

MTT assay is designed for spectrophotometric quantification of cell growth and viability without using the radiactive isotopes. The aim of this study is comparison of LIT and MTT as sensitive poliferation assays in imrnunodeficient patients. 20 immunodeficient and 20 healthy subjects were selected in this study. All of them had normal lymphocytes count, normal CD3 and negative DIR reaction to PPD, DT and candida. The immunodeficient patients regularly suffered from herpes, candida and staph abscess. The lymphocytes of inimunodeficient control groups were treated with PHA as mitogen. The lymphocyte proliferation was evaluated by MTT and LTT. The results showed a strong correlation between LTT and MITT between immunodeficient patients and healthy controls. The sensitivity test for MTT was 90%, and the sensitivity test for LTT was 98%. MTT method can be considered as a non-radiactive evaluation of cell proliferation. Detecting cell proliferation with accurate, sensitive, fast, easy and safe method is more preferable than LTT.