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Biomedical subjects

N Romani

Publications and source records attributed to N Romani.

At least 37 records · Page 2Linked to original sources
PubMed1999

Human immunodeficiency virus type 1 derived from cocultures of immature dendritic cells with autologous T cells carries T-cell-specific molecules on its surface and is highly infectious.

During the budding process, human immunodeficiency virus type 1 (HIV-1) acquires cell surface molecules; thus, the viral surface of HIV-1 reflects the antigenic pattern of the host cell. To determine the source of HIV-1 released from cocultures of dendritic cells (DC) with T cells, immature DC (imDC), mature DC (mDC), T cells, and their cocultures were infected with different HIV-1 isolates. The macrophage-tropic HIV-1 isolate Ba-L allowed viral replication in both imDC and mDC, whereas the T-cell-line-tropic primary isolate PI21 replicated in mDC only. By a virus capture assay, HIV-1 was shown to carry a T-cell- or DC-specific cell surface pattern after production by T cells or DC, respectively. Upon cocultivation of HIV-1-pulsed DC with T cells, HIV-1 exclusively displayed a typical T-cell pattern. Additionally, functional analysis revealed that HIV-1 released from imDC-T-cell cocultures was more infectious than HIV-1 derived from mDC-T-cell cocultures and from cultures of DC, T cells, or peripheral blood mononuclear cells alone. Therefore, we conclude that the interaction of HIV-1-pulsed imDC with T cells in vivo might generate highly infectious virus which primarily originates from T cells.

Antigen Presentation↗
PubMed1999

Migration of langerhans cells and dermal dendritic cells in skin organ cultures: augmentation by TNF-alpha and IL-1beta.

Migration from sites of antigen encounter to lymphoid organs is essential to the strong immunogenic function of dendritic cells (DC). In the skin, migration proceeds through dermal lymphatic vessels and is regulated in an incompletely understood way by inflammatory mediators. We studied the effects of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in mouse skin organ cultures by direct enumeration of migrating DC and by immunohistochemistry. (1) Neutralizing antibodies to TNF-alpha and IL-1beta inhibited migration of DC, also in human skin explants (TNF-alpha). (2) TNF-alpha at low concentrations (50 U/mL) and IL-1beta (50-3000 U/mL) augmented migration to about 150% of spontaneous migration. (3) High concentrations of TNF-alpha (5000 U/mL) inhibited migration by approximately 50%. (4) DC migration from skin explants of TNF-alpha/lymphotoxin-alpha double-deficient mice and TNF-receptor type 1 and 2 double knockout mice was not impaired. (5) TNF-alpha effects were neutralized by anti-IL-1beta, and vice versa. We conclude that in normal animals both TNF-alpha and IL-1beta are required for DC migration to occur. In the complete absence of one cytokine (TNF-alpha), however, backup mechanisms step in.

Animals↗
PubMed1998

Expression of maturation-/migration-related molecules on human dendritic cells from blood and skin.

Progress in dendritic cell research has been overwhelming in the past few years. This was made possible by the recent development of simple methods to generate large numbers of dendritic cells. These methods use as starting populations for culture either CD34+ progenitor cells from cord blood or bone marrow, or monocytes from peripheral blood. The latter approach is critically dependent on the combination of GM-CSF and interleukin 4. Such "priming cultures" yield populations of immature dendritic cells (CD83-/CD86 +/- /CD115+/antigen uptake high/antigen processing high/T cell sensitization low). In order to generate mature dendritic cells a subsequent "differentiation culture" has to be added whereby monocyte-conditioned medium appears to be the optimal stimulus for maturation. This results in terminally mature dendritic cells (CD83+/CD86++/CD115-/antigen uptake low/antigen processing low/T cell sensitization high). We investigated the expression of some molecules involved in maturation and migration on human monocyte-derived dendritic cells from blood in comparison with dermal dendritic cells and epidermal Langerhans cells. We present a method to highly enrich epidermal Langerhans cells. Survival of purified Langerhans cells in culture is dependent on the presence of GM-CSF and TNF-alpha. During maturation a substantial part of the Langerhans cells loses expression of the cutaneous lymphocyte antigen (CLA); mature dendritic cells from the dermis are completely devoid of CLA. Similarly, CLA as well as CD15s (Sialyl Lewis x) and CD31 (PECAM-1) that can be readily detected on immature monocyte-derived dendritic cells are down-regulated upon maturation. CD68 expression is very low in cutaneous dendritic cells; in monocyte-derived dendritic cells this molecule is abundantly present. Subsets of monocyte-derived dendritic cells express E-cadherin; CD87 (urokinase plasminogen activator receptor) is weakly expressed on both immature and mature monocyte-derived dendritic cells. Taken together, these data suggest that the phenotype of monocyte-derived dendritic cells (E-cadherin low to negative, CD68++) is not indicative for a cutaneous destiny. Furthermore, the downregulation upon maturation of molecules involved in migration through vessel walls (CD31, CLA, CD15s) indicates that the entry of mature dendritic cells into lymphatic vessels may not be as rigidly regulated by adhesion molecules as the process of extravasation from blood vessels.

Antigens, CD↗
PubMed1998

Entry into afferent lymphatics and maturation in situ of migrating murine cutaneous dendritic cells.

An important property of dendritic cells (DC), which contributes crucially to their strong immunogenic function, is their capacity to migrate from sites of antigen capture to the draining lymphoid organs. Here we studied in detail the migratory pathway and the differentiation of DC during migration in a skin organ culture model and, for comparison, in the conventional contact hypersensitivity system. We report several observations on the capacity of cutaneous DC to migrate in mouse ear skin. (i) Upon application of contact allergens in vivo the density of Langerhans cells in epidermal sheets decreased, as determined by immunostaining for major histocompatibility complex class II, ADPase, F4/80, CD11b, CD32, NLDC-145/DEC-205, and the cytoskeleton protein vimentin. Evaluation was performed by computer assisted morphometry. (ii) Chemically related nonsensitizing or tolerizing compounds left the density of Langerhans cells unchanged. (iii) Immunohistochemical double-staining of dermal sheets from skin organ cultures for major histocompatibility complex class II and CD54 excluded blood vessels as a cutaneous pathway of DC migration. (iv) Electron microscopy of organ cultures revealed dermal accumulations of DC (including Birbeck granule containing Langerhans cells) within typical lymphatic vessels. (v) Populations of migrating DC in organ cultures upregulated markers of maturity (the antigen recognized by monoclonal antibody 2A1, CD86), but retained indicators of immaturity (invariant chain, residual antigen processing function). These data provide additional evidence that during both the induction of contact hypersensitivity and in skin organ culture, Langerhans cells physically leave the epidermis. Both Langerhans cells and dermal DC enter lymphatic vessels. DC mature while they migrate through the skin.

Animals↗
PubMed1997

Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CML-specific primary cytotoxic T-cell response.

Dendritic cells (DC) are professional antigen-presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM-CSF, IL-4, and monocyte-conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)-induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 +/- 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 +/- 7.3%), whereas only 11.4 +/- 1% of the B cells and 4.4 +/- 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen-presenting cells, a primary CML-directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML-directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T-cell response or by in vitro generation of CML-specific cytotoxic autologous or HLA-matched normal T-cell clones for use in vivo.

Cell Line, Transformed↗
PubMed1997

Maturation and migration of murine dendritic cells in situ. Observations in a skin organ culture model.

Dendritic cells reside in tissues such as skin in an immature state. Upon antigenic challenge they begin to mature and migrate to the draining lymph nodes. These processes are still poorly understood. One way to study in situ aspects of maturation and migration are skin organ culture models. In an attempt to learn more about the relationship between maturation and migration we investigated the expression of several marker molecules by immunohistochemistry. Sheets from normal murine ear skin and from skin that had been cultured for three days were compared. During culture the numbers of epidermal Langerhans cells decreased and accumulations of strongly MHC class II-positive cells ("cords") were found in the dermis. As compared to untreated skin, the few Langerhans cells remaining in the epidermal sheets after 2-3 days expressed increased levels of MHC class II and had also upregulated B7-2 (CD86) as described. They did not express the antigen recognized by mAb 2A1, a marker for mature dendritic cells. Double-staining of dermal sheets after 3 days of culture showed that dendritic cells in the "cords" expressed high levels of MHC class II and CD86 but were also reactive with mAb 2A1. This pattern is identical to those dendritic cells that had emigrated into the culture medium over the period of 3 days. Invariant chain (mAb In1) was detected at all stages of culture as opposed to isolated epidermal Langerhans cells in suspension where invariant chain expression disappears after 3 days of culture. We conclude that the up- (class II, B7-2, 2A1) and down-regulation (invariant chain) of dendritic cell molecules during migration does not happen in a synchronized manner. The molecule recognized by mAb 2A1 seems to appear late in maturation.

Animals↗
PubMed1997

Intravenous immunoglobulin treatment in therapy-resistant epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita is an uncommon autoimmune bullous disease of the skin and mucous membranes. It is chronic, disabling, and difficult to treat. We describe a case of severe epidermolysis bullosa acquisita of 7 years' duration that had been treated with azathioprine, corticosteroids, chlorambucil, plasma exchanges, cyclophosphamide, cyclosporine, and colchicine without any lasting effect. Seven cycles of treatment were administered with immunoglobulin given intravenously at a low dose, 40 mg/kg body weight daily for 5 days. The patient was free of disease for 10 months after the initiation of therapy. We suggest that low-dose regimens of immunoglobulins may be as effective in this disease as the high-dose regimens suggested in the literature, and at much lower cost.

Adult↗
PubMed1997

Dendritic cells: from ignored cells to major players in T-cell-mediated immunity.

Dendritic cells form a system of leukocytes specialized to stimulate resting T cells in vivo. Dendritic cells are crucial for the initiation of primary immune responses of both helper and cytotoxic T lymphocytes, and thus act as 'nature's adjuvant'. The manifold specializations underlying this in vivo immunostimulatory function are becoming increasingly clear. Methods have been developed to generate large numbers of dendritic cells from hematopoietic precursors in vitro. These techniques now allow molecular studies as well as the use of antigen-charged dendritic cells to vaccinate patients against tumors (e.g. B-cell lymphoma or melanoma) and infection (e.g. HIV-1). Recent data suggest that besides the classical immunostimulatory dendritic cells which belong to the myeloid lineage, there exist regulatory dendritic cells related to the lymphoid lineage. These lymphoid-derived dendritic cells which at least in part express Fas-ligand appear to be involved in the induction of central as well as peripheral tolerance, and in the future might allow a novel approach to induce tolerance in transplantation, autoimmunity, and allergy.

Animals↗
PubMed1996

Human renal-cell carcinoma tissue contains dendritic cells.

Immune surveillance of cancer requires antigen-presenting cells which activate T cells specific for tumor-associated antigens. We show here that substantial numbers of dendritic cells, which are the most potent antigen-presenting cells, emigrate from renal-tumor explants in organ culture. Tumor-derived dendritic cells presented with all characteristics of mature dendritic cells. Dendritic cells could be identified by typical cytoplasmic projections (=veils). They expressed high levels of MHC products and of the co-stimulator CD86 (B7-2). Dendritic cells expressed the CD45RO isoform but not CD45RA. The most important point was that up to 9% of the emigrating leukocytes expressed the CD83 antigen, a specific marker for mature dendritic cells. CD83+ cells were approximately 40-fold enriched in the tumor tissue as compared to the peripheral blood. In contrast to cultured blood dendritic cells, tumor-emigrant dendritic cells had a reduced potential to capture soluble antigen, as shown by the exclusion of fluoresceinated Dextran molecules. Finally, in mixed leukocyte reactions, tumor-derived dendritic cells were able to stimulate naive T cells from cord blood, which is a unique feature of dendritic cells. This study demonstrates that genuine dendritic cells reside in or infiltrate renal-cell carcinoma tissue. The failure of patients with renal-cell carcinoma to mount an anti-tumor immune response despite the presence of professional antigen-presenting cells in the tumor tissue suggests that tumor-associated dendritic cells are suppressed in situ, in a similar way to that described for tumor-infiltrating lymphocytes.

Antigens, CD↗
PubMed1996

Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability.

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34+ precursors and GM-CSF plus TNF-alpha. The other method makes use of the more abundant CD34- precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day 'maturation culture' to the initial 6-7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes ('veils'), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34+ progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig. cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8-3.3 x 10(6) mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83+ cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

Cell Culture Techniques↗
PubMed1996

High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.

We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.

Animals↗
PubMed1996

An improved isolation method for murine migratory cutaneous dendritic cells.

Dendritic cells are highly specialized accessory cells for the initiation of primary immune responses. They occur as trace populations in non-lymphoid and lymphoid organs. Therefore, the isolation and enrichment of primary dendritic cells is difficult and time-consuming. This applies also to dendritic cells from skin, i.e. epidermal Langerhans cells and dermal dendritic cells. Recently introduced skin organ cultures serve as a convenient source for primary cutaneous dendritic cells. We report here a refinement of such cultures in which cutaneous dendritic cells emigrate spontaneously into the culture medium. Murine ear skin is cultured for a total of 3 days in three sequential 24 h steps. This simple modification doubles or triples the yields of dendritic cells that can be obtained and up to 30,000 dendritic cells can be recovered from one ear half. This represents 50-70% (range 30-80%) of all viable cells. The cells are mature dendritic cells that possess potent T cell stimulatory function. Compared to the classical methods of preparing epidermal Langerhans cells by trypsinization this technique is easier and quicker; it does not require enzymes such as trypsin, and it yields similar numbers of mature dendritic cells. It should prove useful for further studies of dendritic cells of the skin.

Animals↗
PubMed1996

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells.

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.

Animals↗
PubMed1996

Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model.

The capacity to migrate from peripheral tissues, where antigen is encountered, to lymphoid organs, where the primary immune response is initiated, is crucial to the immunogenic function of dendritic cells (DC). The skin is a suitable tissue to study migration. DC were observed to gather in distinct nonrandom arrays ("cords") in the dermis upon culture of murine whole skin explants. It is assumed that cords represent lymphatic vessels. Using a similar organ culture model with human split-thickness skin explants, we investigated migration pathways in human skin. We made the following observations. 1) Spontaneous emigration of Langerhans cells took place in skin cultured for 1-3 d. Nonrandom distribution patterns of strongly major histocompatibility complex class II-expressing DC (cords) occurred in cultured dermis. A variable, yet high (>50%) percentage of these DC coexpressed the Birbeck granule-associated antigen "Lag." Ultrastructurally, the cells corresponded to mature DC. 2) Electron microscopy proved that the dermal structures harboring the accumulations of DC (i.e., cords) were typical lymph vessels. Moreover, markers for blood endothelia (monoclonal antibody PAL-E, Factor VIII-related antigen) and markers for cords (strong major histocompatibility complex class II expression on nonrandomly arranged, hairy-appearing cells) were expressed in a mutually exclusive pattern. 3) On epidermal sheets we failed to detect gross changes in the levels of expression of adhesion molecules (CD44, CD54/ ICAM-1, E-cadherin) on keratinocytes in the course of the culture period. The reactivity of a part of the DC in the dermal cords with Birbeck granule-specific monoclonal antibody "Lag" suggests that the migratory population is composed of both epidermal Langerhans cells and dermal DC. We conclude that this organ culture model may prove helpful in resolving pathways and mechanisms of DC migration.

Cell Movement↗
PubMed1996

Immunoglobulin A (IgA) deposits in lesional skin of a patient with blepharochalasis.

We describe a 21-year-old male patient with blepharochalasis, a form of localized acquired cutis laxa. He had a 13-year history of recurrent swelling attacks of the eyelids of unknown origin leading to periocular localized cutis laxa. Histology of lesional skin confirmed almost complete loss of elastic fibres in the reticular and papillary dermis. Immunofluorescence and immunoelectron microscopy studies showed abundant immunoglobulin A (IgA) deposits around the remaining elastic fibres. Control skin of the forearm was negative. These findings support the hypothesis that immunopathogenetic mechanisms may contribute to the elastolytic process of blepharochalasis.

Adult↗
PubMed1995

Tumor-infiltrating T lymphocytes from renal-cell carcinoma express B7-1 (CD80): T-cell expansion by T-T cell co-stimulation.

B7-1 (CD80) provides co-stimulation for T-cell activation by interacting with CD28 or CTLA4. Here we demonstrate the expression of B7-1 in freshly isolated and cultured lymphocytes from renal-cell carcinoma. In fresh preparations of lymphocytes infiltrating renal-cell-carcinoma tissue, B7-1 mRNA could readily be detected by reverse transcription PCR, and 2-color flow-cytometry analysis revealed substantial B7-1 expression on T cells from these isolates. As expected, tumor-derived T cells also expressed CD28, the B7 receptor. While B7-1 expression of tumor-derived T cells was maintained during culture in interleukin-2-supplemented medium, CD28 expression was further enhanced. We also show that B7-1 is functionally involved in T-cell expansion: anti-B7-1 MAb inhibited the PHA-induced proliferation of tumor-derived B7-1+ T cells (35%) in the absence of exogenous antigen-presenting cells, indicating that B7-1 mediates T-T cell co-stimulation (self-co-stimulation). Our data demonstrate that T cells infiltrating renal-cell carcinoma express B7-1, and that mutual co-stimulation via the B7-1/CD28 pathway contributes to the interleukin-2-driven expansion of tumor-derived T cells in vitro. The frequency of B7-1+ T cells in tumor lesions and the level of B7-1 on these cells may determine the time course of T-cell expansion in vivo. Self-co-stimulation, however, might also represent one mechanism leading to the state of suppression or anergy characteristic of tumor-infiltrating lymphocytes.

B7-1 Antigen↗