The effect of preservative-free HP-Guar on dry eye after phacoemulsification: a flow cytometric study.

PURPOSE: To assess the effect of hydroxypropyl (HP)-Guar added to regular post-phacoemulsification treatment in dry eye signs and symptoms, and its influence on the expression of various inflammatory markers by flow cytometry (FCM) in impression cytology specimens. METHODS: This prospective, interventional, single-centre study included 48 eyes of 48 patients with age-related cataract. After phacoemulsification, patients were randomised to the usual treatment group (UT), with 21 patients who received tobramycin and dexamethasone eye drops (Tobradex, Alcon Cusí, Spain), and the HP-Guar group, with 27 patients who received the UT plus preservative-free artificial tears (Systane UD, Alcon Cusí, Spain). Corneal and conjunctival staining with fluorescein and lissamine green, tear film break-up time (TBUT), Schirmer's I test with anaesthesia (Jones test), tear clearance, and ocular surface disease index (OSDI) were assessed preoperatively and 1 month after surgery. Besides, conjunctival impression cytology was performed in order to investigate inflammatory markers (CD3, CD11b, and HLA-DR) using FCM. RESULTS: HP-Guar group shows statistical better results compared with the UT group in TBUT (6.4+/-0.7 vs 9+/-2.5, P=0.0004), OSDI (11.5+/-8.2 vs 3.3+/-2.5, P=0.0002), ocular symptoms subscale (7.3+/-6.1 vs 1.7+/-1.8, P=0.0004), vision-related function subscale (2.2+/-1.8 vs 0.4+/-0.6, P=0.0002), CD3 (2.5+/-1.4 vs 1.1+/-1.1, P=0.011), and HLA-DR (6.8+/-4.5 vs 1.8+/-1.7, P=0.0002). CONCLUSION: The addition of HP-Guar to regular treatment after cataract surgery reduces ocular surface inflammation and dry eye signs and symptoms.

BCL6 represses NFkappaB activity in diffuse large B-cell lymphomas.

BCL6 is a transcriptional repressor whose deregulated expression plays a key role in diffuse large B-cell lymphomas (DLBCLs). BCL6 expression characterizes one of the two main subtypes (GC type) of DLBCL, while the other (ABC type) is recognized by increased NFkappaB activation. The mechanistic basis of this distinction remains unclear and the BCL6 targets have been only partially explored. Here we describe how NFkappaB activity is increased after BCL6 silencing by shRNA in DLBCL cells, leading us to propose that BCL6 represses NFkappaB activity. We also demonstrate that this repression is brought about by a mechanism involving protein-protein interaction between BCL6 and NFkappaB members, both in vitro and in vivo. Analysis of a series of DLBCLs shows a negative correlation between the expression of NFkappaB target genes and BCL6. This combined approach using silenced cells and a series of human DLBCL samples leads us to a better understanding of the role of BCL6 as an NFkappaB regulator in B-cells.

Methylmalonic acidaemia leads to increased production of reactive oxygen species and induction of apoptosis through the mitochondrial/caspase pathway.

Methylmalonic acidaemia (MMA) is a heterogeneous group of rare genetic metabolic disorders caused by defects related to intracellular cobalamin (vitamin B(12)) metabolism. Increasing evidence has emerged suggesting that free radical generation is involved in the pathophysiology of neurodegenerative diseases, including some inborn errors of metabolism. We have previously identified in MMA patients several differentially expressed proteins involved in oxidative stress [mitochondrial superoxide dismutase (MnSOD) and mitochondrial glycerophosphate dehydrogenase (mGPDH)] and apoptosis by a proteomic approach. We have now extensively evaluated various parameters related to oxidative stress and apoptosis in cultured fibroblasts from a spectrum of patients with methylmalonic acidaemia. Fibroblasts from several MMA patients showed a significant increase in intracellular reactive oxygen species (ROS) content and in MnSOD expression level with respect to controls, suggesting a cellular response to intrinsic ROS stress. Moreover, we have demonstrated, using siRNA, that mGPDH is an important ROS generator in MMA patients. Cells from patients with MMA had a higher rate of apoptosis than those of controls and there was evidence that this process primarily involves the mitochondrial/caspase-dependent pathway. ROS level-phenotype correlation revealed that patients with severe neonatal cblB disorder had elevated intracellular ROS content. These findings support the possible role of oxidative stress in the pathophysiology of methylmalonic acidaemia.

Heat-induced programmed cell death in Leishmania infantum is reverted by Bcl-X(L) expression.

An increasing number of reports indicate that single-celled organisms are able to die following what seems to be an ordered program of cell death with strong similarities to apoptosis from higher eukaryotes. DNA degradation and several other apoptotic-like processes have also been described in the parasitic protozoa Leishmania. However, the existence of an apoptotic death in this parasite is still a matter of controversy. Our results indicate that most of the processes of macromolecular degradation and organelle dysfunction observed in mammalian cells during apoptosis can also be reproduced in promastigotes of the genus Leishmania when incubated at temperatures above 38 degrees C. These processes can be partially reversed by the expression of the anti-apoptotic mammalian gene Bcl-X(L), which suggests that this family of apoptosis-regulating proteins was present very early in the evolution of eukaryotic cells.

Identification of a nuclear export signal in the KSHV latent protein LANA2 mediating its export from the nucleus.

LANA2 is a latent protein detected in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected B cells that inhibits p53-dependent transcriptional transactivation and apoptosis and PKR-dependent apoptosis, suggesting an important role in the transforming activity of the virus. It has been reported that LANA2 localizes into the nucleus of both KSHV-infected B cells and transiently transfected HeLa cells. In this study, we show that LANA2 is a nucleocytoplasmic shuttling protein that requires a Rev-type nuclear export signal located in the C-terminus to direct the protein to the cytoplasm, through an association with the export receptor CRM1. In addition, a functional protein kinase B (PKB)/Akt phosphorylation motif partially overlapping with the nuclear export signal was identified. Nuclear exclusion of LANA2 was negatively regulated by the phosphorylation of threonine 564 by Akt. The ability of LANA2 to shuttle between nucleus and cytoplasm has implications for the function of this viral protein.

Evidence for discoordinate regulation of the HLA-DPB1 gene.

Characterization of cell lines derived from patients with type II bare lymphocyte syndrome, a pathological state in which the constitutive and inducible expression of HLA class II antigens is lacking, has permitted the identification of several trans-acting factors involved in the coordinated regulation of HLA class II genes. Although an increasing body of evidence has pointed to the existence of a discoordinate regulation of HLA class II loci, the mechanisms underlying such regulation are essentially unknown. In the present study, 45.EM2, a mutant lymphoblastoid cell line with a new pattern of HLA discoordinate expression is characterized. 45.EM2 expresses HLA-DR and -DQ but fails to express HLA-DP. The absence of HLA-DP expression in 45.EM2 is the result of a transcriptional defect, leading to a lack of DPB1 mRNA. By contrast, DPA1 transcription in this LCL is not impaired. The characteristics of 45.EM2 described here suggest the existence of a specific trans-acting factor involved in the control of DPB1 gene expression.

Use of flow cytometry and confocal microscopy techniques to investigate early CdCl(2)-induced nephrotoxicity in vitro.

CdCl(2) is a well-known toxic compound for the kidney in vivo and in vitro. We report here part of the results of an ECVAM (European Centre for the Validation of Alternative Methods) contract study, aimed at establishing and assessing several flow cytometric and confocal microscopic endpoints for use in an in vitro nephrotoxicity model. Three renal tubule cell lines, OK (opossum, proximal tubule origin), LLC-PK1 (pig, proximal tubule origin) and MDCK (dog, distal tubule origin) were exposed for 1, 5 and 24 h to 25 microM and 100 microM CdCl(2). The results obtained for mitochondrial membrane potential showed a decrease in all the cell lines after 5 h of treatment with both CdCl(2) concentrations. In some cases, this decrease was detected by flow cytometry after a 1-h exposure. On the contrary, intracellular Ca(2+) increased in a time-dependent and concentration-dependent fashion. This increase was especially high in the MDCK cell line after a 24-h exposure to 100 microM CdCl(2). However, cell viability was not affected by 25 microM CdCl(2). Our results demonstrate early changes in mitochondrial membrane potential and cytoplasmic Ca(2+) levels in renal tubular epithelial cell lines treated with CdCl(2).

Applications of flow cytometry to clinical microbiology.

Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.