Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens

Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-gamma was induced after stimulation with A. otitidis. Although the release of IFN-gamma started within 18 h after stimulation with A. otitidis, intracellular production of IFN-gamma was not observed in either CD4+ T cells or CD8+ T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens.     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

Physical changes of β-sitosterol crystals in oily suspensions during heating

The aim of this research was to describe the thermal behavior of β-sitosterol crystals in oil-suspensions with a focus on the role of water during heating. The suspensions were prepared by recrystallization in order to achieve a microcrystalline particle size. The structural changes together with the mechanical properties of the suspensions during heating were studied by using variable temperature X-ray powder diffractometry (VT-XRPD), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). Hydrated β-sitosterol crystals in an oil-suspension, dehydrated, despite the composition of the suspensions, at low temperatures. At high β-sitosterol concentration, the monohydrate crystal form changed partially to a hemihydrated form, and when only a small amount of water was initially incorporated, the hemihydrate crystal form dehydrated to a mostly anhydrate crystal form. The released water, which was immiscible in the surrounding oil, caused the recrystallization of hydrated β-sitosterol during cooling. This procedure indicated a reversible dehydration process. Structural and thermal analysis of β-sitosterol crystals in suspensions, together with mechanical analysis made it possible to understand various physical changes during heating. Published: October 19, 2005     

Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.     

CD2AP contributes to cell migration and adhesion in cultured gastric epithelium

The potential association of CD2AP with the adherens junction protein E-cadherin, co-localization with the actin cytoskeleton, and involvement in cell migration was investigated in cultured rat gastric mucosal cells. In stationary cells, CD2AP was localized perinuclearly while E-cadherin was expressed along cell-cell contacts and F-actin formed a branched network and adhesion belts. In migrating cells, CD2AP appeared as thread-like accumulations in the leading edges, colocalizing with F-actin and occasionally with E-cadherin. Intracellular injection of anti-CD2AP significantly retarded the migration speed of the cells suggesting a crucial role for CD2AP in mucosal cell migration, possibly as a scaffolding protein between cell membrane proteins and actin cytoskeleton. Co-immunoprecipitation assays revealed that CD2AP and E-cadherin are in a complex in HGF stimulated cells. It is concluded that CD2AP interacts with E-cadherin and co-localizes with F-actin in the leading edge of migrating cells, and significantly contributes to cell migration in restituting gastric epithelium.     

Mechano-acoustic determination of Young's modulus of articular cartilage

The compressive stiffness of an elastic material is traditionally characterized by its Young's modulus. Young's modulus of articular cartilage can be directly measured using unconfined compression geometry by assuming the cartilage to be homogeneous and isotropic. In isotropic materials, Young's modulus can also be determined acoustically by the measurement of sound speed and density of the material. In the present study, acoustic and mechanical techniques, feasible for in vivo measurements, were investigated to quantify the static and dynamic compressive stiffness of bovine articular cartilage in situ. Ultrasound reflection from the cartilage surface, as well as the dynamic modulus were determined with the recently developed ultrasound indentation instrument and compared with the reference mechanical and ultrasound speed measurements in unconfined compression (n=72). In addition, the applicability of manual creep measurements with the ultrasound indentation instrument was evaluated both experimentally and numerically. Our experimental results indicated that the sound speed could predict 47% and 53% of the variation in the Young's modulus and dynamic modulus of cartilage, respectively. The dynamic modulus, as determined manually with the ultrasound indentation instrument, showed significant linear correlations with the reference Young's modulus (r(2)=0.445, p<0.01, n=70) and dynamic modulus (r(2)=0.779, p<0.01, n=70) of the cartilage. Numerical analyses indicated that the creep measurements, conducted manually with the ultrasound indentation instrument, were sensitive to changes in Young's modulus and permeability of the tissue, and were significantly influenced by the tissue thickness. We conclude that acoustic parameters, i.e. ultrasound speed and reflection, are indicative to the intrinsic mechanical properties of the articular cartilage. Ultrasound indentation instrument, when further developed, provides an applicable tool for the in vivo detection of cartilage mechano-acoustic properties. These techniques could promote the diagnostics of osteoarthrosis.     

Lucigenin and coelenterazine as superoxide probes in mitochondrial and bacterial membranes

The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans. Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction. Therefore, calibration of measurements on complex I, capable of efficient lucigenin prereduction with reduced nicotinamide adenine dinucleotide, against xanthine oxidase, which in the presence of hypoxanthine is not able to reduce the probe to a significant rate compared to complex I, may give results in error by one order of magnitude. Coelenterazine, although susceptible of storage-dependent high background chemiluminescence, does not require prereduction and is thus a more reliable probe.     

Aminobisphosphonate-pretreated dendritic cells trigger successful Vγ9Vδ2 T cell amplification for immunotherapy in advanced cancer patients

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vγ9Vδ2 T lymphocytes represents an attractive strategy. Indeed, Vγ9Vδ2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vγ9Vδ2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vγ9Vδ2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vγ9Vδ2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vγ9Vδ2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vγ9Vδ2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vγ9Vδ2 T cells as measured by IFN-γ production. Moreover, we demonstrated that the cytotoxic level of Vγ9Vδ2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vγ9Vδ2 T cells leads eligible for Vγ9Vδ2 T cell adoptive immunotherapy the HCC and mCRC patients.     

Nε-Modified lysine containing inhibitors for SIRT1 and SIRT2

Sirtuins catalyze the NAD⁺ dependent deacetylation of N^ε-acetyl lysine residues to nicotinamide, O′-acetyl-ADP-ribose (OAADPR) and N^ε-deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala sequence has been used as a probe for the screening of novel N^ε-modified lysine containing inhibitors against SIRT1 and SIRT2. N^ε-Selenoacetyl and N^ε-isothiovaleryl were the most potent moieties found in this study, comparable to the widely studied N^ε-thioacetyl group. The N^ε-3,3-dimethylacryl and N^ε-isovaleryl moieties gave significant inhibition in comparison to the N^ε-acetyl group present in the substrates. In addition, the studied N^ε-alkanoyl, N^ε-α,β-unsaturated carbonyl and N^ε-aroyl moieties showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small changes in electronic and steric properties of the N^ε-modification. These results are applicable for further screening of N^ε-acetyl analogues.