Pre-clinical safety testing supporting clinical use of allogeneic multipotent adult progenitor cells.

BACKGROUND: Successful clinical development of novel cellular therapeutics requires the evaluation of clinical acute toxicity endpoints in scoring patient adverse events (AE) contributing to dose-limiting toxicity (DLT) for establishment of the maximum-tolerated dose (MTD). However, many clinical pathology parameters are not routinely evaluated in pre-clinical safety testing. The objective of this pre-clinical study was to investigate thoroughly the acute toxicity of single- and multiple-dose administrations of allogeneic multipotent adult progenitor cells (MultiStem), which represent a class of stromal stem cells with therapeutic potential. METHODS: MultiStem were tested as an adjunct treatment in a rat myeloablative hematopoietic stem cell transplantation (HSCT) model for impact on clinical parameters, clinical chemistry, hematology, immunology and histopathology parameters. Animals received MultiStem in a single dose of 12.5 million cells/kg on day 2 after HSCT or in five infusions at this dose on days 2, 9, 16, 23 and 30. Controls received phosphate-buffered saline injections and all animals were killed on day 37. RESULTS: There were no significant differences between tests and controls regarding evaluation of respiratory distress upon infusion, clinical assessment and hematology and clinical chemistry analysis. Gross necropsy and histopathology analysis showed no organ profile alterations. There was no significant evidence for allogeneic antibody production or T-cell sensitization upon MultiStem infusion. DISCUSSION: These studies demonstrate the safety of administration of allogeneic stromal stem cells in repeat dosing regimens in bone marrow transplant settings, and define pre-clinical safety testing standards relevant to the development of cellular therapeutics using allogeneic adherent adult stem cells.

Secondary amyloidosis: a severe complication of ankylosing spondylitis. Two case-reports.

STUDY OBJECTIVE: To report two cases of amyloidosis secondary to ankylosing spondylitis. PATIENTS AND RESULTS: Of the 47 ankylosing spondylitis patients who have received follow-up at our department over the last few years, two have developed AA amyloidosis. Both have extremely severe, long-standing joint disease, with virtually complete spinal ankylosis and destructive peripheral arthritis of the hips and wrists; one also has tarsal joint destruction. Renal dysfunction was the first manifestation of amyloidosis in both cases. One patient required chronic hemodialysis and developed peritonitis due to colonic perforation, probably at a site of amyloid deposition. CONCLUSIONS: Secondary amyloidosis is a rare complication of ankylosing spondylitis that can cause severe renal and gastrointestinal complications. No treatment capable of clearing established amyloid deposits is available to date.

[Thromboangiitis obliterans (Buerger disease) discovered by symptoms of arthralgia in a young woman]. / Thromboangéite oblitérante (maladie de BP6rger) révélée par des arthralgies chez une jeune femme.

Thromboangiitis obliterans (TAO) or Buerger's disease is an inflammatory disease that affects the small and medium caliber arteries and veins. It affects predominantly young, smoking males. The clinical manifestations are usually ischemic lesions of the extremities or limb claudication. The physiopathology is yet unknown. However, several hypotheses involving a dysregulation of the immune response are discussed. Here, we report a case of a young woman, smoker, that presented with diffuse arthralgia of the lower extremities. We were able to diagnose a thromboangiitis obliterans using criteria established by Papa et al. They include anamnestic, clinical and angiographic data. The pharmacological treatment of TAO is disappointing. The only way to prevent the extension of the disease is to stop smoking cigarettes.

Analysis of MHC class II presentation of particulate antigens of B lymphocytes.

To generate Ab responses to most protein Ags, B cells must first degrade proteins in endocytic compartments and then display antigenic peptides bound to MHC class II molecules. T helper lymphocytes recognize these complexes and stimulate the B cell to synthesize Ab. Although Ab play a key role in host defense against bacteria, it is believed that B cells are incapable of internalizing particulate Ags. However, we find that B lymphoblastoid cell lines and LPS-activated B lymphocytes can present particulate Ag up to 10(5)-fold more efficiently compared with soluble Ag. Moreover, particulate Ags are presented efficiently by unstimulated B cells when they bind to surface Ig. In comparison to B cells, macrophages in general presented particulate Ags 10- to 1000-fold more efficiently and could also present Ag from particles of a much wider range of sizes. We document by ultrastructural and immunofluorescence analysis that B lymphoblastoid cell lines bind and internalize these particles. The internalization and presentation of the particulate Ag is inhibited by cytochalasin B. In contrast, a similar morphologic analysis of normal lymphocytes demonstrated that while Ag beads are bound to the cell surface, they are internalized only rarely. These results suggest there may be both surface and intracellular pathways for the presentation of particulate Ags by B cells. Interestingly, for both macrophages and B cells, the epitopes generated from particulate and soluble Ags were not identical quantitatively or qualitatively, indicating that there are differences in how these forms of Ag are processed and presented.

Targeting antigen into the phagocytic pathway in vivo induces protective tumour immunity.

Cytotoxic T lymphocytes (CTLs) kill neoplastic or virally infected cells after recognizing on their surface antigenic peptides bound to major histocompatibility complex class I molecules. These peptides are derived from antigens that are degraded in the cytosol of the affected cell. Because exogenous proteins cannot enter the cytosol, immunizations with killed pathogens or their proteins do not generally elicit CTLs. However, antigens that are internalized into phagocytic cells can enter the cytosol and be processed for class I presentation. Here we show that immunization with a purified antigen on an avidly phagocytized particle primes CTLs, which in turn protect animals from subsequent challenge with tumours transfected with the antigen gene. Interestingly, these animals also become immune to other antigens expressed by the tumour. This approach could be exploited to develop tumour and viral vaccines.

A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules.

Peptides from endogenous proteins are presented by major histocompatibility complex class I molecules, but antigens (Ags) in the extracellular fluids are generally not. However, pathogens or particulate Ags that are internalized into phagosomes of macrophages (M phi s) stimulate CD8 T cells. The presentation of these Ags is resistant to chloroquine but is blocked by inhibitors of the proteasome, a mutation in the TAP1-TAP2 transporter, and brefeldin A. Moreover, phagocytosis of a ribosomal-inactivating protein inhibited M phi protein synthesis. These results demonstrate that M phi s transfer Ags from phagosomes into the cytosol and that endogenous and exogenous Ags use a final common pathway for class I presentation.

Presentation of exogenous antigens by macrophages: analysis of major histocompatibility complex class I and II presentation and regulation by cytokines.

There is an antigen presenting cell (APC) in the lymphoid organs capable of presenting exogenous antigen (Ag) with major histocompatibility complex (MHC) class I molecules. This study was initiated to isolate clones of these APC to definitively establish their phenotype and to further study their properties. Murine bone marrow macrophages (BM M psi) were immortalized by overexpression myc and raf oncogenes. Five BM M psi cell lines were generated that are phagocytic and expressed at their surface M psi differentiation Ag. All five cell lines processed and presented exogenous ovalbumin (OVA) with MHC class I molecules. They all presented OVA-linked to a phagocytic substrate 10(2)-10(4)-fold more efficiently than soluble Ag. Clonal isolates of two of the M psi cell lines had an identical phenotype and functional properties as the uncloned lines. These results definitively establish that M psi are APC with the capacity of presenting exogenous Ag with MHC class I molecules. Interferon (IFN)-gamma interleukin-4, granulocyte-macrophage colony stimulating factor and lipopolysaccharide either alone or in combination induced little or no augmentation and in some cases decreased presentation of exogenous OVA with MHC class I. In contrast, all of M psi activating factors increased MHC class I expression. Moreover, IFN-gamma increased the presentation of cytosolic OVA, demonstrating differences between the presentation of cytosolic Ag versus exogenous Ag with MHC class I. Finally, some lines constitutively processed and presented exogenous OVA with MHC class II while others only presented after stimulation with IFN-gamma. These results demonstrate that the pathways involved in the presentation of exogenous Ag with MHC class I and class II are independently regulated and that a cloned cell is capable of presenting exogenous Ag through both pathways.

Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages.

Antigens in extracellular fluids can be processed and presented with major histocompatibility complex (MHC) class I molecules by a subset of antigen presenting cells (APCs). Chicken egg ovalbumin (Ova) linked to beads was presented with MHC class I molecules by these cells up to 10(4)-fold more efficiently than soluble Ova. This enhanced presentation was observed with covalently or noncovalently linked Ova and with beads of different compositions. A key parameter in the activity of these conjugates was the size of the beads. The APC that is responsible for this form of presentation is a macrophage. These cells internalize the antigen constructs through phagocytosis, since cytochalasin B inhibited presentation. Processing of the antigen and association with MHC class I molecules appears to occur intracellularly as presentation was observed under conditions where there was no detectable release of peptides into the extracellular fluids. When injected in vivo in C57BL/6 mice, Ova-beads, but not soluble Ova, primed CD4- CD8+ cytotoxic T lymphocytes (CTLs). Similar results were obtained in BALB/c mice immunized with beta-galactosidase-beads. The implications of these findings for development of nonliving vaccines that stimulate CTL immunity are discussed.

Isotypic distribution of HIV-1-specific antibodies in individuals from central Africa.

Individuals infected with human immunodeficiency virus type 1 (HIV-1) develop a humoral immune response to the virus's major structural gene products env, gag, and pol. The distribution of antibodies to env, gag, and pol proteins in Central African populations is of interest as they have a high level of immune system activation compared to non-African populations. Using the Western blot technique, we analyzed the isotypic distribution of anti-HIV antibodies in 45 HIV-1-infected individuals from Central Africa that were either symptomatic or asymptomatic. We observed two basic differences between the isotypic profile of individuals from Central Africa and non-African populations. Central African individuals had a strong polyisotypic response to gag and pol, which has only been observed for gag in American and European populations. In addition, individuals from Central Africa had a high frequency of IgG4 to gag and pol, 75 and 51%, respectively, as compared to 29 and 6% in a non-African population. The elevated IgG4 response may result from the high basal level of immune stimulation seen in Africans due to multiple and frequent exposures to viral, bacterial, and parasitic antigens.