Ia Mutant Research Articles

MOLECULAR SCREENING OF FOUR CANINE HEREDITARY DISEASES IN TAIWAN

Animals that have inherited a mutant gene from their parents (father and/or mother) may acquire hereditary diseases. Most mutations are autosomal recessive. Usually, hereditary diseases go unnoticed until symptoms arise. The objective of this study is to screen four canine hereditary diseases in Taiwan. The diseases include persistent Müllerian duct syndrome (PMDS) and myotonia congenita (MC) in Miniature Schnauzers, Golden Retriever muscular dystrophy (GRMD) in Golden Retrievers and glycogen storage disease type Ia (GSD Ia) in Maltese. We collected 98 samples from Miniature Schnauzers, 57 samples from Golden Retrievers and 54 samples from Maltese. All molecular tests are based on restriction fragment length polymorphism (RFLP), which consists of a genomic polymerase chain reaction (PCR) plus specific restriction enzyme digestion. Results showed that the 98 samples, which were collected from Miniature Schnauzers, were all MC wild type (WT), with 78 being PMDS WT and 20 (20%) being PMDS heterozygous type. None of the samples were MC mutant type (MT) or PMDS MT; on the other hand, no GRMD mutant gene or GSD Ia mutant gene was found in Golden Retrievers and Maltese samples.

Role of the sulfhydryl redox state and disulfide bonds in processing and assembly of 11S seed globulins.

Seed legumins contain two conserved disulfide bonds: an interchain bond (IE) connecting the acidic and basic chains and an intrachain bond (IA) internal to the acidic chain. Mutant subunits were constructed in which these disulfide bonds were disrupted. Oxidized glutathione stimulated the rate of assembly of trimers with unmodified prolegumin subunits. Stimulation was not detected during assembly of IE mutant subunits and was diminished for the IA mutant. Hexamer assembly with trimers of mature unmodified subunits required oxidizing conditions. Trimers composed of mature IE mutants did not form hexamers. Both mutant and non-mutant subunits accumulated in hexamers when the cDNAs were expressed in tobacco. Hexamer assembly in seeds probably involved trimers with a mixture of mutant and non-mutant subunits. Similarly, mixed trimers that were a mixture of mutant and non-mutant subunits assembled into hexamers in vitro. The results demonstrate the importance of disulfide bonds during the assembly of 11S globulins.

Abnormalities of phosphoprotein gene expression in three osteopetrotic rat mutations: elevated mRNA transcripts, protein synthesis, and accumulation in bone of mutant animals.

Osteoclast abnormalities that characterize osteopetrosis, a disorder of bone resorption, may derive from aberrant signals from the osteoblast or the bone matrix. In the present studies, both synthesis and the bone matrix content of the major bone phosphoprotein component, osteopontin, were found to be elevated in three osteopetrotic rat mutations (ia, op, and tl). In whole bone, a twofold increase in the content of the characteristic amino acid O-phosphoserine for osteopontin occurred in op and tl mutant long bone, but a smaller (15%) and more variable increase was observed in ia mutant rat long bone. Extraction of the bone matrix components and partial purification by reverse phase chromatography showed a twofold increase in a phosphoprotein fraction relative to other noncollagenous components. Amino acid analysis and staining characteristics of SDS-PAGE fractionated proteins indicated this to be osteopontin. Organ cultures of calvarial bone from 4 day ia osteopetrotic mutant and normal rats in the presence of 3H-proline showed increased synthesis of this 60 kD protein, which was stimulated by vitamin D. Preparation of total cellular RNA from bone of 2- and 6-week-old mutants and normal rats supported increased synthesis of osteopontin as reflected by hybridization with osteopontin cDNA probe, showing significantly higher levels of mRNA transcripts in ia (3-5 fold), tl (1.4-2 fold), and op (6-25 fold) mutant bone compared to normal littermates. The changes in osteopontin mRNA levels in mutant bone were also examined in relation to other growth and phenotype-expressed genes. The findings of increased accumulation of osteopontin in osteopetrotic bone and increased synthesis by osteoblasts are interesting in light of the previously reported decrease in bone osteocalcin content (Endocrinology, 126:966, 1990), confirmed here by decreased osteocalcin mRNA transcripts. Such aberrations in the composition of skeletal extracellular matrix could be a reflection of or a contributing factor to the osteoclast abnormalities of some of these osteopetrotic disorders.

Adoptive immunotherapy is suppressed in C57BL/6J AND B6.C‐H‐2bm12 mice following recognition of congenic class II MHC antigen determinants

In previous reports, we have shown that adoptive transfer of tumor-sensitized T (immune) cells to tumor-bearing mice that have received a prior injection of cyclophosphamide (CY) results in the induction of permanent tumor regression in syngeneic strains. It has also been shown that adoptive immunotherapy results in an increased expression of class II MHC antigens (Ia) by macrophages at the tumor site and in the peritoneal cavity and is associated with expansion of CD4+ and CD8+ T cells at the site of tumor regression. In this report, we use the Ia mutant strain, B6.C-H-2bm12, and congenic C57BL/6J (B6) mice to determine the relative importance of Ia expression in regulating amplification of immune responses following adoptive immunotherapy and to test the hypothesis that recognition of congenic Ia determinants will result in the induction of suppressor mechanisms that down-regulate active immunity. The data indicated that the adoptive transfer of immune congenic T cells (B6 immune cells into CY-treated tumor-bearing bm12 mice and vice-versa) down-regulated active immunity, while the transfer of syngeneic immune cells resulted in permanent tumor regression. By using radiation-chimeric mice, it was shown that down-regulation was associated with incompatibility of the transferred immune T cells and bone-marrow-derived cells (putatively expressing the Ia haplotype of donor-derived macrophages) and the appearance of long-lived splenic suppressor cells. Suppression per se was shown to be induced in response to the Ia difference between the two strains and not in response to the MCA/76-9 sarcoma, which appears to be one of the few tumors that can induce active immunity in both the syngeneic and congenic strains without obvious subsequent down-regulation by suppression.

Pluripotent hemopoietic stem cells give rise to osteoclasts

Osteopetrosis in the ia (incisors absent) rat is the result of reduced bone resorption due to abnormal osteoclasts. The mutant osteoclasts lack a ruffled border--the membrane specialization involved in osteolysis. Studies in the ia mutant have shown that when pluripotent hemopoietic stem cells from normal littermates are transplanted into ia recipients, normal osteoclasts are formed and the skeletal sclerosis is eventually cured. The present study was conducted to provide evidence for the mechanism of the cure. Do the transplanted stem cells provide a helper function, i.e. secrete soluble factor(s) which transform pre-existing osteoclasts, or do they fuse with each other or pre-existing osteoclasts, or do they fuse with each other or pre-existing osteoclasts to form functional osteoclasts? Using the procedures described by Gold-schneider and co-workers, and fluorescence-activated cell sorting (FACS), pluripotent hemopoietic stem cells were isolated from normal rat bone marrow, labeled with saturated FITC, and injected intravenously into irradiated ia rats. After 48 hr, the recipients' long bones were removed and split longitudinally, and the endosteal surface was scraped. The resulting cellular suspension containing osteoclasts was examined by phase contrast and fluorescence microscopy. Fluorescing mononuclear cells of donor origin that had homed to the bone marrow demonstrated moderate cytoplasmic fluorescence. Approximately 30% of the osteoclasts observed demonstrated light cytoplasmic fluorescence. When cellular pools incapable of curing osteopetrosis (thymocytes) were labeled and injected into ia recipients, no labeled osteoclasts were observed. These studies indicated that pluripotent hemopoietic stem cells, when transplanted into ia hosts, fuse with each other and differentiate into osteoclasts or fuse with pre-existing osteoclasts.

Characterization of a dominant anti-Ia idiotype using the Ia mutant mouse strain B6.C- H-2 bm12

Initial studies of antibody recognition of Ia molecules using the IA mutant mouse strain bm12 suggested that two anti-Ia monoclonal antibodies (mAbs), 25−9−17 and 34−5−3, share several features: (1) indistinguishable serologic specificity including a lack of reactivity with Ia bm12, (2) binding of the same spatial epitope (cluster), and (3) definition of a cross-reactive idiotype (CRI) as defined by xenogeneic antisera. In the present study we characterize a rabbit anti-idiotype (anti-Id) to 25−9−17 by affinity chromatography, and demonstrate that it detects at least two distinct idiotopes, one shared by 25−9−17 and 34−5−3 designated CRI (25−9−17) and one unique for 25−9−17 molecules. Experiments were also undertaken to determine whether CRI (25−9−17) represents a measurable component of allogeneic humoral responses to Ia b antigens. By both absorption analyses of a polyspecific antiserum and production of antigenically-restricted antisera using bm12 mice, CRI (25−9−17) was found to represent a significant proportion of the antibodies to la b. By several criteria it was shown that the CRI (25−9−17) + molecules were among the antibodies defining the serologic lesion of bm12 mice. In preparation for future studies to alter in vivo T-cell responses involving recognition of la (e.g. graft vs host disease and allogeneic transplant rejection), various immunization protocols and mouse strains were tested for induction of Id (25−9−17) following in vivo administration of various anti-idiotypic reagents. Rabbit anti-Id (25−9−17) successfully induced CRI (25−9−17) positive molecules in all strains tested regardless of IA or Ig genotype. Moreover, some of these treated mice produced antibodies to an la determinant missing on bm12 cells, suggesting that they recognize the same serologic determinant as mAb 25−9−17.

Cellular specificity of the cure for osteopetrosis: Isolation of and treatment with pluripotent hemopoietic stem cells

Osteopetrosis in the ia rat is the result of reduced bone resorption due to abnormal osteoclasts. Studies in the ia mutant have shown that mononuclear cells from normal littermates could cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia rats. Recent studies demonstrated that the Ficoll-Hypaque isolates of spleen, bone marrow, and newborn liver were effective in curing the skeletal disease. These results suggest that the cellular source of the cure is a pluripotent hemopoietic stem cell (PHSC). Goldschneider et al. (1980) demonstrated that stem cells from rat bone marrow relatively large mononuclear cells that are strongly positive for Thy 1.1 antigen. Splenic and bone marrow samples from normal rats were treated with a monoclonal antibody developed against the rat Thy 1.1 antigen and guinea pig complement. This procedure was cytotoxic for 47% of the mononuclear spleen cells and 74% of the mononuclear bone marrow cells. When the Thy 1.1 depleted samples were transplanted into ia littermates, they were ineffective in reversing the osteoclast and bone resorption defects. Using the parameters described by Goldschneider, and fluorescence-activated cell sorting (FACS), the PHSC were isolated from normal rat bone marrow. The isolated cells were assayed for colony forming units — spleen (CFU-S) and were found to contain approximately 65–70% PHSC. Two, 3, and 5-week-old ia rats were given an intraperitoneal injection of normal bone marrow stem cells (1.4 × 10 4 to 7.0 × 10 4 cells). The effects of treatment were evaluated by radiography, by measuring the size of the tibial marrow cavity, and by identifying active (phenotypically normal) osteoclasts at 3 weeks after transplantation. At all of the ages transplanted and at all of the cell numbers tested, the stem cell suspensions were effective in curing the osteopetrotic condition. These positive findings and the fact that the Thy 1.1 depleted cells were ineffective suggests that the PHSC is a cellular source of the cure in the ia mutation.

Immunological Competence in Osteopetrotic ( ia) Rats

Osteopetrosis in is rats is characterized by excessive skeletal mass and reduced bone resorption. The skeletal defects can be corrected by the transfer of mononuclear spleen cells from normal littermates. These studies suggest that osteopetrotic mutants may also have defective immune functions. The op, osteopetrotic, rat demonstrates early thymic atrophy and immune function which decreases with age. Several studies have shown significantly reduced responses to T and B cell mitogens by spleen cells from osteopetrotic mutant mice. The problem with these latter studies is that different populations of cells have been compared in mutants and normal littermates because the spleen is a focus of extramedullary hemopoiesis in osteopetrotic animals. To circumvent this problem, the Ficoll-Hypaque, mononuclear isolate of spleen and mesenteric lymph node from 5-week-old ia and normal littermates were compared. Under appropriate culture conditions the cells were exposed to Con A, PHA, and LPS for 3 days and 3H-thymidine for the last 24 hours. In all cases, the response to optimal concentrations of the 3 mitogens was similar for is and normal spleen and lymph node cells ( ia/control ratios ranged from 0.6 to 1.2). The cellular composition of the samples tested in the mitogen assays were also evaluated by fluorescent microscopy using FITC-conjugated monoclonal antibodies directed against specific cell surface markers. The percentage of B cells, macrophages, total T cells, and helper T cells were found to be similar in the Ficoll-Hypaque isolate of ia and normal spleen and lymph nodes. Likewise, the ia mutant does not show any signs of abnormal thymic involution. These results indicate normal immune function in the ia mutant when similar populations of cells are compared.

Selective loss of antigen-specific Ir gene function in IA mutant B6.C-H-2bm12 is an antigen presenting cell defect.

Immune responses to several soluble antigens were compared between B6.C-H-2bm12 mutant and wild-type B6 mice by using a lymph node T-cell proliferation assay. B6.C-H-2bm12 mice failed to respond to beef insulin whereas other IA gene-controlled responses, such as response to poly(L-Tyr, L-Glu)--poly(DL-Ala, L-Lys) and collagen, were indistinguishable between mutant and wild-type mice. The responses to multideterminant antigens such as ovalbumin and purified protein derivative of tuberculin were also found to be comparable in B6.C-H-2bm12 and B6 mice, thus indicating that this mutation resulted in a selective loss of the ability to respond to a certain antigen(s)--e.g., beef insulin. Populations depleted of adherent cells have been used to examine the mechanism by which Ia molecules mediate Ir gene control of antigen recognition. We show that the nonresponsiveness to beef insulin in the mutant mouse is the result of defective antigen presentation. In addition, we find that F1 hybrids between two nonresponders--B6.C-H-2bm12 and B10.A or B10.AKM (IAk) mice--become responders to beef insulin, thus demonstrating gene complementation. These findings taken together with other serologic and biochemical studies in the B6.C-H-2bm12 present convincing genetic evidence for the direct association of the A beta polypeptide chain of the Iab molecules with the expression of immune responsiveness to beef insulin. Study of the B6.C-H-2bm12 mouse should provide new insight into the cellular and molecular mechanisms by which Ir genes determine the nature of the immune response.

The role of lymphoid cells in bone resorption: cellular immunological competence in ia rats.

AbstractThe ia (incisors absent) rat is a naturally occurring mutant that has osteopetrosis resulting from reduced bone resorption. A number of in vestigators have reported that other osteopetrotic mutant rats (op) and mice (mi and op), in addition to demonstrating the skeletal manifestations of the disease, have cellular immunological defects. In light of the fact that bone resorption has been demonstrated to be controlled by hematopoietic tissues and more specifically by mononuclear cells isolated from lymphoid organs, it seemed appropriate to investigate immunological competence in the ia rat, to determine whether or not it had this defect in common with the op rat.Contrary to the results presented for other osteopetrotic mutants, we found that the thymuses in our ia mutants had a normal cellular composition and the T‐cell‐dependent zones in the lymph nodes were not deficient in lymphocytes. Therefore, this study was undertaken to investigate the response of ia rats to oxazolone, which produces one type of T‐cell‐dependent immune response, delayed hypersensitivity.The results of these studies indicate that ia rats at 18 days of age have the ability to develop delayed hypersensitivity, as shown by both a blastogenic response in the lymph node draining the site of sensitization and a positive skintest. The magnitudes of these responses were similar in ia and normal littermates, both histologically and quantitatively. Therefore, the ia mutant does not appear to have a defect of the thymus‐dependent immune system, and further studies should be carried out to investigate other parameters of immunological competence in these animals.