Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg²⁺, Ca²⁺, Mn²⁺, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg²⁺, 7 mM for Sr²⁺, 6 mM for Ca²⁺, 3.5 mM for Ba²⁺ and 1.8 mM for Mn²⁺. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn²⁺ > Ca²⁺ > Mg²⁺ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn²⁺ and Ca²⁺, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The     

Cord blood cytokine levels in neonates born to mothers with prolonged premature rupture of membranes and its relationship with morbidity and mortality

The purpose of this study was to determine cord blood cytokine levels and their relationship with morbidity and mortality in neonates with prolonged, premature rupture of membranes (PPROM). Forty two premature neonates of 29-35 weeks gestational age with PPROM exceeding 24 hours were considered as the PPROM group and simultaneously, 41 premature neonates without PPROM were considered as the control group. All the neonates were admitted to the Neonatology Unit for further evaluation of subsequent complications such as early neonatal sepsis, pneumonia, intraventicular haemorrhage (IVH), respiratory distress syndrome (RDS), necrotizing enterocolitis (NEC) and chronic lung disease (CLD). Cord blood and mothers' blood samples were obtained during delivery in both groups and tested for IL-6, IL-8 and TNF-alpha levels. Twenty one percent of patients with PPROM had histological chorioamnionitis. The risk for developing early neonatal sepsis increased significantly in neonates whose mothers had histological chorioamnionitis (p < 0.05). There was a statistically significant relationship between PPROM and risk of developing NEC (p < 0.05); no significant increase was seen as regards early neonatal sepsis, IVH, RDS, pneumonia, or BPD. The mean IL-8 levels in cord blood and mothers' serum were significantly higher in the PPROM group (p < 0.001, p< 0.005). In addition, IL-6 levels found in mothers' serum were significantly higher than those found in the control group (p < 0.01). However, levels in cord blood were similar (p > 0.05). TNF-alpha levels were similar in both groups (p > 0.05). Neonates who developed NEC had higher IL-8 levels in their cord blood when compared to those without NEC (p < 0.05). In conclusion, the presence of PPROM increases the risk of chorioamnionitis. In addition, PPROM increases the risk of NEC, and patients who developed NEC had significantly higher cord blood IL-8 values. We may conclude that patients with PPROM and higher IL-8 levels in cord blood might be considered as at possible risk of NEC.     

Geometric characterization of cell membrane of mouse oocytes for ICSI

Intracytoplasmic sperm injection (ICSI) is a broadly utilized assisted reproductive technology. A number of technologies for this procedure have evolved lately, such as the most commonly utilized piezo-assisted ICSI technique (P-ICSI). An important problem with this technique, however, is that it requires a small amount of mercury to stabilize the tip of the penetration micropipette. A completely different and mercury-free injection technology, called the rotationally oscillating drill (Ros-Drill) (RD-ICSI), was recently developed. It uses microprocessor-controlled rotational oscillations of a spiked micropipette after the pipette deforms the membrane to a certain tension level. Inappropriate selection of this initiation instant typically results in cell damage, which ultimately leads to unsuccessful ICSI. During earlier manual clinical tests of Ros-Drill, the technicians' expertise determined this instant in an ad hoc fashion. In this paper, we introduce a computer-vision-based tool to mechanize this process with the objective of maintaining the repeatability and introducing potential automation. Computer images are used for monitoring the membrane deformations and curvature variations as the basis for decision making. The main contribution of this paper is in the specifics of the computer logic to perform the monitoring. These new tools are expected to provide a practicable means for automating the Ros-Drill-assisted ICSI operation.     

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patients T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patients PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.     

Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability

Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times.     

IL-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo

Tumor-associated macrophages (TAMs) play a major role in promoting tumor growth and metastasis and in suppressing the antitumor immune response. Despite the immunosuppressive environment created by the tumor and enforced by tumor-associated macrophages, treatment of tumor-bearing mice with IL-12 induces tumor regression associated with appearance of activated NK cells and activated tumor-specific CTLs. We therefore tested the hypothesis that IL-12 treatment could alter the function of these tumor-associated suppressor macrophages. Analysis of tumor-infiltrating macrophages and distal TAMs revealed that IL-12, both in vivo and in vitro, induced a rapid (<90 min) reduction of tumor supportive macrophage activities (IL-10, MCP-1, migration inhibitory factor, and TGFbeta production) and a concomitant increase in proinflammatory and proimmunogenic activities (TNF-alpha, IL-15, and IL-18 production). Similar shifts in functional phenotype were induced by IL-12 in tumor-infiltrating macrophages isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen, and peritoneal cavity. Therefore, although TAMs display a strongly polarized immunosuppressive functional profile, they retain the ability to change their functional profile to proinflammatory activities given the appropriate stimulus. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive antitumor immune response.     

Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells

Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett. 10 (2005) 455–470). Here, we examined the efficacy of these vectors to deliver β-galactosidase (β-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer. We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%) on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer are highly effective in transfecting SCCVII cells. Approximately 60–70% of SCCVII cells transfected with pCMV.lacZ were positive for β-gal staining. The expression of β-galactosidase was essentially not affected by serum. Mouse serum (20–60%) reduced both Metafectene-and GeneJammer-mediated luciferase expression by ∼30–45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over 90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes is the primary cause of serum inhibition of transfection.     

Unexpected heterogeneity due to recessive and de novo dominant mutations of GJB2 in an Iranian family with nonsyndromic hearing loss: implication for genetic counseling

Mutations in the GJB2 gene are the most common cause of nonsyndromic autosomal recessive sensorineural hearing loss (HL). A few mutations in GJB2 have also been reported to cause dominant nonsyndromic HL. Here we report a large inbred family including two individuals with nonsyndromic sensorineural hearing loss. A dominant GJB2 mutation, c.551G>A (p.R184Q), was detected in the proband, yet his parents were negative for the mutation. The second affected person had heterozygous c.35delG mutation, which was inherited from his father. Large deletions of the GJB6 gene were not detected in this family. This study highlights the importance of mutation analysis in all affected cases within a pedigree.