Possible role of cellular immunity against tubular basement membrane antigen (TBM) in gold nephropathy in rheumatoid arthritis patients]

Autoantibody formation and lymphocytes proliferative response to tubular basement membrane (TBM) antigen were examined to clarify the pathogenesis of gold nephropathy, in rheumatoid arthritis patients. The existence of tubulopathy was ascertained by urine protein analysis, electrophoresis and urine TBM antigen titration. Circulating antibody to human TBM antigen titrated by enzyme immunoassay was significantly elevated in patients with gold tubulopathy, and mitogenic stimulation with TBM antigen of peripheral lymphocytes specifically responded in the early stage after receiving gold, but then clearly decreased after the cessation of gold. But, when the lymphocytes had been passed through a nylon wool column, the reaction was remarkably high even in the later stage after receiving gold, suggesting that another suppressive population of lymphocytes became trapped in the nylon wool column. This evidence suggests that gold compounds definitely act as initiating and promoting agents, and the development of tubular disorders induced by gold are likely related to the cellular recognition of effector T cells to the TBM antigen, following the strong effect of gold on the cellular immune system.     

Detection of nephritogenic antigen from the Lewis rat renal tubular basement membrane

Immunopathogenicity of trypsin-solubilized or non-solubilized renal tubular basement membrane (TBM) of the Lewis (LEW) rat was investigated. Autoimmune tubulointerstitial nephritis (TIN) was induced in BALB/c mice by immunization with trypsin-solubilized LEW rat TBM, while immunization with non-solubilized TBM did not produce the disease. Based on this preliminary experiment we studied the characterization of immunogenic and nephritogenic TBM antigen of the LEW rat. TIN was characterized by severe mononuclear cell infiltrates with multi-nucleated giant cells in the interstitium, tubular destruction and intensive IgG and C3 deposits along the TBM. Anti-TBM antisera and eluate from the nephritic mouse kidneys reacted with the TBM of normal LEW rat kidney by immunofluorescence. LEW rat TBM was also detected immunofluorescently by using antisera from BALB/c mice immunized with autologous trypsin-solubilized TBM. A competitive inhibition test revealed a higher titer of anti-TBM antibody in the eluate than in the adsorption-treated antisera per microgram IgG. Immunoblotting showed one reactive band with a molecular weight of 45,000 daltons, and the blotting patterns in tryptic TBM of the Brown Norway (BN) and LEW rats appeared similar. Amino acid analysis of nephritogenic LEW rat tryptic TBM showed that it contained no hydroxyproline and hydroxylysine, suggesting that this TBM preparation was not collagenous. These findings suggest that tryptic digestion contributes to the release of nephritogenic antigen from the LEW rat TBM and that this antigen system might participate in the immune system involved in the anti-TBM associated TIN that is well known to be induced by non-digested TBM of TBM antigen positive animals.     

Anti-prostate specific membrane antigen designer T cells for prostate cancer therapy

BACKGROUND: Designer T cells are T lymphocytes engineered toward specific antibody-type membrane antigens through chimeric immunoglobulin-T-cell receptor (IgTCR) genes that have been used for adoptive cellular immunotherapy. We have extended this approach to prostate specific membrane antigen (PSMA) as a means to attack prostate cancer. METHODS: A chimeric anti-PSMA IgTCR gene was constructed based on an anti-PSMA monoclonal antibody, 3D8. Both T-cell lines and primary cultured human T lymphocytes were transduced with the chimeric anti-PSMA IgTCR construct and were analyzed for IgTCR expression, specific activation by PSMA, cytotoxicity against PSMA-expressing tumor cells in vitro, and retardation of tumor growth in an animal model. RESULTS: The IgTCR was incorporated into the TCR-CD3 complex and formed a functional chimeric complex. The IgTCR-modified T cells were specifically activated through the chimeric receptor with PSMA as measured by IL-2 production and increased CD25 expression and specifically lysed the PSMA-expressing prostate cancer cells in vitro as well as retarded tumor growth in an animal model. CONCLUSIONS: The anti-PSMA designer T cells exhibit an antibody-type specificity that can recognize PSMA expressing tumor cells in a MHC-independent fashion, resulting in T-cell activation, target cell lysis in vitro and inhibition of tumor growth in vivo.     

Morphometric study of glomerular basement membrane and proximal tubular basement membrane in adult thin basement membrane disease

Background. Thin basement membrane disease (TBMD) is a benign hereditary glomerulopathy with a diffuse attenuation of glomerular basement membrane (GBM). Whether the development of renal basement membranes other than GBM is normal in TBMD has not yet been resolved. Methods. We performed a morphometric study to measure the thickness of GBM and proximal tubular basement membrane (P-TBM) in 44 adult patients with TBMD and in 10 adult diseased controls confirmed to have minor glomerular abnormalities. Results. There was a significant difference between the patients with TBMD and the diseased controls in the thickness of the GBM; however, there was no significant difference between the two groups in the thickness of the P-TBM. In the patients with TBMD, the thickness of the GBM was unchanged with age, but the thickness of the P-TBM increased with age, as did that in the diseased controls. Conclusion. Our morphometric study clarified that the development of P-TBM was normal in the patients with TBMD. Received: October 7, 1998 / Accepted: May 27, 1999     

Differences in suppressor of cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c and spontaneously-diabetic NOD recipient mice

BACKGROUND: The ability to block interferon signaling represents an important strategy in designing therapies to prevent beta-cell destruction during islet allograft rejection. METHODS: The SOCS proteins regulate cytokine signaling by blocking activation of JAK/STAT proteins. Using islets isolated from SOCS-1 transgenic mice (SOCS-1-Tg; these mice express SOCS-1 under the control of the human insulin promoter and are on the C57BL6/J background), we investigated whether SOCS proteins can prevent the destruction pancreatic islet cells transplanted beneath the kidney capsule of major histocompatibility complex mismatched normal BALB/c and spontaneously-diabetic NOD mouse recipients. RESULTS: Immunohistochemical staining for insulin confirmed the presence of donor SOCS-1-Tg islets in islet allografts harvested at 22 days posttransplant, whereas grafts of control non-Tg islets were destroyed by 14 days. In contrast, SOCS-1-Tg allogeneic islets were not protected from beta-cell destruction in clinically diabetic NOD mice. The islet allografts functioned for 1 week posttransplant; however, hyperglycemia returned after 2 weeks and the grafts were destroyed. Rejection of SOCS-1-Tg and non-Tg islets in autoimmune diabetic NOD mice was associated with an infiltrate of both CD4+ and CD8+ T cells and a T2-type cytokine response (IL-4) rather than the conventional T1-type cytokine response observed during islet allograft rejection. Self-antigen upregulation in response to IFN-gamma stimulation did not appear to be a factor in rejection of the islet allografts. CONCLUSIONS: These results demonstrate that expression of SOCS-1 in islets delays islet allograft rejection but cannot circumvent destruction of the islets by the recurrence of the tissue-specific autoimmune process of spontaneous diabetes.     

BALB/c小鼠精原干细胞长期培养和移植的实验研究

目的 建立小鼠精原干细胞(SSC)长期培养体系,探讨SSC体外增殖分化的关键因子.方法 收集出生4～6 d BALB/c绿色荧光小鼠睾丸,采用改良两步消化法获得细胞悬液,3次差速贴壁去除体细胞获得富集的精原细胞,采用添加生长因子的无血清基础培养液重悬,种植到小鼠胚胎成纤维细胞饲养层上培养.基础培养液为StemPro-34 SFM干细胞培养基并补充15种添加成分;生长因子为10 ng/ml碱性成纤维细胞因子、20 ng/ml胶质细胞源性神经营养因子和200 ng/mlGDNF家族受体a1.取4～5周龄BALB/c雄性小鼠15只,腹腔注射40 mg/kg的白消安建立受体模型,采用三维显微注射系统将培养的SSC移植到受体左侧睾丸精曲小管内,右侧睾丸作为自身对照;分别采用体视荧光显微镜观察和HE染色检测细胞移植后睾丸生精功能恢复情况.结果 改良消化富集法消化后细胞活性>98%,SSC富集约18.5倍.饲养层培养1～2 d后细胞成对称或线形排列,细胞间可见明显的胞质桥连接.3～4 d后精原细胞增殖形成典型的克隆,为边缘不清楚的团块;小鼠SSC能在该培养体系中稳定培养、传代3个月.移植后2个月,体视荧光显微镜下受体睾丸内可见明显绿色阳性克隆,HE染色证实移植的SSC在受体睾丸内克隆增殖并分化产生成熟的精子.结论 成功建立了BALB/c小鼠SSC培养体系,为研究SSC增殖分化调控机制及SSC移植治疗男性不育提供了实验依据.     

Induction of human cytotoxic T lymphocytes specific for prostate-specific antigen

Prostate-specific antigen (PSA), a tissue-specific protein expressed by most adenocarcinomas of the prostate, might be a useful target for T-cell-mediated immunotherapy of prostate cancers. The current study examined whether it is possible to elicit human cytotoxic T lymphocytes (CTL) with specificity for PSA. A synthetic nonamer peptide, corresponding to residues 146–154 of PSA and containing a canonical HLA-A2-binding motif, was shown to stabilize the expression of HLA-A2 on the T2 antigen-processing mutant cell line. Repeated in vitro stimulation of peripheral blood lymphocytes from a normal HLA-A2⁺ donor induced CTL with specificity for the PSA 146–154 peptide. The peptide-induced CTL expressed the CD4⁻ CD8⁺ cell surface phenotype and were restricted by HLA-A2. A large portion of patients with prostate cancer express the HLA-A2 phenotype, implying that many prostate cancers might be targeted by HLA-A2-restricted CTL with specificity for the PSA 146–154 epitope. Prostate 30:73–78, 1997. © 1997 Wiley-Liss, Inc.     

Multi-organ inflammation after hepatic cryoablation in BALB/c mice

BACKGROUND: There is increasing evidence that injury to the liver can precipitate or exaggerate lung injury. We have previously shown that hepatic cryoablation (cryo) causes activation of nuclear factor (NF)-kappaB, cytokinemia (tumor necrosis factor-alpha, Mouse Macrophage Inflammatory Protein-2 [MIP-2]), and lung inflammation in transgenic HLL (5'HIV-LTR-Luciferase gene) mice and in Sprague-Dawley rats. It has been reported that BALB/c mice are susceptible to traumatic injury and are active immune responders. We tested whether activation of NF-kappaB and the development of multiple-organ inflammation in response to hepatic injury from 35% cryo were demonstrable in the BALB/c mouse. METHODS: BALB/c mice (n = 9) were anesthetized, and midline laparotomy was performed. Cryoablation was performed with careful isolation of adjacent structures to avoid inadvertent organ injury to the gastrointestinal tract. A freeze-thaw cycle of the left lobe of the liver was induced, encompassing approximately 35% (by weight). Animals were sacrificed at 1, 2, 4, and 24 h after cryoablation. Serum was collected via IVC puncture and liver, lungs, and kidneys were harvested and freeze-clamped. Two animals were sacrificed without undergoing cryo surgery to serve as a baseline control. NF-kappaB activity was monitored by electrophoretic mobility shift assays. MIP-2 levels and Mouse KC levels from tissue and serum were measured using enzyme-linked immunosorbent assay. Organs were submitted for histological review. We characterized lung inflammation induced by cryosurgery by measuring total and differential cell counts in lung lavage fluid 4 h after hepatic cryoablation. RESULTS: After cryo, NF-kappaB activation was demonstrated in the 1, 2, and 4-h time points by electrophoretic mobility shift assay in the liver and lungs. Mouse KC and MIP-2 levels increased from baseline, peaked at the 4-h time point, and returned to baseline after 24 h in both liver and lung. Lung lavage 4 h after cryoablation showed increased total cells and neutrophilic lung inflammation. CONCLUSIONS: BALB/c mice demonstrate evidence of multi-organ inflammation in response to 35% hepatic cryo. These data demonstrate that this model provides for assessment of liver-mediated multi-system inflammation after direct liver injury.     

小鼠肝星状细胞的分离、培养及鉴定方法

目的：探索BALB／c小鼠肝星状细胞（HSCs）分离、纯化的可行方案．并评价依该法分离所得HSCs的生物学特性。方法：经肝门静脉先用不含钙镁离子的Hank液充分灌洗,再以浓度为1mg／mL的Ⅳ型胶原酶灌注BALB／c小鼠肝脏。取肝后．完整分离后碾碎,37℃水浴振荡30min,Percoll连续密度梯度（60％）离心法分离、纯化HSCs．苔盼蓝染色检测HSCs活性,结蛋白免疫细胞化学鉴定HSCs,光镜观察体外培养HSCs的形态学变化。结果：纯化后每只小鼠HSCs收获量约为（5．5±0．4）×10^5个．HSCs纯度〉90％．HSCs细胞活率〉90％。结论：本实验建立的的分离纯化方案可获得高纯度高活率的小鼠HSCs。     

Induction of suppressor T cells by donor-specific blood transfusion (DST): establishment of a human T cell hybridoma producing an MLR suppressing factor]

We established a human T cell hybridoma producing a mixed lymphocyte reaction (MLR) suppressing factor. Three weeks after DST, peripheral blood lymphocytes (PBL) were obtained from a recipient and were cultured for 3 days with mitomycin C (MMC)-treated donor PBL. These lymphocytes were fused with an azaguanine-resistant mutant of a human T cell leukemic cell line (CCRF-CEMAG). Four weeks after fusion, approximately 30% of the wells showed hybridoma cell growth. To select hybridoma clones with suppressive activity, irradiated hybridoma clones were added as regulator cells to the mixed lymphocyte culture. After the cloning, one clone causing suppression of the donor-specific MLR was established (termed HK40: %MLR suppression = 38.9%). Unstimulated supernatant of HK40 showed no suppressive effect on the specific MLR. In contrast, supernatant of HK40 cultured with donor PBL for 24 hrs, suppressed the donor-specific MLR dose-dependently. This primed supernatant of HK40 markedly suppressed the specific MLR when added added at the culture initiation. These findings indicate that functional clones causing suppression of the alloantigen-specific MLR can be generated in patients receiving DST, and suggest that these clones may be essential to the prolongation of kidney allograft survival.     

Immunohistochemical study of a tubular basement membrane antigen in normal human urinary sediment by a monoclonal antibody

This preliminary study concerns murine monoclonal antibodies (Mabs) against tubular basement membranes (TBM) of human kidney which were produced by the classical hybridization technique. One of these Mabs (PP8-1) specifically recognized an antigen present in the tubular basement membranes of various portions of tubule and in the Bowman's capsule. By immunocytochemical techniques, this Mab was used to study the presence and the distribution of the related antigen in urinary sediment from 12 normal healthy subjects. This TBM and Bowman's capsule antigen was demonstrated along hyaline urinary cast surface and, in some cases, in free extracellular fibrillar structures morphologically unrelated to the casts. This suggests that, in some normal conditions, catabolism of TBM and/or Bowman's capsule could release material in urine, as it has been described for glomerular basement membrane (GBM) constituents. Such an immunocytochemical approach could be useful to detect components from tubular renal basement membranes in urinary sediment during pathological conditions.     

Helper-inducer T-lymphocytes mediate diabetes in EMC-infected BALB/c ByJ mice

Diabetes mellitus developing in BALB/c ByJ mice infected with the M variant of encephalomyocarditis (EMC) virus is dependent on thymic immune mechanisms. We treated mice with the anti-T-lymphocyte monoclonal anti-L3T4 and anti-Lyt2.2 antibodies before virus inoculation and monitored the infection and occurrence of diabetes thereafter. Mice depleted of L3T4+ cells exhibited a reduced incidence and severity of diabetes compared with both untreated and anti-Lyt2.2-treated animals. All mice sustained pancreatic infections, but islet lesions with beta-cell degranulation only occurred in control infected and anti-Lyt2.2-treated animals. These data support the conclusion that the induction of diabetes in EMC-infected BALB/c mice is immune mediated and controlled by the L3T4 T-lymphocyte subpopulation.     

Histopathology of humorally mediated anti-glomerular basement membrane (GBM) glomerulonephritis in mice.

BACKGROUND: From a diagnostic point of view it would be important to learn more about the relationship between the immune responses underlying glomerulonephritis and the patterns of glomerular lesions. A murine model of anti-GBM glomerulonephritis in which inflammation is driven by delayed-type hypersensitivity (DTH) has been studied extensively. The aim of this study was to uncover histological features that might be specific for anti-GBM glomerulonephritis driven by a humoral immune response. METHODS: BALB/c mice were immunized with rabbit IgG in incomplete Freund's adjuvant. Six days later, on day 0, they received rabbit anti-GBM serum intravenously. Proteinuria was assessed with dipsticks. Mice were killed on days 4, 8 or 14. Kidneys from days 4 and 8 were processed for immunofluorescence and histology. On day 14 mice were perfusion-fixed for electron microscopy. RESULTS: Proteinuria started on day 3. Autologous IgG and of C3 were found along the GBM. There was only slight infiltration with macrophages and no measurable infiltration by CD4 T cells, indicating the virtual absence of DTH. Besides infiltration with neutrophils there were little histological alterations on day 4. On day 8 many loops were hyalinized. On day 14, cellular crescents were found in 23% of glomeruli. Subendothelial spaces contained hyaline material, cells and fibrin. Podocytes displayed effacement of foot processes and apical microprotrusions. Podocyte bridges were common. These alterations were identical to those reported in the standard model that produces a DTH-like inflammation. CONCLUSION: The qualitative pattern of histological damage in a murine model of anti-GBM glomerulonephritis does not depend on the underlying immunological process.     

Induction of transplantation tolerance in rats by spleen allografts. II. Evidence that W3/25+ T suppressor/inducer and OX8+ T suppressor/effector cells are required to mediate specific unresponsiveness

The results presented in this report demonstrate that T cells, isolated from AGUS rats bearing long-term (WAG X AGUS)F1 spleen allografts adoptively transferred to irradiated AGUS recipients could not mediate the rejection of WAG hearts but rejected PVG. A hearts in acute fashion. Further, unresponsive T cells were able to suppress the capacity of adoptively transferred (40 X 10(6) normal T cells to reject WAG but not PVG.A heart allografts. We also studied the role of W3/25+ and OX8+ T cells subsets in the maintenance of unresponsiveness. Isolated W3/25+ or OX8+ unresponsive T cells were not able to mediate acute rejection, but were less effective in prolonging WAG allograft survival than the unresponsive whole T cell population, suggesting that both W3/25+ Ts1 and OX8+ Ts2 subsets were required for effective suppression in vivo. When, however, unresponsive W3/25+ T cells were infused simultaneously with normal OX8+ T cells, they could produce indefinite survival of WAG heart allografts. These results indicate that the unresponsive state induced by (WAG X AGUS)F1 spleen allografts transplanted to AGUS rats is maintained by the interaction of W3/25+ T suppressor/inducer and OX8+ T suppressor/effector cells.     

Development of suppressor T cells by antilymphocyte serum treatment in mice

Administration of rabbit anti-mouse lymphocyte serum (ALS) in mice results in the development of suppressor cells which can be detected by coculture mixed lymphocyte culture experiments. The putative suppressor cells inhibit nonspecifically the proliferative response as well as generation of cytotoxicity of normal responder cells. Suppressor activity is dose dependent and is not attributable to cell crowding, shifting of peak activity, or release of cell-bound ALS. Additional antigenic stimulation by skin allografting in ALS-treated mice shifts the specificity of suppressor cells from nonspecific to specific for skin donor alloantigen. ALS-induced suppressor cells are Lyt-1+2- T cells while suppressor cells present in ALS-treated, skin allograft-bearing mice are Lyt-1-2+ T cells. Both types of suppressor cells appear to bear I-J determinants. The possible mechanisms of suppressor cell induction by ALS and skin allografting are discussed.     

Induction of suppressor T cells by intravenous administration of monoclonal anti-I-A antibody

Intravenously administered monoclonal anti-I-A antibodies successfully induced suppressor T (Ts) cells specific for alloantigen-specific delayed-type hypersensitivity (DTH) responses in mice. These Ts cells exerted their effects in the effector phase and had no H-2 restrictions. Phenotypic analysis revealed L3T4 antigens on their cell surface but failed to reveal Lyt-2 antigen. Ts cell activity was abrogated by a 30-min incubation with the anti-I-A antibodies used for Ts cell induction. Incubation in the anti-I-A-antibody-coated plate also abrogated the Ts cell activity. Since anti-I-A antibody is idiotypic for the I-A antigen, it is suggested that these Ts cells might express antiidiotypic receptors for I-A antigens. These findings are considered to be consistent with previous observations of hapten-specific systems, in which antiidiotypic Ts cells are inducible by idiotypic antibodies.