Multiple allogeneic progenitors in combination function as a unit to support early transient hematopoiesis in transplantation.

Cord blood (CB) is a valuable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in CB transplantation (CBT) is often delayed because of low graft cell numbers. This limits the use of CB. To overcome this cell dose barrier, we modeled an insufficient dose CBT setting in lethally irradiated mice and then added hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) derived from four mouse allogeneic strains. The mixture of HSPCs rescued recipients and significantly accelerated hematopoietic recovery. Including T cells from one strain favored single-donor chimerism through graft versus graft reactions, with early hematopoietic recovery unaffected. Furthermore, using clinically relevant procedures, we successfully isolated a mixture of CD34(+) cells from multiple frozen CB units at one time regardless of HLA-type disparities. These CD34(+) cells in combination proved transplantable into immunodeficient mice. This work provides proof of concept that when circumstances require support of hematopoiesis, combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft.

Inhibitory function of NKT cells during early induction phase of nickel allergy.

Until now, metal allergies have been regarded as a Th1-type immune response.　However, because the contribution of a Th2-type immune response has been suggested by clinical findings, we previously examined the Th2-type immune response during the development of metal allergies using a GATA-3 transgenic (GATA-3 Tg) mouse model. As a result, a Th2-type immunization reaction was suggested to be involved in the early phase of metal allergies. Recently, the involvement of NKT cells in metal allergies has been suggested. We examined this possibility using the activation of NKT cells and an NKT cell-deficient mouse model to determine the contribution of NKT cells to nickel allergy in the present study. In NKT cell-deficient mice, ear swelling was remarkably increased, compared with that in control mice. Also, in mice that had been treated with α-galactosylceramide (α-GalCer) to activate NKT cells, the ear swelling response was remarkably inhibited, compared with that in untreated mice. These facts show that NKT cells are involved in the inhibition of nickel allergy-induced ear swelling responses.

A possible clue for the production of anti-glomerular basement membrane antibody associated with ureteral obstruction and hydronephrosis.

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) antibody-mediated glomerulonephritis (anti-GBM GN) is an autoimmune disease with rapidly progressive glomerulonephritis. Based on a case report of anti-GBM GN following hydronephrosis, we hypothesized that hydronephrosis may act as a trigger for the development of anti-GBM antibodies. PATIENTS AND METHODS: We evaluated 11 patients who were diagnosed with hydronephrosis. It was measured with serum anti-GBM antibody. These patients' medical histories as well as risk factors for the development of anti-GBM antibodies and causes of hydronephrosis were reviewed. Renal function and hematuria were also considered. The serum anti-GBM antibody was measured with enzyme-linked immunosorbent assays (ELISA) or chemiluminescent enzyme immunoassays (CLEIA). Histopathological findings of renal biopsy specimens were also evaluated. RESULTS: No patient had a medical history of renal disease. Five patients had a history of smoking. Ten of the 11 patients had renal dysfunction as evidenced by serum creatinine levels of 0.85-13.8 mg/dl, while 8 patients had RBCs in their urinary sediment at the time of diagnosis for hydronephrosis. Two of the patients assessed by ELISA and CLEIA were positive for anti-GBM antibodies. In 1 of these 3 patients, anti-GBM antibodies and renal dysfunction improved upon treatment for hydronephrosis. Another of the 3 patients developed anti-GBM GN, but anti-GBM antibodies and renal dysfunction improved dramatically upon treatment. In the 3rd patient without improved hydronephrosis, anti-GBM antibodies and renal dysfunction remained unchanged. CONCLUSION: Our results provide insights into the development of anti-GBM antibodies in patients with ureteral obstruction and hydronephrosis.

Curative haploidentical BMT in a murine model of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder characterized by defective microbial killing in phagocytes. Long-term prognosis for CGD patients is generally poor, highlighting the need to develop minimally toxic, curative therapeutic approaches. We here describe the establishment of a mouse model in which X-linked CGD can be cured by allogeneic bone marrow transplantation. Using a combination of non-myeloablative-dose total body irradiation and a single injection of anti-CD40 ligand monoclonal antibody, transplantation of whole bone marrow cells achieved long-lasting mixed chimerism in X-linked CGD mice in a haploidentical transplantation setting. Stable mixed chimerism was maintained for up to 1 year even at a low range (<20 % donor cells), indicating induction of donor-specific tolerance. The regimen induced mild myelosuppression without severe acute complications. Stable chimerism was therapeutic, as it suppressed cutaneous granuloma formation in an in vivo test suited for evaluation of treatment efficacy in murine CGD models. These results warrant future development of a simplified allogeneic hematopoietic cell transplantation regimen that would benefit CGD patients by allowing the use of haploidentical donor grafts without serious concerns of severe treatment-related toxicity.

Cognate interaction plays a key role in the surveillance of autoreactive B cells in induced mixed bone marrow chimerism in BXSB lupus mice.

The effects of bone marrow transplantation (BMT) as a treatment for and/or preventive measure against autoimmune diseases in mice were investigated extensively. The reconstitution of the hematopoietic system with a mixture of autologous and heterologous bone marrow cells was reported to suppress the development of autoimmune diseases. However, the pathological mechanism through which mixed chimerism results in the suppression of disease development is still unknown. We have previously reported that the induction of fully major histocompatibility complex (MHC)-mismatched allogeneic mixed chimerism can prevent the disease development in BXSB mice. Interestingly, serum anti-dsDNA IgM antibody (anti-DNA IgM) levels were not significantly decreased in these chimeric mice, though other symptoms of autoimmune disease were ameliorated. In this study, we showed that self-reactive anti-DNA IgM production was mainly attributable to genetically normal B cells from the donor rather than genetically deficient B cells from the host. Host-type B cells responded normally to foreign antigens and produced the appropriate antibodies. BMT from fully MHC-matched or haplo-identical donors could suppress the production of anti-DNA antibodies. Our present study suggests the existence of a surveillance system dependent on the recognition of MHC molecules on B cells.

Allogenic mixed chimerism induced by nonlymphoablative regimen including donor BMT with low-dose TBI and anti-CD40L cured proliferative glomerulonephritis in lupus mice.

Allogeneic mixed chimerism achieved by low-dose total body irradiation (TBI) and anti-CD40L monoclonal antibody (mAb) with donor bone marrow transplantation (BMT) and host T cell depletion overcomes both allo- and autoimmunity. We investigated whether a similar regimen without T cell depletion cured diffuse proliferative glomerulonephritis. Male BXSB mice (H-2b) were injected with 20 x 10(6) BALB/c (H-2d) BM cells. When indicated, 3 Gy TBI on day -1 and anti-CD40LmAb (2 mg) on day 0 of BMT was given. Skin grafting was performed 1 day after BMT. BXSB mice were divided into four groups--I: BMT with TBI and anti-CD40LmAb; II: TBI; III: TBI and anti-CD40LmAb; and IV: no treatment. Chimerism in peripheral blood was analyzed. The kidney was examined histologically. TBI with anti-CD40LmAb and BMT allowed induction of multilineage mixed chimerism and donor-specific tolerance to skin grafts without graft-versus-host disease (GVHD). There was significant decrease in glomerular PAS-positive material deposition score, glomerular cell numbers, IgG, and C3 deposition in chimeric mice. All chimeric mice survived. Allogeneic mixed chimerism induced by a less toxic, nonlymphoablative regimen achieved allograft tolerance and cured glomerulonephritis in BXSB lupus mice.

Prominent dominant negative effect of a mutant Fas molecule lacking death domain on cell-mediated induction of apoptosis.

Using a panel of transfectant B lymphoma cells expressing varying amounts of the mutant Fas together with the endogenous wild type Fas, semi-quantitative studies on the dominant negative effect of a murine mutant Fas molecule lacking death domain were carried out. In anti-Fas antibody-mediated induction of apoptosis, the mutant molecules exerted significant dominant-negative effect only when their expression level was comparable to or higher than that of wild type molecules, or when exposed to low amounts of the antibody. The inhibitory effect was accompanied by the failure in DISC formation in spite of Fas aggregation. When they were subjected to T cell-mediated Fas-based induction of apoptosis, however, the dominant negative effect was prominent such that the expression of even a small amount of the mutant molecules resulted in significant inhibition. Such a strong inhibitory effect explains the dominant phenotype of this type of mutant Fas molecules in ALPS heterozygous patients and also implies that the physiological effectors for Fas in vivo are cells, i.e., FasL-expressing activated T cells.

Complement mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy.

BACKGROUND: The onset of proteinuria in passive Heymann nephritis, (PHN), a rat model of human membranous nephropathy (MN), is complement-dependent and is associated with altered podocyte slit diaphragm integrity and dissociation of nephrin from the actin cytoskeleton. These studies examined if complement is responsible for these podocyte changes. METHODS: PHN was induced with sheep anti-Fx1A. Controls were injected with normal sheep globulin. A third group was injected with anti-Fx1A and depleted of complement with cobra venom factor. Four days later, proteinuria was measured, slit diaphragm integrity was examined by electron microscopy, nephrin distribution was studied by immunofluorescence, and the glomerular content of nephrin and its association with actin were assessed by sequential extraction of isolated glomeruli and Western blotting. RESULTS: Four days after immunization, seven out of eight PHN rats were proteinuric, whereas none of the complement depleted group had proteinuria despite similar levels of antibody deposition. Complement depletion preserved slit diaphragm morphology. Immunofluorescence microscopy with an antibody to the extracellular domain of nephrin showed a normal staining pattern in the rats depleted of complement and a shift to a more dispersed and clustered pattern in the PHN group. Western blot analysis of the glomerular extracts showed a significant reduction in the total amount of nephrin and in the fraction of actin-associated nephrin in the PHN group, whereas the amounts in the complement-depleted rats were similar to normal controls. CONCLUSION: The onset of proteinuria in the PHN model of MN is coincident with complement-dependent alterations in the association of nephrin with the actin cytoskeleton and loss of podocyte slit diaphragm integrity.

Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton.

Nephrin is an Ig-like transmembrane protein. It is a major component of the podocyte slit diaphragm and is essential for maintaining normal glomerular permeability. CD2-associated protein (CD2AP) is also necessary for normal glomerular permeability and is a putative nephrin adapter molecule. Here, we document that nephrin and CD2AP are linked to the actin cytoskeleton. As detected by Western blot analysis, nephrin and CD2AP were both insoluble when cell membranes from normal rat glomeruli were extracted with 0.5% Triton X-100 (TX-100) at 4 degrees C in the presence of divalent cations, but they were solubilized when the extraction included potassium iodide (KI) to depolymerize F-actin. In addition, a small fraction of the solubilized nephrin and CD2AP was recovered in the low-density fractions of OptiPrep flotation gradients, which indicates that a portion of nephrin, possibly associated with CD2AP, resides in a cholesterol- or sphingolipid-rich region of the plasma membrane. Immunofluorescent staining of unfixed sections of normal rat kidney for nephrin, CD2AP, and F-actin was unaltered by treatment with TX-100 but was greatly diminished by addition of KI. Nephrin staining was slightly reduced by cholesterol depletion with methyl-beta-cyclodextrin in the presence of TX-100 but was nearly absent after addition of KI. These results document that nephrin anchors the slit diaphragm to the actin cytoskeleton, possibly by linkage to CD2AP, and that nephrin traverses a relatively cholesterol-poor region of the podocyte plasma membrane. In addition, a small pool of actin-associated nephrin and CD2AP resides in lipid rafts, possibly in the cholesterol-rich apical region of the podocyte-foot processes.

Nephrin dissociates from actin, and its expression is reduced in early experimental membranous nephropathy.

These studies examined the expression of the podocyte slit diaphragm protein nephrin and its association with actin at the onset of proteinuria in passive Heymann nephritis (PHN), a rat model of human membranous nephropathy. Four days after immunization, 58% of PHN rats had mild proteinuria. At that time, most slit diaphragms were still visible on electron microscopy; however, in those locations where the deposits encroached on the filtration slits, the slit diaphragms were either displaced or absent. On day 7, the PHN rats were severely proteinuric, and most slit diaphragms were either absent, displaced, or replaced by occluding-type junctions. Immunofluorescence microscopy with antibodies to the external and cytoplasmic domains of nephrin showed a progressive loss of staining and a change in the distribution of nephrin from an interrupted linear pattern in normal controls to a more dispersed and clustered pattern in PHN. In contrast, the intensity of staining for ZO-1 and CD2-associated protein (CD2AP), two other proteins that are located on the cytoplasmic face of the slit diaphragm, was undiminished. Immunogold electron microscopy confirmed the progressive disappearance of nephrin from podocyte foot processes and retention of CD2AP. Glomeruli and glomerular cell membranes were extracted sequentially with Triton X-100, followed by DNase I or potassium iodide to depolymerize actin. Western blot analysis of the extracts showed a progressive decline of total nephrin on days 4 and 7 of PHN as well as a reduction in the actin-associated fraction. These findings show that nephrin partly dissociates from actin at the onset of podocyte injury in PHN. This is accompanied by a progressive loss of nephrin from the podocyte foot processes and prominent changes in the morphology of the slit diaphragms. These events may underlie the loss of podocyte barrier function in membranous nephropathy.