In vitro growth of human urinary tract smooth muscle cells on laminin and collagen type I-coated membranes under static and dynamic conditions.

This study investigates in vitro growth of human urinary tract smooth muscle cells under static conditions and mechanical stimulation. The cells were cultured on collagen type I- and laminin-coated silicon membranes. Using a Flexcell device for mechanical stimulation, a cyclic strain of 0-20% was applied in a strain-stress-time model (stretch, 104 min relaxation, 15 s), imitating physiological bladder filling and voiding. Cell proliferation and alpha-actin, calponin, and caldesmon phenotype marker expression were analyzed. Nonstretched cells showed significant better growth on laminin during the first 8 days, thereafter becoming comparable to cells grown on collagen type I. Cyclic strain significantly reduced cell growth on both surfaces; however, better growth was observed on laminin. Neither the type of surface nor mechanical stimulation influenced the expression pattern of phenotype markers; alpha-actin was predominantly expressed. Coating with the extracellular matrix protein laminin improved in vitro growth of human urinary tract smooth muscle cells.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Human urothelial cells grown on collagen adsorbed to surface-modified polymers.

OBJECTIVES: Tissue engineering methods can be applied to regenerate diseased, or congenitally missing, urinary tract tissues. Urinary tract tissue cell cultures must be established in vitro and adequate matrices, acting as cell carriers, must be developed. Although degradable and nondegradable polymer matrices offer adequate mechanical stability, they are not optimal for cell adherence and growth. To overcome this problem, extracellular matrix proteins, permitting cell adhesion and regulation of cell proliferation and differentiation, can be adsorbed to the surface-modified polymer. METHODS: In this study, nondegradable polymer films, poly(ethylene terephthalate), were used as an experimental model. Films were modified by graft polymerization of acrylic acid to subsequently allow collagen type I and III immobilization. The following adhesion, proliferation of human urothelial cells, and induction of their stratification were analyzed. RESULTS: Collagen adsorption on 0.2 microg/cm2 poly(acrylic acid)-grafted polymer films rendered the matrix apt for human urothelial cell adhesion and proliferation. Furthermore, stratification of urothelial cells was demonstrated on these surface-modified matrices. CONCLUSIONS: These results have shown that surface-modified polymer matrices can be used to act as cell carriers for cultured human urothelial cells. Such a cell-matrix construct could be applied in reparative surgery of the urinary tract.

Compressed collagen gel: a novel scaffold for human bladder cells.

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo-tissue level. This controlled, cell-independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell-seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation.

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Adult human adipose tissue contains several types of multipotent cells.

Multipotent mesenchymal stromal cells (MSCs) are a type of adult stem cells that can be easily isolated from various tissues and expanded in vitro. Many reports on their pluripotency and possible clinical applications have raised hopes and interest in MSCs. In an attempt to unify the terminology and the criteria to label a cell as MSC, in 2006 the International Society for Cellular Therapy (ISCT) proposed a standard set of rules to define the identity of these cells. However, MSCs are still extracted from different tissues, by diverse isolation protocols, are cultured and expanded in different media and conditions. All these variables may have profound effects on the selection of cell types and the composition of heterogeneous subpopulations, on the selective expansion of specific cell populations with totally different potentials and ergo, on the long-term fate of the cells upon in vitro culture. Therefore, specific molecular and cellular markers that identify MSCs subsets as well as standardization of expansion protocols for these cells are urgently needed. Here, we briefly discuss new useful markers and recent data supporting the rapidly emerging concept that many different types of progenitor cells are found in close association with blood vessels. This knowledge may promote the necessary technical improvements required to reduce variability and promote higher efficacy and safety when isolating and expanding these cells for therapeutic use. In the light of the discussed data, particularly the identification of new markers, and advances in the understanding of fundamental MSC biology, we also suggest a revision of the 2006 ISCT criteria.

In vitro infection of human dendritic cells by Aspergillus fumigatus conidia triggers the secretion of chemokines for neutrophil and Th1 lymphocyte recruitment.

Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7+ naïve T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.

Cytotoxic effects of combination of oxidosqualene cyclase inhibitors with atorvastatin in human cancer cells.

Ten oxidosqualene cyclase inhibitors with high efficacy as cholesterol-lowering agents and of different chemical structure classes were evaluated as potential anticancer agents against human cancer cells from various tissue origins and nontumoral human-brain-derived endothelial cells. Inhibition of cancer cell growth was demonstrated at micromolar concentrations, comparable to the concentrations of statins necessary for antitumor effect. Human glioblastoma cells were among the most sensitive cells. These compounds were also able to decrease the proliferation of angiogenic brain-derived endothelial cells, as a model of tumor-induced neovasculation. Additive effects in human glioblastoma cells were also demonstrated for oxidosqualene cyclase inhibitors in combination with atorvastatin while maintaining selectivity against endothelial cells. Thus, not only statins targeting the 3-hydroxy-3-methylglutaryl coenzyme A reductase but also inhibitors of oxidosqualene cyclase decrease tumor growth, suggesting new therapeutic opportunities of combined anti-cholesterol agents for dual treatment of glioblastoma.

Quantification, self-renewal, and genetic tracing of FL1+ tumor-initiating cells in a large cohort of human gliomas.

Evidence has emerged that the initiation and growth of gliomas is sustained by a subpopulation of cancer-initiating cells (CICs). Because of the difficulty of using markers to tag CICs in gliomas, we have previously exploited more robust phenotypic characteristics, including a specific morphology and intrincic autofluorescence, to identify and isolate a subpopulation of glioma CICs, called FL1(+). The objective of this study was to further validate our method in a large cohort of human glioma and a mouse model of glioma. Seventy-four human gliomas of all grades and the GFAP-V(12)HA-ras B8 mouse model were analyzed for in vitro self-renewal capacity and their content of FL1(+). Nonneoplastic brain tissue and embryonic mouse brain were used as control. Genetic traceability along passages was assessed with microsatellite analysis. We found that FL1(+) cells from low-grade gliomas and from control nonneoplasic brain tissue show a lower level of autofluorescence and undergo a restricted number of cell divisions before dying in culture. In contrast, we found that FL1(+) cells derived from many but not all high-grade gliomas acquire high levels of autofluorescence and can be propagated in long-term cultures. Moreover, FL1(+) cells show a remarkable traceability over time in vitro and in vivo. Our results show that FL1(+) cells can be found in all specimens of a large cohort of human gliomas of different grades and in a model of genetically induced mouse glioma as well as nonneoplastic brain. However, their self-renewal capacity is variable and seems to be dependent on the tumor grade.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines.

PURPOSE: The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. METHOD: Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. RESULTS: Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. CONCLUSIONS: Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.

NPY regulates catecholamine secretion from human adrenal chromaffin cells.

The aim of the present work was to find out whether NPY synthesized in human adrenal chromaffin cells controls in an autocrine/paracrine fashion the release of catecholamines by these cells. Accordingly, the constitutive and regulated release of both NPY and catecholamines was measured simultaneously in cultured human chromaffin cells. In addition, by using both RT-PCR and a combination of specific agonists and antagonists, we characterized the expression of NPY receptors on these cells as well as their pharmacology. Our results were as follows. 1) Human chromaffin cells constitutively secrete NPY. 2) Nicotine elicits a rapid increase in the release of both catecholamines and NPY; this release of NPY is more sustained than that of catecholamines. 3) RT-PCR shows expression of Y1, Y2, Y4, and Y5 receptor mRNA by chromaffin cells; these receptors are functional, as various receptor specific agonists elicit an increase in intracellular calcium. 4) Peptide YY, in contrast to NPY, is not able to stimulate the release of catecholamines. This finding was corroborated by the observation that no receptor-specific antagonists were able to reduce constitutive catecholamine release, whereas an NPY-immunoneutralizing antibody markedly attenuated the secretion. Taken together, these data suggest that NPY originating from the adrenal medulla locally enhances the secretion of catecholamines, presumably by acting via the putative y3 receptor.