Lymphocytes are a major source of circulating soluble dipeptidyl peptidase 4.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease that is expressed constitutively by many haematopoietic and non-haematopoietic tissues. It exists as a membrane-associated protein, as well as in an active, soluble form (herein called sDPP4), present at high concentrations in bodily fluids. Despite the proposed use of sDPP4 as a biomarker for multiple diseases, its cellular sources are not well defined. Here, we report that individuals with congenital lymphocyte immunodeficiency had markedly lower serum concentrations of sDPP4, which were restored upon successful treatment and restoration of lymphocyte haematopoiesis. Using irradiated lymphopenic mice and wild-type to Dpp4-/- reciprocal bone marrow chimeric animals, we found that haematopoietic cells were a major source of circulating sDPP4. Furthermore, activation of human and mouse T lymphocytes resulted in increased sDPP4, providing a mechanistic link between immune system activation and sDPP4 concentration. Finally, we observed that acute viral infection induced a transient increase in sDPP4, which correlated with the expansion of antigen-specific CD8+ T cell responses. Our study demonstrates that sDPP4 concentrations are determined by the frequency and activation state of lymphocyte populations. Insights from these studies will support the use of sDPP4 concentration as a biomarker for inflammatory and infectious diseases.

Abnormalities of acid-base balance and predisposition to metabolic acidosis in Metachromatic Leukodystrophy patients.

Metachromatic Leukodystrophy (MLD; MIM# 250100) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The enzymatic defect results in the accumulation of the ARSA substrate that is particularly relevant in myelin forming cells and leads to progressive dysmyelination and dysfunction of the central and peripheral nervous system. Sulfatide accumulation has also been reported in various visceral organs, although little is known about the potential clinical consequences of such accumulation. Different forms of MLD-associated gallbladder disease have been described, and there is one reported case of an MLD patient presenting with functional consequences of sulfatide accumulation in the kidney. Here we describe a wide cohort of MLD patients in whom a tendency to sub-clinical metabolic acidosis was observed. Furthermore in some of them we report episodes of metabolic acidosis of different grades of severity developed in acute clinical conditions of various origin. Importantly, we finally show how a careful acid-base balance monitoring and prompt correction of imbalances might prevent severe consequences of acidosis.

Dendritic cell functional improvement in a preclinical model of lentiviral-mediated gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was(-/-) mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector (LV). The aim of this study was to investigate whether GT can correct DC defects in was(-/-) mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium. When BMDCs from the treated mice (GT BMDCs) and the was(-/-) mice were injected into wild-type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared with mice injected with was(-/-) BMDCs. Finally, we found that ovalbumin (OVA)-pulsed GT BMDCs or vaccination of GT mice with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T-cell activation in vivo. These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of LV-mediated WAS GT.

The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood.

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38- or CD34+/DR-) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)

Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.

The 5' sequence of human factor XII gene contains transcription regulatory elements typical of liver specific, estrogen-modulated genes.

The human Factor XII gene codes for a serine proteinase synthesized in liver that activates both the coagulation and the fibrinolytic cascades. The nucleotide sequence analysis of a HincII-HincII 3129 bp fragment was performed showing that the FXII promoter region contains neither CAAT and TATA regulatory elements, nor GC islands, but revealing the presence of two tandemly repeated sequences in opposite orientation, two LF-A1 elements typical of the liver specific genes and one estrogen responsive element, that substantiates the observation of Factor XII gene modulation by estrogens.

Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation.

Using a novel collection of conditionally immortalized mouse stromal cell clones, we evaluated the role of distinct elements of the hematopoietic microenvironment in supporting and regulating the growth, division, and differentiation of a candidate human stem cell population (CD34+/CD38-). We found functional diversity in the capacity of different stromal cell clones to support the growth of primitive (CD34+/CD38-) and committed (CD34+/CD38+) hematopoietic progenitors and their differentiation into mature hematopoietic cells (CD34-/CD45+). Among the stromal cell clones that supported long-term hematopoiesis, we identified two clones that induced expansion of CD34+ progenitor/stem cells during the first 4 weeks of coculture and that supported the maintenance of this CD34+ population for up to 10 weeks in vitro. However, these two clones appeared to represent two different microenvironments with regard to the signals they provide to the different CD34+ progenitor subpopulations: One stromal clone preserved a pool of undifferentiated, relatively quiescent (CD34+/CD38-) progenitor cells, allowing their differentiation at a low rate into more committed (CD34+/CD38+) progenitors; the other fostered a more extensive and rapid differentiation of all CD34+/CD38- progenitors into CD34+/CD38+ cells, preferentially maintaining this committed population at a higher rate of cell division. These stromal cell clones were also able to support the proliferation and differentiation of CD34+/CD38- cells in conditions in which progenitor-stroma contact was prevented. This collection of stromal cell clones may represent a unique tool for the study of stromal regulators of hematopoiesis as well as for the support of gene transfer into hematopoietic progenitor cells.

Lack of evidence for a superantigen in lymphocytes from HIV-discordant monozygotic twins.

OBJECTIVE: An HIV-associated superantigen (SAg) has been hypothesized. Here we test whether an SAg is functionally detectable in peripheral blood mononuclear cells (PBMC) from monozygotic twins discordant for HIV infection. DESIGN AND METHODS: The V beta selective T-cell depletion found in minor lymphocyte stimulation (Mls)-positive mice is caused by an SAg encoded by the mouse mammary tumour virus. Mls is a locus whose gene product stimulates a mixed lymphocyte reaction (MLR) in mice strains identical at the major histocompatibility complex locus. If an SAg is present in PBMC and/or sorted CD4+ cells from one HIV-infected monozygotic twin, it would stimulate PBMC from the corresponding healthy monozygotic twin in an MLR. In addition, if an SAg causes V beta-selective T-cell depletion in AIDS patients, a differential proliferation to a panel of staphylococcal enterotoxins (SE) of T lymphocytes from healthy and HIV-infected monozygotic twins should become measurable. RESULTS: No positive MLR or significant differences in the SE-driven proliferation between the healthy and the HIV-infected twins were observed. CONCLUSIONS: Our results suggest that PBMC from the two HIV-infected twins do not express a functionally detectable SAg.

A novel human packaging cell line with hematopoietic supportive capacity increases gene transfer into early hematopoietic progenitors.

The hematopoietic stem/progenitor cell (HSPC) represents the ideal target for gene therapy of disorders of the hematopoietic system, but still faces problems related to ex vivo manipulation and gene transfer efficiency. We demonstrate that soluble factors from the human endothelial-like cell line ECV 304/T24 support the growth of human CD34(+) progenitor cells as primary human bone marrow stroma and increase the rate of gene transfer into progenitor cells up to 5-fold. ECV 304/T24 was used to generate split-function amphotropic packaging cell lines (named APEX) with the purpose of combining, in the same cells, hematopoietic support and gene transfer vehicle functions. The APEX cell lines were negative for the presence of replication-competent retroviruses and produced complement-resistant vector particles. When mobilized peripheral blood or umbilical cord blood CD34(+) cells were exposed once to APEX supernatants, the level of gene transfer was equivalent to that observed with GP + Am12, in spite of the lower titer of the APEX producers. More importantly, APEX supernatants gave rise reproducibly to a 2-fold increase in transduction of early progenitors (long-term culture-initiating cells), reaching on average 50% gene transfer. This novel packaging cell represents a significant advance in HSPC genetic modification technology, combining both a beneficial hematopoietic supportive effect and the gene transfer vector function in a human-based system.

Cell-surface marking of CD(34+)-restricted phenotypes of human hematopoietic progenitor cells by retrovirus-mediated gene transfer.

Human CD34+ cells lacking detectable levels of HLA-DR antigens (CD34+ DR-) are highly enriched in hematopoietic pluripotent progenitors with long-term marrow repopulating ability. We investigated the feasibility of transducing and marking CD34+ DR- progenitor cells from bone marrow (BM) or mobilized peripheral blood samples (MPB) of 13 patients undergoing BM transplantation with the purpose of developing a protocol for a large-scale clinical application. A new retroviral vector coding for the truncated form (delta) of the low-affinity nerve growth factor receptor (LNGFR) was used to quantitate the level of gene transfer into CD34+ cells and their progeny by multiparameter cytofluorimetry and immunocytochemistry. Light-density mononuclear cells as well as purified CD34+ cells were transduced either by direct incubation with retroviral supernatants or prestimulated in vitro with various combinations of growth factors prior to transduction. Transduction efficiency, assessed as G418-resistant growth of granulocyte-macrophage colony-forming units (CFU-GM) progenitors from MPB, was 1.7-fold higher (14.9% +/- 4.5%) than those from BM (8.5% +/- 3.9%) and it was further improved (26.9% +/- 3.1%) using a purified CD34+ population as target cells. Three-color fluorescence-activated cell sorting (FACS) analysis demonstrated the presence of transduced delta LNGFR+ cells within the CD34+ DR- subpopulation. In the absence of growth factors, gene transfer into BM or MPB CD34+ DR- cells was generally poor, but following a 72-hr prestimulation it peaked at 38% of total CD34+ DR- bone marrow (BM) cells in the presence of the c-kit ligand (KL) and at 31% in the presence of IL-3. Furthermore, KL gave, compared to the other cytokines, the highest absolute yield of BM delta LNGFR+ CD34+ DR- cells recovered after transduction (p = 0.05 compared to 24 hr). Gene transfer into in vitro primitive progenitor cells was further confirmed by expression of the delta LNGFR marker on CD34+ cells and CFU-GM derived from 5-week long-term culture on stroma.

Spectrotype of anti-gp120 antibodies remains stable during the course of HIV disease.

Human immunodeficiency virus (HIV) infection is characterized by a progressive decline in immune functions. The behavior of B-cell clones specifically engaged in the anti-HIV response could play a relevant role in the pathogenesis of such impairment. The spectrotype observed on isoelectric focusing and reverse blotting after antigen challenge is the serum image of antigen-specific B-cell activity and may provide some insight into Ag-dependent B-cell clone recruitment. In this study, we examined the spectrotype of anti-gp120 antibodies in a group of sera from 56 HIV-infected patients, belonging to groups II, III, and IV of the Centers for Disease Control classification, as well as in a group of 31 sera from 12 patients in a 21-month follow-up evaluation (range 7-36 months). All tested sera were positive for gp120 antibodies on Western blot. In the first group of 56 HIV-infected subjects, only 19 displayed well-focused banding patterns. Among these, the spectrotype was found to be consistently oligoclonal, thus confirming clonal restriction of anti-gp120 antibodies previously described by other investigators. No correlation could be established between a particular spectrotype and phase of the disease. The follow-up evaluation in the second group of 31 sera revealed the tendency in each patient to maintain the same spectrotype throughout the course of the disease. These findings confirm clonal restriction of anti-gp120 antibodies in HIV infection and suggest that the number of B-cell clones recruited in the anti-gp120 response remains stable over the course of the disease, at least in the time range explored by us.

Recovery of hematopoietic activity in bone marrow from human immunodeficiency virus type 1-infected patients during highly active antiretroviral therapy.

The mechanisms responsible for the hematopoietic failure in human immunodeficiency virus type 1 (HIV-1)-infected patients are still unknown. Several findings indicate that the in vitro proliferative potential of precursor cells from AIDS patients is reduced. The changes seen in bone marrow (BM) morphology and the defective BM functions associated with cytopenias have both been proposed as potential explanations. In patients treated with highly active antiretroviral therapy (HAART) an immune reconstitution associated with increased whole blood cell counts has been described. We have investigated the effects of HAART on the number of colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs), using long-term BM cell cultures (LTBMC) in a group of subjects with HIV-1 infection enrolled in an open study to evaluate the mechanisms of immune reconstitution during HAART. In each patient, the increase in colony growth was homogeneous, regardless of the type of hematopoietic progenitor cells assayed; in four subjects an increase in the most primitive progenitor cells (LTC-ICs) was observed. These findings were associated with the in vivo data showing increased numbers of BM mononuclear cells (BMMCs) after HAART and with a rise in peripheral CD4(+) T cell counts and decreased levels of plasma HIV-1 RNA. A decreased number of hematopoietic progenitor cells and/or a defective modulation of progenitor cell growth might be the cause of the hematological abnormalities in AIDS patients. Controlling HIV-1 replication by HAART could determine a restoration of stem cell activity, probably because of the suppression of factors that inhibit normal hematopoiesis.

Evaluation of ADA gene expression and transduction efficiency in ADA/SCID patients undergoing gene therapy.

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients.

Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic-specific cellular defects. In order to develop an optimal lentiviral gene transfer cassette for this application, we compared the performance of several internal promoters in a variety of cell lineages from human WAS patients. Vectors using endogenous promoters derived from short (0.5 kb) or long (1.6 kb) 5' flanking sequences of the WAS gene, expressed the transgene in T, B, dendritic cells as well as CD34(+) progenitor cells, but functioned poorly in non-hematopoietic cells. Defects of T-cell proliferation and interleukin-2 production, and the cytoskeletal anomalies in WAS dendritic cells were also corrected. The levels of reconstitution were comparable to those obtained following transduction with similar lentiviral vectors incorporating constitutive PGK-1, EF1-alpha promoters or the spleen focus forming virus gammaretroviral LTR. Thus, native regulatory sequences target the expression of the therapeutic WAS transgene to the hematopoietic system, as is naturally the case for WAS, and are effective for correction of multiple cellular defects. These vectors may have significant advantages for clinical application in terms of natural gene regulation, and reduction in the potential for adverse mutagenic events.

Progress and prospects: gene therapy clinical trials (part 2).

This is the second part of a review summarizing progress and prospects in gene therapy clinical research. Twenty key diseases/strategies are succinctly described and commented on by leaders in the field. This part includes clinical trials for skin diseases, neurological disorders, HIV/AIDS, ornithine transcarbamylase deficiency, alpha(1)-antitrypsin deficiency, haemophilia and cancer.