Academic literature on the topic 'Mutation lymphoproliferation(lpr)'

MRL-Fas lpr/lpr mice represent an excellent animal model for studying non-malignant lymphoproliferation, regeneration and systemic autoimmunity. Retro-transposon insertion into the second intron of the pro-apoptotic Fas gene appears to be responsible for both lymphoproliferation and autoimmunity, while other genes are more likely to contribute to the regenerative healing characteristic of this mouse strain. Previous studies have shown that neonatal thymectomy can halt the development of abnormal lymphoproliferation. Whereas at four weeks of age primary and secondary lymphoid organs appear to be grossly intact, vigorous lymphoproliferation and autoantibody production subsequently ensues. This is first noticeable at six weeks of age, at which time lymph nodes, spleens and thymuses, but not the bone marrow, become infiltrated with abnormal B220+CD3+CD4−CD8− T cells. Around the same time, thymuses show a significant drop in CD4+CD8+double-positive T cells generating an abnormal ratio between double-positive and single-positive thymocytes. The objective of current study was to evaluate the effect of synthetic oligonucleotides-toll-like receptor antagonists on early lymphoid development in this strain of mice. Herein, we demonstrate the ability of synthetic oligonucleotides made with the nuclease-resistant phosphorothioate backbone to partially reverse abnormal lymphoproliferation and thymic involution in pre-diseased MRL-Fas lpr/lpr mice when administered intraperitoneally starting from week four of age. This curative effect of oligonucleotides was primary sequence/secondary oligonucleotide structure-independent, suggesting an effect through the toll-like receptor 7. A similar approach may potentially benefit patients with autoimmune lymphoproliferative syndrome who, like MRL-Fas lpr/lpr mice, carry a mutation in the Fas gene.

Homozygosity for either of the lymphoproliferation (lpr) or generalized lymphoproliferative disease (gld) mutations of mice causes the development of systemic lupus erythematosus-like autoimmune syndromes that are characterized by severe lymphadenopathy and highly elevated serum immunoglobulin levels. Although the mutations are nonallelic, analysis of homozygous lpr/lpr and gld/gld mice on the same strain background has indicated that the pathology and severity of the autoimmune syndromes induced by these mutations are indistinguishable. To explain this, it has previously been suggested that lpr and gld may represent mutations in molecules involved in sequential steps of an intracellular metabolic pathway of T cells. We have now investigated the behavior of both lpr and gld in a variety of bone marrow chimeras and have found that functional differences between lpr and gld become apparent after bone marrow transfer. Transfer of lpr/lpr bone marrow to irradiated congenic +/+ recipients caused the development of a graft-vs.-host-like lymphoid wasting syndrome, whereas transfer of gld/gld bone marrow to +/+ recipients resulted in development of a gld-like autoimmune syndrome. Additionally, gld/gld hosts behaved like +/+ hosts irrespective of the genotype of the donor bone marrow, whereas lpr/lpr hosts behaved unlike +/+ hosts when reconstituted with either lpr/lpr, gld/gld, or +/+ bone marrow. These are the first clear differences between these two mutations yet described. Our studies indicate that the molecule altered by the gld mutation is expressed only by bone marrow-derived cells, whereas the molecule altered by the lpr mutation is expressed by both bone marrow-derived cells and by one or more peripheral radioresistant cell populations. To reconcile these differences with the fact that homozygous lpr/lpr and gld/gld mice are indistinguishable, we suggest an alternative model for the relationship between the lpr and gld mutations in which the two molecules affected represent an interacting ligand-receptor pair expressed by different cells.

The lpr and gld murine models have been important contributors to our understanding of systemic autoimmune diseases. Mice homozygous for either of these autosomal recessive genes develop a phenotypically identical disease characterized by the accumulation of CD4-CD8- T-cells and the production of a wide spectrum of autoantibodies. The lpr (lymphoproliferation) mutation encodes a defective Fas apoptosis receptor gene. More recently, gld (generalized lymphadenopathy) has been shown to be a point mutation in the Fas ligand gene. Despite the molecular characterization of these mutations, the exact mechanism by which tolerance is lost is still unknown, although in vivo cell transfer studies have provided clues. Chimera studies, in which normal and lpr bone marrow were co-infused into lpr mice, demonstrated not only that the normal Fas receptor is functionally expressed in both T- and B-cells, but that the Fas mutation is required in both for full expression of the lpr phenotype. Conversely, in analogous experiments with gld mice, co-infusion of normal and gld bone marrow largely prevented the development of autoantibodies. Sporadic autoantibody titers were seen in some mice, but were derived from both donors. The effects on T-cells were subtly different: The CD4-CD8- T-cells were also greatly reduced in number, but all were of gld origin. These data indicate that the gld defect is extrinsic to B-cells but only partially extrinsic to T-cells, and suggest that Fas ligand in T-cells may have an autocrine and paracrine function.

Abstract ALPS is an inherited disorder of lymphocyte apoptosis leading to childhood onset of chronic lymphadenopathy, hepatosplenomegaly, autoimmunity, and an increased risk of lymphoma in a subset of patients with mutations in the intracellular domain of Fas (ALPS Type Ia). Similarly, MRL/lpr −/− mice are homozygous for Fas mutations and develop massive lymphadenopathy and splenomegaly associated with hypergammaglobulinemia, glomerulonephritis, and expansion of TCR αβ+, CD4−/CD8−double-negative (DN) T cells that are pathognomonic of ALPS. There remain no proven therapies for the lymphoproliferation underlying ALPS itself, although investigators in Europe reported that some children with ALPS showed reductions in spleen and lymph node sizes while on a weekly regimen of pyrimethamine/sulfadoxine (Fansidar@) (Br J Haematol.2002; 117:176). However, in vitro studies using stimulated human PBMCs, revealed that lymphocyte apoptosis is induced by the antifolate agent, pyrimethamine (Pyr) at an EC50 of 2.5ug/mL, and not sulfadoxine. This suggested that Pyr alone might prove clinically beneficial, thus avoiding risks of allergy and hypersensitivity associated with sulfa drugs. Hence, studies were conducted in the MRL/lpr−/− murine model of ALPS with Pyr alone given 2mg/kg twice weekly by gavage for 8 weeks, and in combination with sulfadoxine. Neither Pyr nor Fansidar yielded significant shrinkage of lymphoid mass in mice. A pilot study of the safety and efficacy of Pyr was conducted under an FDA IND in 6 children with ALPS Type 1a and one with ALPS Type III (with no identifiable mutation). Aged 6 to 17 yrs (median; 13 y), these 6 males and 1 female patient were enrolled following informed consent to be treated with oral Pyr at escalating doses (25–50mg) given twice weekly for 12 weeks. Computerized tomography was done before and after treatment to determine whether these patients would show 40 and 50% reductions in the sizes of their lymphadenopathy and/or splenomegaly, respectively. Lymph node sizes were assessed as sums of the bi-dimensional products of 3 to 5 nodes. Spleen sizes were measured as products of their craniocaudal and anterioposterior dimensions at the superior mesenteric artery level, as well as spleen volumes computed by digital image processing. The effects of treatment on other well-defined laboratory features of ALPS were also assessed. Pyr was discontinued after 11 days in one patient due to skin rash. Dose escalation was interrupted transiently in 3 others because of neutropenia, aphthous ulcer, or leucopenia. There were no significant changes in study outcome measures. The aggregate experience with Pyr and Fansidar in mice, as well as with Pyr alone in ALPS patients (Table) lead us to conclude that these agents do not manifest sufficient effectiveness to warrant larger trials.

Fas is a cell surface protein of the tumor necrosis factor receptor, nerve growth factor receptor, CD40 family, and is involved in the control of lymphocyte apoptosis. A mutation in the Fas gene in MRL/lpr mice results in massive lymphoproliferation (lpr) and accelerated autoimmunity. To further study the nature of this defect, Fas mRNA expression was evaluated by reverse transcriptase polymerase chain reaction as well as by Northern blotting. These studies revealed that the wild-type Fas message was produced at approximately 10-fold lower levels in the lpr compared with the ++ substrain of MRL mice. In addition to the wild-type transcript, lpr mice also synthesized chimeric transcripts containing an insertion of the early retrotransposon (ETn). Molecular cloning and nucleotide sequencing of a Fas-ETn chimeric cDNA suggested that the striking reduction in wild-type Fas mRNA levels and the presence of aberrant transcripts in MRL/lpr mice are most likely explained by the insertion of the ETn retrotransposon into an intron of the Fas gene and induction of alternative splicing involving the 5' ETn long terminal repeat.

Hemopoietic cells have been reciprocally transferred between two lines of mice (MRL lpr/lpr and MRL +/+) that are congenic, differing only at the lpr (lymphoproliferation) and possibly closely linked genes. The lpr strain develops a significantly more severe and fast-paced lupus-like syndrome than +/+ strain, along with a substantially larger lymphoid mass. The results showed that: (a) hemopoietic cells of such mice were sufficient to induce the respective disease phenotypes in lethally irradiated syngeneic recipients; (b) cells of MRL +/+ mice maturing in an MRL lpr/lpr environment essentially retained the disease-producing characteristics of the donor, i.e., they induced late-life lupus without lymphadenopathy; but (c) MRL lpr/lpr cells transferred into irradiated MRL +/+ recipients unexpectedly failed to induce the early-life severe lupus and lymphoid hyperplasia of the donor, instead they caused a severe wasting syndrome resembling, in many respects, graft-vs.-host disease (GVHD). This GVHD-like syndrome developed after transfer of MRL lpr/lpr fetal liver, bone marrow, or spleen cells, and was not abrogated by elimination of T cells from the inocula. Thymectomy of the MRL +/+ recipients retarded, but did not prevent, the wasting disease. The unidirectional nature of this disease suggests that the lpr mutation conferred either a structural or regulatory defect that interfered, blocked, or altered the expression or structure of certain lymphocyte antigen(s). As a result, the MRL +/+ cells that did express this antigen(s) were recognized as foreign, and stimulated a graft-vs.-host reaction. These findings may allow definition of a new kind of rejection phenomenon caused by non-H-2 products, and may extend our understanding of the means by which the lpr gene adversely affects lymphocyte regulation and homeostasis.

Fas is a cell surface protein that mediates apoptosis. A mouse mutant, lpr (lymphoproliferation), has a mutation in the Fas gene. In this report, we studied the expression and function of Fas in various subpopulations of mouse thymocytes. Abundant expression of Fas was detected on CD4+CD8+ double positive as well as CD4+ or CD8+ single positive thymocytes in wild-type mice. Little or low levels of Fas were expressed in CD4-CD8- double negative thymocytes except for the CD4-CD8-CD3+ phenotype, which expresses Fas as abundantly as double positive or single positive subsets. On the other hand, no Fas expression was detected in any population of thymocytes from lpr mice. When the wild-type thymocytes were treated with the agonistic anti-Fas antibody, double positive cells from the wild-type mice were selectively killed by apoptosis, whereas, the single positive cells were resistant to its cytolytic activity despite their abundant expression of Fas. Unlike the apoptosis of thymocytes induced by glucocorticoid or T cell activator, the Fas-induced apoptosis of thymocytes was enhanced by metabolic inhibitors such as cycloheximide. Furthermore, intraperitoneal administration of the anti-Fas antibody into mice caused rapid apoptosis of thymocytes in vivo.

Abstract Abstract 4696 Autoimmune lymphoproliferative syndrome (ALPS) is a rare immunologic disorder caused by defects in the Fas-induced programmed cell death pathway. Impaired lymphocyte apoptosis results in gradual lymphocyte accumulation and dysregulation of lymphocyte homeostasis. ALPS patients usually suffer from persistent generalized lymphadenopathy, hepato-splenomegaly, immune-mediated cytopenias, and other autoimmune phenomena. A 15-month-old boy was diagnosed with ALPS when he presented with typical symptoms. Lab exams showed a pancytopenia, elevated serum immunoglobulin levels, a peripheral expansion of double-negative T lymphocytes of up to 40% of TCRab+ T cells and impaired lymphocyte apoptosis. Molecular analysis confirmed the diagnosis of a type Ia ALPS by identifying a heterozygous Fas gene mutation (D260H). Despite treatment with repeated pulses of high-dose methylprednisolone, intravenous immunoglobulins, and mycophenolate mofetil (MMF) to control lymphoproliferation and recurrent pancytopenia his disease progressed. At three years of age he developed numerous arterial aneurysms of the iliac, mesenterial, renal, hepatic, right middle meningeal, brachial and femoral arteries up to 2.6 cm in diameter and lymphoproliferation resulting in paraplegia and right arm paresis. Stem cell transplantation was considered, and BMT from a 9/10 matched unrelated donor (MUD) after a reduced intensity conditioning regimen (CR) with Fludarabin (150 mg/m2), Melphalan (140 mg/m2/d) and ATG (60mg/kg) was performed using 5.22 × 106 CD34+ cells/kg body weight. However, graft failure had to be diagnosed on day +27. 53 days after the first BMT a PBSCT from the same donor after myeloablative CR using Busulfan (19.2 mg/kg/d i.v.), Etoposid (30 mg/kg/d), Cyclophosphamid (120 mg/kg/d) and ATG (60mg/kg) was performed (24.5 × 106 CD34+ cells/kg bw). Again, graft failure was seen. At day +55 a third HSCT using PBSCT from another 9/10 MUD after CR with Fludarabin i.v. (160 mg/m2), Thiotepa (5 mg/kg/d), 4 Gy total body irradiation (TBI) and campath (1 mg/kg) was performed. 10.8 × 106 CD34+ cells/kg bw were given intra osseous, 9.8 × 106 CD34+ cells/kg bw were given i.v. Engraftment was slow (Leukos 980 day +35), but chimerism showed 99 % donor cells. Two years later the patient is alive and well, with persistent engraftment and good hematological and immunological function. Arterial aneurysms stopped growing and some have thrombosed. This case illustrates some interesting points. Atypical and unusually severe manifestations of ALPS forced us to perform a HSCT in this patient. In severe ALPS stem cell engraftment is difficult to achieve as previously reported in the Fas deficient lpr mouse model. One reason might be the reduced ability of cytotoxic drugs to induce apoptosis in the Fas deficient recipient T cells. Thus, recipient T cells could persist and kill donor cells resulting in graft failure or rejection. Additionally, increased FasL expression on recipient cells could induce apoptosis in Fas bearing donor stem cells as shown in lpr mice. Also, trapping of infused stem cells in the extremely enlarged liver and spleen could have played a role. A 3rd attempt was therefore designed to overcome graft resistance and proved finally successful. The use of TBI together with campath possibly induced more T cell apoptosis than chemotherapy alone. The intra osseous application probably increased engraftment efficiency by avoiding trapping of stem cells in liver and spleen and possibly by induction of tolerance by intra osseous application as described before. Disclosures: No relevant conflicts of interest to declare.

Abstract Hydroxychloroquine (HCQ) is an anti-malarial drug in clinical use for decades that is finding further use as a steroid sparing agent in the treatment of immune disorders such as chronic GVHD, lupus and rheumatoid arthritis. HCQ is a lysosomotropic agent with more recent evidence showing immunomodulatory anti-TNF activity. It is currently being explored as a cytotoxic antineoplastic/antimicrobial agent. Hence, studies were conducted to determine the efficacy of HCQ to control the lymphoproliferation associated with ALPS. ALPS is an inherited disorder of apoptosis leading to lymphoproliferation and autoimmunity. The majority of ALPS patients are classified as Type Ia (>70%), having germline mutations in Fas. Other ALPS patients are classified as Type Ib, II, IV or III, if they have mutations in FasL, casapases, NRAS, or no identified mutations, respectively. They often present with childhood onset autoimmune cytopenias with lymphadenopathy, splenomegaly, increased circulating double negative T cells (DNT; TCRa/b+CD3+CD4−CD8−), defective apoptosis in vitro, and have an increased risk of lymphoma. Similarly, MRL/lpr−/− mice homozygous for Fas mutations develop an ALPS-like disease with massive lymphadenopathy, splenomegaly, hypergammaglobulinemia, autoimmune glomerulonephritis, and expansion of DNT cells secondary to defective lymphocyte apoptosis leading to lymphomagenesis. Currently, there are no proven therapies for the lymphoproliferation underlying ALPS itself. PBMCs from normal controls and ALPS Type Ia patients were cultured in vitro with 0–120 ug/mL HCQ in the presence or absence of 50 uM of the pan-caspase inhibitor Z-VAD-FMK, the caspase 9 inhibitor Z-LEHD-FMK, or the caspase 8 inhibitor Z-IETD-FMK. A dose response was observed with a high degree of cell death noted at 120 ug/mL after 48 hours, with an LD50 of 40 ug/mL. HCQ induced cell death was through a caspase-independent mechanism based on no inhibition of cell death by Z-VAD-FMK, Z-LEHD-FMK, or Z-IETD-FMK. Further preclinical studies were conducted in the MRL/lpr−/− murine model of ALPS. Forty five, 8-week old female MRL/lpr−/− mice were treated with 30 or 60 mg/kg of HCQ by gavage in sterile PBS or PBS alone three days per week for up to 14 weeks. Reductions of the spleen weight (p = 0.045, 437±85 vs 240±27) and cellularity (p = 0.08, 217±32 vs 148±18) were observed in high dose HCQ treated mice compared to controls at 7 weeks. Reductions in cellularity were also noted in the lymph nodes (p = 0.032, 118±33 vs 38±6). There was a trend towards decreasing DNT percentages in the spleen (21±5 vs 14±2), LN (70±4 vs 67±2) and blood (57±6 vs 51±5). Additionally, the CBC showed only marginal reductions in the WBC count (4.2±0.5 vs 3.5±0.3), hematocrit (49±0.3 vs 46±1.0), polymorphonuclear cells (33±3.7 vs 29±3.5) and monocytes (23±3.2 vs 17±2.5). Future studies are planned to more clearly determine the effect of HCQ on autoimmune kidney disease. Based on our in vitro and in vivo data, HCQ is effective at reducing lymphoproliferative activity in Fas deficient MRL/lpr−/− mice. It is being further explored in early phase clinical trials as a lympholytic agent to shrink lymph nodes and abrogate hypersplenism in ALPS patients. HCQ may also prove beneficial for long term use, especially in children, as a steroid sparing agent for treating refractory autoimmune cytopenias in ALPS.

Abstract Programmed cell death (or apoptosis) is a physiological process essential to the normal development and homeostatic maintenance of the immune system. The Fas/Apo-1 receptor plays a crucial role in the regulation of apoptosis, as demonstrated by lymphoproliferation in MRL-lpr/lpr mice and by the recently described autoimmune lymphoproliferative syndrome (ALPS) in humans, both of which are due to mutations in the Fas gene. We describe a novel family with ALPS in which three affected siblings carry two distinct missense mutations on both the Fas gene alleles and show lack of Fas-induced apoptosis. The children share common clinical features including splenomegaly and lymphadenopathy, but only one developed severe autoimmune manifestations. In all three siblings, we demonstrated the presence of anergic CD3+CD4−CD8− (double negative, [DN]) T cells; moreover, a chronic lymphocyte activation was found, as demonstrated by the presence of high levels of HLA-DR expression on peripheral CD3+ cells and by the presence of high levels of serum activation markers such as soluble interleukin-2 receptor (sIL-2R) and soluble CD30 (sCD30).