Mortality and predictors in pulmonary tuberculosis with respiratory failure requiring mechanical ventilation.

OBJECTIVE: To analyse the predictors and mortality rate among patients receiving mechanical ventilation (MV) for respiratory failure due to pulmonary tuberculosis (TB). DESIGN: We retrospectively compared patients who required MV for TB with patients who required MV for community-acquired pneumonia (CAP). RESULTS: In-hospital mortality was significantly different between the two groups: 95.1% in TB vs. 62.7% in CAP (P < 0.001 using the χ(2) test). TB patients had a higher 30-day mortality (P = 0.040 using log-rank test), although the median sequential organ failure assessment (SOFA) (7.0 vs. 6.0, P = 0.842) and mean Acute Physiology and Chronic Health Evaluation (APACHE) II scores (20.0 ± 6.7 vs. 21.2 ± 6.7, P = 0.379) for TB and CAP patients were not different. TB patients were more likely to have increased lung lesion intrusions (OR 1.307, 95%CI 1.042-1.641, P = 0.021), and reduced albumin (OR 0.073, 95%CI 0.016-0.335, P = 0.001), C-reactive protein (OR 0.324, 95%CI 0.146-0.716, P = 0.005) and CURB-65 score (confusion, uraemia, respiratory rate, blood pressure and age ⩾65 years) (OR 0.916, 95%CI 0.844-0.995, P = 0.037). CONCLUSIONS: TB patients showed identical SOFA and APACHE II scores, but higher mortality than CAP patients. The higher mortality was not related to severity, but suggested an association with the extent of destructive lung lesions.

Characterization and safety assessment of bioengineered limbal epithelium.

Transplantation of cultivated limbal epithelium on substrates such as amniotic membrane is an established treatment for severe ocular surface disease with limbal stem cell deficiency. In this study, we adapted an established method to generate sheets of limbal epithelium on amniotic membrane and characterized the cells contained in these sheets and tested them for safety with regard to microbial contamination. Human limbal biopsies were cultivated on denuded amniotic membranes. After three weeks of culture, the phenotypes of cultivated cells were analyzed by immunohistochemistry and real-time RT-PCR for the expression of a panel of specific markers. Cultivated limbal epithelial cell sheets were also analyzed by scanning (SEM) and transmission (TEM) electron microscopy. Sterility tests and mycoplasma assays were conducted for the safety of product. A confluent layer of polygonal cells was formed in 2 weeks and 1-3 stratified layer of cells were observed after three weeks of culture. Cultivated cells were positive for p63, K3, K19, and involucrin but negative for K14, integrin alpha9 and ABCG2 when analyzed by immunohistochemistry. Expression of molecular markers was detectable with real-time RT-PCR. SEM showed multilayer of flat squamous polygonal epithelial cells. Desmosomal and hemidesmosomal attachments were evident. Our study showed that cultivated limbal epithelium consists of limbal progenitors as well as differentiated corneal epithelial cells. SEM and TEM analysis showed cultivated cells demonstrated typical features of corneal epithelium. The risk of contamination is low and can be prevented by culturing the cells in a clean room facility complying to Good Manufacturing Practice standard.

Modulating multidrug resistance gene in leukaemia cells by short interfering RNA.

INTRODUCTION: The multidrug resistance gene, MDR1, is one of the genes responsible for resistance to chemotherapy in the treatment of leukaemia and other cancers. The discovery of RNA interference in mammalian cells has provided a powerful tool to inhibit the expression of this gene. However, very little is known about the transfection of leukaemia cells with short interfering RNA (siRNA) targeted at MDR1. This study aims to evaluate the effectiveness of two chemically-synthesised siRNA in modulating MDR1 gene and inhibiting P-glycoprotein expression in leukaemic cells. We also evaluated two siRNA delivery methods in this study. METHODS: K562/Adr was transfected with two MDR1-targeted siRNA or negative control siRNA, by using cationic lipid-based transfection reagents or electroporator. Gene expression of MDR1 was quantified by real-time polymerase chain reaction and calculated as a percentage relative to the negative control siRNA. P-glycoprotein expression was evaluated via flow cytometry and drug sensitivity after treatment was assessed by cytotoxicity assays. RESULTS: The percentage of MDR1 gene knockdown from cells transfected with an electroporator was significantly higher (84.4 percent, p-value is 0.094) compared to cells transfected with cationic lipid-based transfection reagents (52.8 percent). Both siRNA significantly reduced the expression of MDR1 by 84.9 percent (p-value is 0.001) and 86.0 percent (p-value is 0.011), respectively. P-glycoprotein expression was down-regulated and drug sensitivity was increased after treatment with the siRNA. CONCLUSION: This study shows that the two siRNA sequences are capable of modulating MDR1 and P-glycoprotein expressions and increased drug sensitivity. Transfection with an electroporator was superior to chemical transfection for leukaemia cells.

Generation of dendritic cells from acute myeloid leukaemia cells and monocytes: our local experience.

Dendritic cells (DC) are efficient and potent antigen-presenting cells. Pilot clinical trials indicated that DC loaded with tumour antigen could induce tumour-specific immune responses in various cancers including B-cell lymphoma, melanoma and prostate cancer. Owing to extensively low number of DC in the blood circulation, a variety of sources have been used to generate DC including monocytes, CD34+ stem cells and even with leukaemic blast cells. We demonstrate here a simple method to generate DC from acute myeloid leukaemia (AML) cells and monocytes from healthy donor or remission samples. AML cells or monocytes were cultured in RPMI 1640 media supplemented with foetal bovine serum or autologous serum where possible and different combinations of cytokines GM-CSF, IL-4 and TNF-alpha. The generated DC were evaluated for their morphology by phase contrast microscopy and May Grunwald Giemsa staining. Viability of cells was determined by trypan blue dye exclusion. Percentage of yields and immunophenotypes were carried out by flow cytometry. We found that cultured AML cells and monocytes developed morphological and immuno-phenotypic characteristics of DC. Monocytes are better than AML blast in generating DC and serve as a ready source for dendritic cell vaccine development.

Prognostic value of laparoscopic ultrasound in patients with gastro-esophageal cancer.

Forty-four patients with gastro-esophageal tumors regarded as resectable by conventional staging underwent laparoscopic ultrasonography (LUS). Following LUS, seven were found to be irresectable and were managed by palliative therapies. Thirty-seven patients proceeded to surgical exploration and 36 were resected (R0 80%, R1 11%, and R2 9%). All patients were reviewed until death or for a minimum of 24 months. Patients undergoing resection had a 62% 1-year survival (median 17 months; confidence intervals, CI 6-28). LUS defined nodal status indicated a trend toward prolonged survival in the node-negative group, median 22 months (CI 5-39), compared with 13 months (CI 6-20) in the node-positive group. Disease-free survival was greater in LUS node-negative patients at 29 months (CI 23-35) compared with node-positive patients at 13 months (CI 5-21) P=0.0083. LUS staging allows prediction of the likelihood of recurrence of gastro-esophageal malignancies. This may prove useful for the appropriate allocation of patients to primary and adjuvant therapies.

Cardiac abnormalities in systemic lupus erythematosus: prevalence and relationship to disease activity.

We conducted a prospective longitudinal study to determine the nature and prevalence of cardiac abnormalities in systemic lupus erythematosus and to study their natural history and relationship with disease activity. Forty consecutive inpatients with systemic lupus erythematosus were studied during their admission and subsequently 6 to 12 months later. On each occasion a clinical cardiovascular examination was carried out, disease activity was scored using the "Lupus Activity Criteria Count" and a Doppler echocardiographic examination was carried out. 72.5% of patients had an abnormal echocardiogram in the first study while 51.7% were abnormal during the follow-up study. Valvar disease occurred in 37.5% of patients. The mitral valve was most commonly affected. Libman-Sacks endocarditis was rare (2.5%). Pericardial effusions were seen in 36.2% of echocardiograms. The majority (76.0%) of these were associated with hypoalbuminaemia. 80.0% of patients had active disease during the first examination and 41.4% at follow-up. There was no correlation between activity of disease and prevalence of cardiac abnormalities at either examination. We conclude that cardiac disease is common in systemic lupus erythematosus. Prevalence of cardiac abnormality did not correlate with disease activity.

Improved sperm concentration, motility, and fertilization rates following Ficoll treatment of sperm in a human in vitro fertilization program.

Duplicate sperm samples from the spouses of 54 patients admitted to an in vitro fertilization program were prepared by the swim-up and a simplified procedure using Ficoll. Cellsoft (CRYO Resources, New York, NY) sperm curvilinear velocities (microns/sec) and mean amplitude of lateral head displacement values (microns) equivalent to grade 1 and 2 visual sperm motility were significantly higher for Ficoll as compared with swim-up samples (P less than 0.01). Fertilization rates were significantly higher in the Ficoll as compared with the swim-up group for poor semen samples (grade less than 2: 58% versus 24%; P less than 0.01) and normal semen samples (grade greater than or equal to 2: 85% versus 78%; P less than 0.05). Ficoll sperm separation appears to be an excellent method of yielding increased fertilization rates in in vitro fertilization programs.

Effect of sperm motility on human in vitro fertilization.

Several sperm motility parameters in semen prepared by the swim-up technique were compared with IVF rates in 84 patients. The patients were either on clomiphene + human menopausal gonadotrophin or follicle stimulating hormone + human menopausal gonadotrophin stimulation regimens. Motility ratings were assessed both manually according to World Health Organization guidelines as well as computer-automated semen analysis (Cellsoft, Cryoresources, USA). Motility ratings of greater than or equal to 2 yielded significantly higher fertilization rates (78-82%) than ratings below 2 (20-23%) (p less than 0.001) for patients on both regimens. Velocity (41, 55, 78 microns/sec) and mean amplitude of lateral head displacement (1.96, 3.29, 4.91 microns) correlated significantly with and between manual ratings of 1, 2, and 3, respectively (r = 0.83; p less than 0.01). No significant differences were observed in linearity and beat/cross frequency between the manual ratings, although beat/cross frequencies tended to reduce linearly with increases in intensity of motility. The velocity of sperm motility has a significant effect on fertilization rates, and cut-off points of greater than or equal to 2 or greater than or equal to 50 microns/sec predict the actual potential and likely success of in vitro fertilization. These criteria on the swim-up semen should be used in the selection of patients admitted to IVF programs, and they justify the necessity of research investigations to improve motility in those patients with sluggish motility.

Evaluation of Chang's culture medium for mouse in vitro fertilization and embryonic development.

Chang's medium [with and without human serum (HS)] was compared with T6 medium [with and without bovine serum albumin (BSA)] for in vitro fertilization (IVF) and development of two-cell mouse embryos to the blastocyst stage. Chang's medium without any supplementation gave significantly better fertilization rates (83.3%) than Chang's with 10% HS (76.4%) or T6 and BSA (76.6%) (P less than 0.01). In a separate experiment 87.7% of the two-cell mouse embryos developed to the blastocyst stage in Chang's medium, compared to 90.6% for T6 with BSA and 93.6% without BSA, respectively (P greater than 0.01). In Chang's medium supplemented with 10% HS, 76.6% of the embryos developed to the blastocyst stage and 17.2% stopped development after the morula stage. After 72 hr in vitro hatched trophoblast and inner-cell-mass cells from 26.5 and 30.8% of the embryos grown in Chang's medium (with and without HS) attached to the plastic culture dishes and grew to form a mixed monolayer of epithelioid and fibroblastic cells. Chang's medium can thus be successfully used for IVF and growth of mammalian embryos. Further, inner cell mass and trophoblast cell lines could be established for various reproductive studies using this medium.