Respiratory Influenza Virus Infection Induces Memory-like Liver NK Cells in Mice.

Although NK cells are classified as innate immune cells, recent studies have demonstrated the transformation of NK cells into long-lived memory cells that contribute to secondary immune responses in certain mouse models. However, whether NK cells mount an Ag-specific memory response to acute influenza virus infection has not yet been examined. Here, we show that, consistent with previous studies, lung NK cells play an important role in controlling viral proliferation after primary influenza virus infection. However, although lung NK cells display a memory phenotype at the late stage of infection, these cells do not protect mice against secondary influenza virus infection. Interestingly, liver NK cells from influenza virus-infected mice possess a memory phenotype and protect mice against secondary influenza virus infection. Memory-like liver NK cells display a CD49a+DX5- phenotype, and the adoptive transfer of purified liver CD49a+DX5- NK cells into naive mice followed by viral infection results in protective immunity and decreased viral titer. Moreover, we demonstrate that primary inactivated influenza virus induces memory NK cells residing in the liver of Rag1-/- mice. Collectively, these data suggest that liver CD49a+DX5- NK cells remember encountered Ag from influenza virus after primary infection and are more protective upon subsequent infection.

Trifluoroethanol-containing RP-HPLC mobile phases for the separation of transmembrane peptides human glycophorin-A, integrin alpha-1, and p24: analysis and prevention of potential side reactions due to formic acid.

Reversed-phase high-pressure liquid chromatography analysis and purification of three hydrophobic, aggregation-prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2-trifluoroethanol (TFE). The addition of 10-16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE-containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711: 181-186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM-peptide purification. Characterization of the potential side reactions using MALDI and ESI-LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE-formic acid possibly induces O-formylation of the Ser residue and N-formylation of the N-terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation-prone TM peptides and proteins.

Increasing the bioactivity of elastomeric poly(ε-caprolactone) scaffolds for use in tissue engineering.

BACKGROUND: Biodegradable polymers used in tissue engineering applications, such as poly(ε-caprolactone) (PCL), are hydrophobic leading to a lack of favorable cell signalization and finally to a poor cell adhesion, proliferation and differentiation. To overcome this problem, scaffolds undergo generally a surface modification. OBJECTIVE: Our laboratory has demonstrated that the grafting of poly(sodium styrene sulfonate) (pNaSS) onto titanium or poly(ethylene terephthalate) surfaces, leads to a more specific protein adsorption and a better control of cell proliferation. The objective of this work is to develop, through a straightforward way, bioactive elastomeric PCL scaffolds by grafting pNaSS. METHODS: Porous elastomeric PCL scaffolds were developed using a particulate-leaching process. pNaSS was grafted into the scaffold by a "grafting from" technique. In vitro tests were carried out to assess cell adhesion and protein expression. RESULTS: pNaSS was grafted homogeneously onto PCL scaffolds without degrading the biodegradable polymer or the porous structure. The in vitro studies have shown that pNaSS grafted onto PCL improves the cell response with a better expression of collagen, fibronectin and integrin α1. CONCLUSIONS: The grafting of pNaSS onto biomaterial surfaces is a versatile method that can provide a new generation of biodegradable scaffolds which could be "biointegrable".

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture.

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell's replication activity and the donor's age factor, and to assess the stem cells as a new source for tissue engineering. hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds: <20 years old, 21-40 years old, 41-60 years old and >61 years old groups). The protein markers (CD29, CD34, CD44, CD45, CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell, and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro. The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula "TD = t x log2/logNt - logN0" was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the <20 years old group was lower than that of the >61 years old group (statistical analysis of variance (ANOVA), P=0.002, P<0.05). These findings suggested that a higher level of hADAS cells replication activity was found in the younger donators, and they represent novel and valuable seed cells for studies of tissue engineering.

Optimal combination chemotherapy and chemoradiotherapy with etoposide for advanced cervical squamous cancer cells in vitro.

Patients with unresectable advanced carcinoma of the uterine cervix are usually treated with chemotherapy or chemoradiotherapy. In the present study, the optimal administration protocol for etoposide in chemotherapy and chemoradiotherapy for advanced cervical cancer patients was investigated in vitro using the radio-sensitive and anticancer drug-sensitive human cervical squamous cell carcinoma cell line ME180. Therapeutic doses of concurrent irradiation reduced the cellular etoposide sensitivity in a dose-dependent manner, while postirradiation-surviving subclones established from repeatedly irradiated ME180 cells showed significantly higher etoposide sensitivities than the non-irradiated parent cells. Of the 6 monoclonal etoposide-resistant subclones established from ME180 cells, 5 were significantly radioresistant. Although the etoposide-resistant subclones were also significantly resistant to other anticancer drugs, such as cisplatin, carboplatin, nedaplatin, pirarubicin, paclitaxel and docetaxel, they were more sensitive to 5-fluorouracil, mitomycin C and SN38 than the parent cells. Flow cytometric analyses revealed that the etoposide-resistant subclones showed significantly increased cell surface expression of CD40 compared to the parent cells, which expressed undetectable levels of CD40. However, the expression of some integrin receptor subunits, such as CD29, CD49a and CD49f, was apparently reduced in the etoposide-resistant subclones. These results indicate that etoposide should be administered to advanced cervical squamous cancer patients after the completion of radiotherapy, rather than as a concurrent chemoradiotherapy. In order to kill surviving etoposide-resistant cancer cells more effectively, 5-fluorouracil, mitomycin C and irinotecan may be candidate combination drugs for use with etoposide. Differential expression of integrin receptors and CD40 may be involved in the acquisition of etoposide resistance by cervical squamous cancer cells.

Wound healing is defective in mice lacking tetraspanin CD151.

The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins alpha6beta4, alpha3beta1, and alpha6beta1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of alpha6 and beta4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on alpha3 and beta1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration.

Dynamic protein expression patterns during intraoral wound healing in the rat.

Wound healing after cleft palate surgery is often associated with impairment of maxillary growth and dento-alveolar development. Wound contraction and scar tissue formation contribute strongly to these effects. In vitro studies have revealed that fibroblasts isolated during different phases of palatal wound healing show phenotypical differences. They change from a quiescent to an activated state and then partly back to a quiescent state. In this study, we evaluated the existence of fibroblast phenotypes at several time-points during palatal wound healing in the rat. Based on cytoskeletal changes (alpha-sma, vimentin, vinculin), integrin expression (alpha1, alpha2, alpha(v) and beta1) and changes in cellularity, we conclude that phenotypically different fibroblast populations are also present during in vivo wound healing. Alpha-sma and the integrin subunits alpha1 and alpha(v) were significantly up-regulated, and vinculin was significantly down-regulated, at early time-points compared to late time-points in wound healing. These changes point to an activated fibroblast state early in wound healing. Later in wound healing, these activated fibroblasts return only partially to the unwounded situation. These results strongly support the idea that different fibroblast populations with specific phenotypes occur in the course of palatal wound healing.

Integrin expression in cells of the intervertebral disc.

In this study, we investigated the profile of integrin expression in human and porcine intervertebral disc tissue. Differences in extracellular matrix composition between anulus fibrosus (AF) and nucleus pulposus (NP) regions of the disc, as well as differences in cellular responses to environmental stimuli, suggest a role for integrins in presenting matrix signals that may mediate these responses. Human disc tissue and porcine AF and NP tissue were stained with antibodies to alpha integrin subunits 1-6, V and IIb, and beta integrin subunits 1-6 and graded for evidence of positive staining on a scale from 0 (no staining) to 3 (high incidence of staining). Human tissue expressed alpha and beta integrin subunits shown to be present in articular cartilage, including alpha(1), alpha(5) and alpha(V). Porcine AF tissue expressed similar integrin subunits to human disc, with both expressing alpha(1), alpha(5), beta(1), beta(3) and beta(5) subunits, whereas porcine NP tissue expressed higher levels of alpha(6), beta(1) and beta(4) than AF tissue. The expressed subunits are known to interact with proteins including collagens, fibronectin and laminin; however, additional studies will be required to characterize the interactions of the integrin subunits with specific matrix constituents, as well as their specific involvement in regulating environmental stimuli.

Expression of integrins and extracellular matrix proteins at the maternal-fetal interface during tubal implantation.

Investigation of the expression pattern of integrins and their extracellular matrix (ECM) ligands in trophoblasts at the maternal-fetal interface during tubal pregnancy may aid better understanding of the adhesion and invasion of acceptable maternal endometrium by trophoblast cells at the very early stage of human gestation. In this study, spatial and temporal alterations of integrins and ECM ligands were examined in specimens of tubal pregnancies during weeks 3-9 of gestation. In situ hybridization and immunohistochemistry revealed that relatively high levels of integrin alpha(1), beta(1), alpha(5) subunits and heterdimer alpha(5)beta(1) as well as ECM ligands, were displayed in trophoblast cells as early as weeks 3-4 of gestation. Expression peaked during weeks 5-7 and then, with the exception of integrin alpha(1), which remained high, declined slightly up to weeks 8-9 of gestation. Immunoreactive fibronectin, laminin and type IV collagen were detected in column cytotrophoblastic cells (CTB) and some invasive extravillous cytotrophoblast (EVCT) cells and the alterations were coincident with those of the corresponding integrin receptors in EVCT cells. Laminin was strongly stained in EVCT cells that had invaded maternal blood vessels and deep into the interstitium. Maternal epithelial, endothelial and stromal cells also expressed these integrins and ECM ligands. The results indicate their involvement in mediating the adhesion of trophoblasts to the epithelium of the maternal Fallopian tube. The upregulated expression of these molecules in column CTB and invasive EVCT cells may also facilitate the invasion of trophoblasts into the maternal interstitium. Moreover, trophoblasts possessed the potential for self-controlled adhesion and invasion and appear to reach peak invasive capability in the second month of tubal implantation.

A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion.

A three-dimensional (3-D) endometrium culture was established, in which human endometrial stromal cells embedded in a mixture of collagen I, a major component of extracellular matrix, and matrigel, a basement membrane material, supports the epithelial cells seeded on top of the collagen/matrigel matrix. The biological growth and differentiation of the epithelial cells were studied microscopically and immunohistochemically. Transmission electron microscopy showed a polarized columnar epithelium in monolayer with basally positioned nuclei. Scanning electron microscopy revealed a confluent epithelium with an abundance of microvilli and cilia as well as pinopodes on the apical surface. An immunohistochemical staining showed that integrin alpha1, alpha4, and beta3 were co-localized with cytokeratin, confirming the epithelial origin of the cells. In contrast, immunoreactivity against cyclooxygenase-1 or -2 was positive in both epithelial and stromal cells. When epithelial cells were replaced by KLE cells, an endometrial cancer cell of epithelial origin, invasion of KLE cells into the stromal fraction was observed. The invasion was closely correlated to expression of matrix metalloproteinases and their tissue inhibitors of metalloproteinases in a manner consistent with paracrine fashion. The present 3-D culture imitates the normal endometrium physiologically as well as morphologically, thus provides an excellent in vitro tissue suitable for reproducing in vivo physiological processes, including endometrial cancer invasion.

Endometrial integrin expression in the early luteal phase in natural and stimulated cycles for in vitro fertilization.

OBJECTIVE: To investigate the effect of ovarian stimulation on integrin expression in the early luteal phase. STUDY DESIGN: Seven endometrial biopsies were taken 2 days after the oocyte retrieval from stimulated cycles for IVF and 23 from natural cycles, 2 days after ovulation. RESULT: Endometrium was in-phase in 22/23 and 7/7 biopsies from the natural and stimulated cycles, respectively. Integrins alpha(1) and alpha(4) were simultaneously positive in 43.4% from the natural and in all (100%) the stimulated cycles (P<0.03). On the day of the endometrial biopsy, progesterone serum values were higher in the stimulated cycles (55.2+/-9.5 microg/l versus 8.5+/-3.8 microg/l) (P<0.001). HSCORE value was significantly higher in stimulated cycles for both integrins. CONCLUSION: Endometrial integrin expression is more consistently present in the early luteal phase in stimulated cycles than in natural cycles and this may be related to the higher serum progesterone concentration and/or the more advanced endometrial histological features.

Fibroblast subpopulations in intra-oral wound healing.

The objective of this study was to characterize fibroblasts at sequential time points during intra-oral wound healing in the rat. Experimental wounds were made at several time points in the mucoperiosteum of the palate of 35-day-old Wistar rats. Fibroblasts were cultured from the biopsies under standard conditions for the same number of passages. The expression of the integrin subunits alpha 1, alpha 6, and beta 1; and the intermediate filaments alpha-smooth muscle actin and vimentin were analyzed by flow cytometry. Western blot analysis was performed at 0, 8, and 60 days postwounding to confirm the expression of both intermediate filaments. The phenotypic profiles of fibroblasts cultured from subsequent stages in the wound healing process differed considerably. We conclude that distinct fibroblast phenotypes can be isolated from different stages in wound healing. These phenotypes remained stable during in vitro culturing. In addition, cryosections of the wound areas were made at identical time points and were immunohistochemically stained for the same antigens. The immunohistochemical staining correlated well to the flow-cytometric data. These results suggest the occurrence of multiple subpopulations of fibroblasts with a specialized function during wound healing. We hypothesize that undesirable consequences of wound healing might be prevented through the modulation of specific fibroblast subpopulations.

Antiphospholid antibodies regulate the expression of trophoblast cell adhesion molecules.

OBJECTIVE: To examine the effect of antiphospholipid antibodies on trophoblast expression of adhesion molecules. DESIGN: Primary cytotrophoblast cell cultures. SETTING: Department of Obstetrics and Gynecology, Catholic University, Rome, Italy. PATIENT(S): Five normal pregnant women underwent uncomplicated vaginal delivery at 36 weeks of gestation. INTERVENTION(S): IgG antibodies were isolated from a patient with antiphospholipid syndrome and from a normal control subject, using protein-G Sepharose columns. Cytotrophoblast cells were dispersed in bicarbonate buffer containing trypsin and DNAse I. MAIN OUTCOME MEASURE(S): We investigated the effects of antiphospholipid antibodies on trophoblast adhesion molecules (alpha1 and alpha5 integrins, E and VE cadherins), both at the protein and mRNA levels. RESULT(S): The alpha1 and alpha5 integrins were present in trophoblast cells from 24 hours of culture. Treatment with IgG that were obtained from the patient with antiphospholipid syndrome significantly decreased alpha1 integrin and increased alpha5 integrin at both the mRNA and protein levels. Furthermore, IgG with antiphospholipid antibodies activities induced VE-cadherin down-regulation and the E-cadherin up-regulation at protein and mRNA levels compared with control IgG or untreated cells. CONCLUSION(S): The results suggest that the inadequate trophoblastic invasion, induced by antiphospholipid antibodies, can be the result of abnormal trophoblast adhesion molecules expression.