Mutation of DNASE1 in people with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.

Association study of TRAF1-C5 polymorphisms with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese.

OBJECTIVE: The primary aim of this study was to investigate the association of polymorphisms of TRAF1-C5, a newly identified rheumatoid arthritis (RA) risk locus in Caucasians, with susceptibility to RA and systemic lupus erythematosus (SLE) in Japanese populations. Gene expression levels of TRAF1 and C5 to assess the functional significance of genotypes were also analysed. METHODS: A multicentre association study consisting of 4 RA case-control series (4397 cases and 2857 controls) and 3 SLE case-control series (591 cases and 2199 shared controls) was conducted. Genotyping was performed using TaqMan genotyping assay for two single nucleotide polymorphisms (SNPs) that showed the best evidence of association in the previous Caucasian studies. Quantifications of TRAF1 and C5 expression were performed with TaqMan expression assay. RESULTS: Significant differences in allele frequency for both SNPs were observed between RA and control subjects (combined odds ratio = 1.09), while no significant difference was detected between patients with SLE and controls. Interestingly, alleles rs3761847 A and rs10818488 G had increased the risk for RA in the present study, while they decreased the risk in the original studies. A significant difference was found between risk allele carriers and non-carriers of rs10818488 for the expression level of TRAF1 in phorbol myristate acetate-stimulated lymphoblastoid cell lines (p = 0.04). CONCLUSION: Association of TRAF1-C5 locus with RA susceptibility was detected in the Japanese populations with modest magnitude, while no significant association was observed for SLE. Significant positive effect of genotype on the expression of TRAF1 might support the genetic association between TRAF1 and RA.

Transduction of retrovirus-mediated NeoR gene into CD34+ cells purified from granulocyte colony-stimulating factor (G-CSF)-mobilized infant and cord blood.

To help establish an effective gene therapy protocol for patients with congenital metabolic diseases, we evaluated retrovirus-mediated transduction and long-term (LT) expression of the NeoR gene in cryopreserved and thawed CD34+ cells purified from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) of infant and cord blood (CB). The results were compared with those in bone marrow (BM) CD34+ cells. The final purity of the CD34+-enriched fraction from PB, CB, and BM, based on FACS analysis, was 88 +/- 14%, 73 +/- 13%, and 68 +/- 19% (mean +/- SEM), respectively. Cells were then cultured for 96 hours with supernatant containing the vector in the presence of interleukin (IL)-3, IL-6, and stem cell factor (SCF). The average efficiency of gene transfer into mobilized PB (n = 5) or CB CD34+ cells (n = 6) was significantly higher than that into BM CD34+ cells, as measured by G418-resistant colony-forming units for granulocyte/macrophage (CFU-GM; 59% or 58% vs. 39%; p < 0.05) and PCR-positive CFU-GM (83% or 79% vs. 53%; p < 0.05). When the evaluation was made in an LT culture system with irradiated allogeneic marrow stroma, these efficiencies were, respectively, 74% or 61% vs. 34% (p < 0.005 or < 0.02) for G418-resistant CFU-GM at week 5 of long-term culture, and 88% or 83% vs. 63% (p < 0.05) for PCR-positive CFU-GM. Fluorometric examination was performed for cell-cycle analysis before and after culture, and the results showed that the fraction of cycling cells was largest in freshly prepared BM (18%), whereas only a small portion of PB (4.6%) and CB (2%) was cycling. However, this value was 17% in BM, 22% in PB, and 13% in CB after culture. These results suggest that mobilized PB from small children and CB cells are suitable and realistic targets for clinical gene therapy and that tandem transduction procedures can be achieved by combining CB and PB.

Streptokinase gene variable region classification in streptococci: lack of correlation with post-streptococcal glomerulonephritis.

To investigate a possible causal role of streptokinase (SKase) in acute post-streptococcal glomerulonephritis (APSGN), the major variable region of SKase genes of Streptococcus pyogenes strains isolated from patients with and without APSGN were analyzed using the polymerase chain reaction, restriction enzyme analysis and the direct sequencing of SKase genes. In the APSGN-associated strains, six of nine revealed mutant classes corresponding to the nephritogenic classes I and II proposed by Johnston et al. [1992], the remaining three belonged to non-nephritogenic classes. In twenty strains not associated with APSGN, seventeen belonged to classes I and II, while three were from other classes. The major variable region of the SKase gene shows no apparent relation with induction of APSGN in humans, suggesting that unique classes of streptococcal SKase do not play a role in the pathogenesis of APSGN.

Detection of hepatitis C virus core protein in the glomeruli of patients with membranous glomerulonephritis.

Two Japanese patients suffered from membranous glomerulonephritis associated with hepatitis C virus (HCV) infection. Renal histologic changes were characterized by granular deposits of IgG and C3 along the capillary wall and numerous subepithelial deposits in glomeruli. Hypocomplementemia was present in one patient, but both cryoglobulins and rheumatoid factors were absent. HCV RNA was detected in both their sera by RT-PCR, both free and in the form of circulating immune complexes. The HCV core protein was found in the glomeruli from both patients by indirect immunofluorescence. These results suggest that in some patients chronic HCV infection causes membranous glomerulonephritis through immune complex deposition involving HCV proteins.

[Clinical toxicity at the infusion of cryopreserved and thawed peripheral blood stem cell grafts in children].

To test the safety of infusing cryopreserved and thawed peripheral blood stem cells (PBSC) in children, toxicity assessment was made at 36 infusions in 33 children with various types of cancer. The mean volume of PBSC graft infused was 224 ml (range, 46-500 ml) or 8.0 ml/kg (1.7-23.8 ml/kg), in containing 10% dimethylsulfoxide (DMSO). Vomiting was the only toxic feature related to the amount of DMSO. However, eight patients developed transient shock, but recovered shortly afterward with or without supportive therapy. Attention should be drawn to increased risk in children receiving thawed blood cell grafts.

Pathological lymphocyte activation by defective clearance of self-ligands in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is one of the autoimmune diseases extensively studied by immunologists and physicians. The main focus regarding SLE pathophysiology has been placed on abnormal cell surface receptor function on lymphocytes. However, recent studies have revealed that defective clearance of apoptotic cells causes self-antigen accumulation, which could trigger the activation of autoreactive lymphocytes. Thus, here we review current findings about the association of the defective clearance of autoantigens and SLE, focusing on mutations in the DNase I locus and their relationship to SLE.

TCR signaling for initiation and completion of thymocyte positive selection has distinct requirements for ligand quality and presenting cell type.

Thymocyte selection involves signaling by TCR engaging diverse self-peptide:MHC molecule ligands on various cell types in the cortex and medulla. Here we separately analyze early and late stages of selection to better understand how presenting cell type, ligand quality, and the timing of TCR signaling contribute to intrathymic differentiation. TCR transgenic CD4+CD8+ thymocytes (double positive (DP)) from MHC-deficient mice were stimulated using various presenting cells and ligands. The resulting CD69high cells were isolated and evaluated for maturation in reaggregate cultures with wild-type or MHC molecule-deficient thymic stroma with or without added hemopoietic dendritic cells (DC). Production of CD4+ T cells required TCR signaling in the reaggregates, indicating that transient recognition of self-ligands by DP is inadequate for full differentiation. DC bearing a potent agonist ligand could initiate positive selection, producing activated thymocytes that matured into agonist-responsive T cells in reaggregates lacking the same ligand. DC could also support the TCR signaling necessary for late maturation. These results argue that despite the negative role assigned to DC in past studies, neither the peptide:MHC molecule complexes present on DC nor any other signals provided by these cells stimulate only thymocyte death. These findings also indicate that unique epithelial ligands are not necessary for positive selection. They provide additional insight into the role of ligand quality in selection events and support the concept that following initiation of maturation from the DP state, persistent TCR signaling is characteristic of and perhaps required by T cells.

B cells are required as APC for antigen-specific T cell proliferation but not for the differentiation or priming of those T cells.

We studied the influences of B cells on functional differentiation of T cells using SCID mice grafted with fetal thymus of C.B-17 mice (TG mice). T cells were shown to be reconstituted in TG mice without B cell development. These mice showed normal DTH response to SRBC and OVA. LN cells of these mice produced cytokines including IL-2, IL-4, IL-6 and IFN-gamma according to Con A stimulation. Thus, majority of T cell functions seem to differentiate in the absence of B cells. However, T cells of TG mice failed to proliferate in response to immunizing antigens in vitro, although they responded well to stimulation with Con A. This unresponsiveness of T cells in TG mice to these antigens was restored when antigen-primed B cells were added to the proliferation assay. Such an inability of T cells in antigen-specific proliferation was not seen in SCID mice grafted with C.B-17 fetal liver cells, in which B cells as well as T cells were efficiently reconstituted (FLT mice). T cell proliferation to immunizing antigen was also abrogated in FLT mice when B cells were depleted from lymphoid population. These results indicate that T cells can functionally differentiate and be primed in the absence of B cells, but they require B cells to proliferate in response to foreign antigens.

CD4+ T cell survival is not directly linked to self-MHC-induced TCR signaling.

T cell receptor (TCR) signaling triggered by recognition of self-major histocompatibility complex (MHC) ligands has been proposed to maintain the viability of naïve T cells and to provoke their proliferation in T cell-deficient hosts. Consistent with this, the partially phosphorylated state of TCR zeta chains in naïve CD4+ and CD8+ T cells in vivo was found to be actively maintained by TCR interactions with specific peptide-containing MHC molecules. TCR ligand-dependent phosphorylation of TCR zeta was lost within one day of cell transfer into MHC-deficient hosts, yet the survival of transferred CD4+ lymphocytes was the same in recipients with or without MHC class II expression for one month. Thus, despite clear evidence for TCR signaling in nonactivated naïve T cells, these data argue against the concept that such signaling plays a predominant role in determining lymphocyte lifespan.

Contribution of extrathymic gamma delta T cells to the expression of heat-shock protein and to protective immunity in mice infected with Toxoplasma gondii.

We demonstrated that gamma delta T cells contribute to protective immunity against Toxoplasma gondii by inducing the expression of a 65,000 MW heat-shock protein (hsp 65) in host macrophages. Here we examined the role of extrathymic and intrathymic gamma delta T cells in protective immunity and hsp 65 expression in mice infected with T. gondii. Intrathymic gamma delta T cells were obtained from severe combined immunodeficiency (SCID) mice grafted with syngeneic fetal thymus (TG-SCID), in which only T cells derived from the donor thymus developed, whereas extrathymic gamma delta T cells were obtained from nude mice that lack thymus. Extrathymic gamma delta T cells from T. gondii-infected nude mice differed from intrathymic gamma delta T cells of infected TG-SCID mice, in terms of Thy1.2 expression and V-region gene usage of T-cell receptor (TCR) gamma delta. Extrathymic gamma delta T cells expressed extremely high levels of Thy1.2, and had V gamma 7 repertoire but lacked V gamma 5,6 and V delta 1,5. On the other hand, intrathymic gamma delta T cells express intermediate and low levels of Thy1,2. These cells possessed V gamma 5,6 and V delta 1,5 but failed to rearrange the V gamma 7 gene. Peritoneal macrophages from infected nude mice contained hsp 65, whereas this protein was scarcely expressed in those of infected TG-SCID mice. Transfer of extrathymic, but not of intrathymic gamma delta T cells to SCID mice enabled their macrophages to express hsp 65. Athymic nude mice were significantly resistant to the infection compared with SCID mice which lack gamma delta T as well as alpha beta T cells. The resistance was dependent upon extrathymic gamma delta T cells, since nude mice depleted of gamma delta T cells using a corresponding monoclonal antibody became extremely susceptible. These results indicated that extrathymic rather than intrathymic gamma delta T cells play some crucial roles in protection against T. gondii and in hsp 65 expression.

Mechanisms of HSP65 expression induced by gamma delta T cells in murine Toxoplasma gondii infection.

We have previously reported that the expression of an endogenous 65-kD heat shock protein (HSP65) in macrophages is closely correlated with the protection against infection by Toxoplasma gondii in mice, and gamma delta T cells play a critical role in the expression of this protein. In this study, we investigated how gamma delta T cells contribute to the protection and HSP65 expression. After intraperitoneal infection with bradyzoites of the Beverley strain of T. gondii, mRNA encoding IFN-gamma and TNF-alpha was detected in the peritoneal gamma delta T cells by RT-PCR technique, and macrophages that produced nitric oxide (NO) and expressed HSP65 were also detected. Depletion of gamma delta T cells resulted in suppression of NO production by macrophages, and it also inhibited HSP65 expression. HSP65 expression, however, does not appear to be induced by stimulation with NO, since treatment with NG-monomethylarginine, an inhibitor of NO synthesis, did not attenuate the expression of HSP65. This expression was completely suppressed when mice were simultaneously treated with anti-IFN-gamma and anti-TNF-alpha although either antibody alone was less effective. The synergistic effect of these cytokines was also demonstrated by an in vitro experiment, in which peritoneal macrophages were cultured with recombinant IFN-gamma and TNF-alpha. These results indicate that gamma delta T cells, which protect against infection with T. gondii induce the expression of HSP65 by secreting IFN-gamma and TNF-alpha and the production of NO, and that the expression of HSP65 is independent of inflammatory chemical compounds like NO and H2O2.

A monoclonal antibody (1F3) to human glomerular epithelial cells: a new marker for renal epithelial cell injury.

In order to identify a new cell surface antigen as a potential marker of renal epithelial cell injury, we produced a monoclonal antibody (Mab), 1F3, by immunizing mice with cultured human glomerular cells. Immunofluorescence (IF) and immunoelectron microscopy (IEM) studies demonstrated that 1F3-recognizing antigen (1F3 antigen) was strongly expressed on the cell surface of glomerular podocytes and very weakly on parietal epithelial cells. 1F3 antigen was not expressed in any other cells in the normal kidney. Immunoprecipitation analysis using metabolically labeled glomeruli revealed that 1F3 recognized a 125-kD protein under reducing conditions. IF studies of biopsy specimens from patients with a variety of glomerular and tubulointerstitial diseases showed that 1F3 antigen was almost negative in cellular crescents but was strongly expressed in fibrocellular crescents. When glomerular sclerosis appeared, the expression of 1F3 antigen decreased in sclerotic areas of glomeruli. 1F3 antigen became positive in atrophic tubules that were seen in diseased kidneys. Severity of tubular atrophy correlated well with the extent of tubular expression of 1F3 antigen. These results indicate that Mab, 1F3 marks phenotypic changes of renal epithelial cells under disease conditions and may be a useful marker for progressive kidney diseases.

The Fas-deficient SCID mouse exhibits the development of T cells in the thymus.

The Fas Ag is a cell surface protein that mediates apoptosis and is highly expressed on thymocytes. However, the role of the Fas system in the thymus is unclear. To study the role of the Fas system in the thymus, we established a novel SCID mouse bearing the lpr (lymphoproliferation) mutation. Thymocytes from these mice differentiated into CD4+ CD8+ T cells and then underwent further differentiation into CD4+ CD8- or CD4- CD8+ single-positive T cells with a low surface expression of CD3, whereas B cell development remained unrescued. These TCR-positive T cells can proliferate in response to stimulation with PMA plus ionomycin, but not with third-party spleen cells. Furthermore, thymocytes from scid/lpr mice had a great variety of TCR Vbeta repertoires. These results suggest that the Fas system plays an essential role in regulating the development of intrathymic T cells as well as peripheral T cells.

The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate.

Signals elicited by binding of the T-cell antigen receptor and the CD4/CD8 co-receptor to major histocompatibility complex (MHC) molecules control the generation of CD4+ (helper) or CD8+ (cytotoxic) T cells from thymic precursors that initially express both co-receptor proteins. These precursors have unique, clonally distributed T-cell receptors with unpredictable specificity for the self-MHC molecules involved in this differentiation process. However, the mature T cells that emerge express only the CD4 (MHC class II-binding) or CD8 (MHC class I-binding) co-receptor that complements the MHC class-specificity of the T-cell receptor. How this matching of co-receptor-defined lineage and T-cell-receptor specificity is achieved remains unknown, as does whether signalling by the T-cell receptors, co-receptors and/or general cell-fate regulators such as Notch-1 contributes to initial lineage choice, to subsequent differentiation processes or to both. Here we show that the CD4 versus CD8 lineage fate of immature thymocytes is controlled by the co-receptor-influenced duration of initial T-cell receptor-dependent signalling. Notch-1 does not appear to be essential for this fate determination, but it is selectively required for CD8+ T-cell maturation after commitment directed by T-cell receptors. This indicates that the signals constraining CD4 versus CD8 lineage decisions are distinct from those that support subsequent differentiation events such as silencing of co-receptor loci.

Acute interstitial nephritis and uveitis with bone marrow granulomas and anti-neutrophil cytoplasmic antibodies.

Acute interstitial nephritis (AIN) and uveitis of unknown etiology developed in a 15-year-old girl. Symptoms and urinary abnormalities improved spontaneously but moderate renal dysfunction and anterior uveitis persisted. A repeat biopsy performed 2 years later showed focal tubulointerstitial changes, while bone marrow examination revealed granulomas. Simultaneously, antineutrophil cytoplasmic antibodies (ANCA) were detected, and decreases in T cell population and lymphocyte function were found. These immunological abnormalities normalized in parallel with clinical improvement with corticosteroid therapy, strongly suggesting that in some patients autoimmune disorders contribute to the pathogenesis of the disease.

Transforming growth factor-beta (TGF-beta) stimulates the expression of beta1 integrins and adhesion by rat mesangial cells.

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of mesangial matrix (MM) accumulation in human and experimental glomerulonephritis. To clarify molecular mechanisms responsible for pathological MM deposition, we examined the effect of TGF-beta on the production of beta1 integrins and on adhesion function of rat mesangial cells (MC). In immunoprecipitation experiments using [35S]methionine-labeled MC, stimulation of MC with TGF-beta for 48 h resulted in an increase in the synthesis of alpha1beta1 (collagen/laminin receptor) and alpha5beta1 (fibronectin receptor) integrins accompanied by increases in the synthesis of their ligands, collagen type I (collagen I), and fibronectin. A time-dependent increase in beta1, alpha1 integrin subunit mRNA peaking 48 h after exposure to TGF-beta was shown by Northern blot analysis. After 48 h of treatment with TGF-beta, MC displayed significant increases in adhesion to fibronectin, collagen I, and laminin as compared to untreated MC. Anti-beta1 antiserum significantly inhibits MC adhesion to fibronectin, collagen I, and laminin. Anti-alpha1 subunit antibody very strongly inhibited adhesion to collagen I and laminin, but not to fibronectin. Synthetic peptides containing RGD sequences specifically blocked adhesion to fibronectin. These data suggest that TGF-beta may promote MM deposition by increasing MC synthesis of both matrix proteins and beta1 integrins which facilitate binding of these proteins to the MC surface and thus enhance their incorporation into MM.