Tibial tunnel widening associated with anterior cruciate ligament reconstruction using autogenous hamstrings: A comparison between antero-medial portal and transtibial techniques. / Valoración de la apertura del túnel tibial tras la reconstrucción del ligamento cruzado anterior empleando isquiotibiales autógenos: Estudio comparativo entre la técnica de portal anteromedial y la técnica transtibial convencional.

OBJECTIVE: Evaluate the enlargement effect of the tibial tunnel emergence of 2 different of anterior cruciate ligament reconstruction techniques: antero-medial portal (AMP) vs. transtibial (TT) technique. METHODS: A prospective, randomized controlled study was performed in 36 consecutive patients who underwent anterior cruciate ligament reconstruction with autologous hamstring tendon grafts employing the AMP and conventional TT techniques. Lateral and antero-posterior radiographs were obtained for each patient at 6 weeks and 12 months postoperatively. The sclerotic margins of the tibial tunnels were measured at the widest dimension of the tunnel as well as the diameter of the tibial emergence and were compared with the initially drilled tunnel size after correction for radiographic magnification. Statistical analysis was performed to compare the 2 groups by use of the independent-samples t test, with significance set at .05. RESULTS: The mean percentage increase in the diameter of tibial tunnel emergence at 6 weeks after surgery was 8.1%±2.9 for the PAM technique and 21.20%±11.87 for the TT technique on the anteroposterior x-ray view. However, the mean percentage increase in the diameter of the tibial tunnel emergence on the lateral view was 7.1%±4.72 for the medial portal technique and 17.64%±11.48 for the transtibial technique. This difference was statistically significant on both anteroposterior and lateral views. CONCLUSIONS: The diameter of the tibial tunnel emergence for hamstring autologous anterior cruciate ligament reconstructions was significantly lower for the medial portal technique when compared with the conventional TT technique.

Retroviral transfer of herpes simplex virus-thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector.

In this study we describe a new retroviral vector utilizing an internal ribosome entry site (IRES) from encephalomyocarditis virus to co-express two genes. One is the herpes simplex virus type 1 thymidine kinase gene (HSV-TK) which induces sensitivity to ganciclovir, and the second is the bacterial beta-galactosidase gene (LacZ) which was revealed by an histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). We engineered the U937 human cell line to co-express both genes and monitored transduced cells using X-Gal staining. Several transduced clones were selected. The clones exhibiting X-Gal positive cells were sensitive to ganciclovir treatment (1 microgram/ml) while X-Gal negative clones were not. Monoclonal cell lines showed a single copy of the provirus integrated in their genome with the TK-IRES-LacZ sequence stably inserted in all clones. The band distribution pattern of the proviral DNA differed only at the long terminal repeat (LTR) level. Northern blot analysis of an X-Gal positive/ganciclovir sensitive clone showed an mRNA band of 6 kb with both LacZ and TK probes. An X-Gal negative/ganciclovir resistant clone was negative with both probes. This report shows: (1) a therapeutic gene can be linked to a marker gene by an IRES element achieving equivalent expression of both proteins; (2) the co-expression of a marker gene makes fluorescein-di-beta-D-galactopyranoside staining possible, and consequently separation of cells expressing the LacZ gene by fluorescence activated cell sorting. Thus the cells expressing the HSV-Tk gene are enriched; (3) the use of a marker gene such as LacZ could open up interesting perspectives in gene therapy protocols because of the opportunity to monitor the transduced cells using a simple cytochemical stain.

Study on selective quantitative determination of barium by sulphonazo III in complex matrices.

Selective quantitative determination of barium by commercially available Sulphonazo III was studied in complex matrices. The application of two more promising methods was tried, but interferences derived from cations and anions present in natural waters and waste waters made them unuseful.

beta-galactosidase transduced T lymphocytes: a comparison between stimulation by either PHA and IL-2 or a mixed lymphocyte reaction.

BACKGROUND: Retroviral-mediated gene transfer stably introduces exogenous genes into normal and neoplastic cells of the hematopoietic system. METHODS: We used two retroviral vectors [the first, FLac, expresses a chimeric protein (Sh-ble::LacZ) between the product of the phleomycin resistance gene (Sh-ble) and the bacterial beta-galactosidase encoded by the LacZ gene; the second, NuNL vector, contains a fusion sequence (LacZ::Neo) that expresses the LacZ and the neomycin resistance genes] to transduce T lymphocytes derived from the peripheral blood of healthy human donors. Two lymphocyte activation procedures were employed: a) phytohemagglutinin/interleukin-2 (PHA/IL-2) polyclonal stimulation; b) allogeneic stimulation in a mixed irradiated or non irradiated lymphocyte reaction, both supplemented with IL-2 (MLR/IL-2). Infection was achieved by co-cultivating activated T cells with the producing amphotropic cell line pretreated with mitomycin C for 96 hours. Infection and transduction efficiency were assayed by LacZ gene expression, which is detected as indigo blue staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X..Gal). RESULTS: The highest percentage of transduced T cells was reached on the 3rd PHA/IL-2 and on 9th MLR/IL-2 activation days. In these conditions with FLac vector we obtained up to 80% X-Gal+ cells after PHA/IL-2 activation and 66% and 44%, respectively, with non irradiated and irradiated MLR/IL-2, respectively. Up to 40% X-Gal+ cells were obtained with NuNL vector after PHA/IL-2 stimulation, 40% with irradiated and 48% with non irradiated MLR/IL-2 activated cells. In term of transduction efficiency, large variability was observed among patients. There were no immunophenotypical differences between FLac or NuNL vector-transduced cells activated by either of the two techniques and the control cells. CONCLUSIONS: Our results indicate that: a) the use of FLac or NuNL vector retroviral-mediated gene transfer into T-lymphocytes derived from peripheral blood and stimulated by either PHA/IL-2 or a MLR produces a high percentage of transduced T cells; b) MLR is a good system for generating a transduced alloreactive lymphocyte population. The combination of high transduction efficiency and the capacity to obtain alloreactive transduced lymphocytes should open up the possibility of generating new in vitro and in vivo studies with selectable genes for in vivo therapeutic use.