B-cell repertoire specific for an unfolded self-determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.     

Bone formation using novel interconnected porous calcium hydroxyapatite ceramic hybridized with cultured marrow stromal stem cells derived from Green rat

The clinical use of cultured marrow stromal stem cells (MSCs) has recently attracted attention in the field of tissue engineering. For the clinical use of the MSCs, a prominent scaffold is needed. A scaffold hybridized with MSCs is transformed into a "bioactive bone substitute," and this provides good osteoconduction. In this study, a novel calcium hydroxyapatite ceramic with an interconnected porous structure (IP-CHA) was used as a scaffold. MSCs were harvested from Green rats containing Green Fluorescent Protein (GFP), and then these hybrids were implanted into the tibias of Sprague-Dawley rats. The purposes of this study were to examine the osteogenic ability of these hybrids without coculture, and to evaluate whether the resulting bone formation originated from the grafted MSCs or the recipient's cells. The hybridized group showed excellent bone formation compared with the IP-CHA-only implant group. Observation of the implanted MSCs revealed that they survived 8 weeks after surgery, and differentiated into osteoblast-like cells, thus providing bone formation. This implantation of the MSCs/IP-CHA composite provides excellent osteoconduction, and is expected to have extensive clinical applications.     

Type IV collagen alpha 5 chain. Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody.

Although the evidence indicates that mutation of the gene for the alpha 5 chain of type IV collagen, alpha 5-(IV), is the primary defect in X-linked Alport syndrome, protein data for the alpha 5(IV) chain with regard to its normal distribution and its distribution in patients with Alport syndrome is lacking. We produced a rat monoclonal antibody (H51) by immunizing rats with a synthetic peptide corresponding to the nonconsensus amino acid sequence of alpha 5(IV) NC1 domain. H51 reacted by Western blotting with 26-kd cationic monomers and associated dimers of human type IV collagen NC1 domain. Immunohistochemical studies demonstrated that in normal human kidney alpha 5(IV) was present in the glomerular basement membrane and basement membranes of the Bowman's capsule and in some tubules (collecting ducts). The alpha 5(IV) chain was also detected in the basement membranes of normal skin, eye, and lung. Male patients with X-linked Alport syndrome revealed no reactivity of renal and epidermal basement membranes with H51, whereas alpha 5(IV) staining was normal in the glomerular basement membrane of patients with other types of glomerular diseases, including benign familial hematuria. The staining was also normal in the skin of nonaffected males in X-linked Alport families. Female heterozygous for Alport syndrome exhibited a discontinuous or mosaic pattern in the immunofluorescent staining of the epidermal basement membrane. These findings confirm that in patients with X-linked Alport syndrome there are abnormalities in alpha 5(IV) in renal and epidermal basement membranes at the protein level. Immunofluorescent staining of skin biopsies with this antibody may be of value in making a diagnosis of Alport syndrome, and, furthermore, may aid in detecting carrier females in whom urinary abnormalities are often mild or silent.     

Clinical significance of antibodies to nonstructural and core proteins of hepatitis C virus in posttransfusion hepatitis patients during long-term follow-up

The prevalence of hepatitis C antibodies (anti- HCV) among multitransfused patients was studied and compared with predicted values obtained from a post-transfusion hepatitis study and from data on the prevalence of anti-HCV among blood donors. The prevalence of hepatitis B core antibodies (anti-HBc) was also studied to determine the routes of transmission of hepatitis C virus. The patients consisted of 65 dialysis patients (57 on haemodialysis and 8 on continuous ambulatory peritoneal dialysis) and 71 leukemia patients in long-term remission [49 with acute myeloid leukemia (AML) and 22 with acute lymphatic leukemia (ALL)]. The presence of anti-HCV was investigated using a second generation enzyme-linked immunosorbent assay. Reactive samples were confirmed by a second generation recombinant immunoblot assay. Anti-HBc was studied in the 65 dialysis patients and in 40 of the leukemia patients. Three (4.6%) of the 65 dialysis patients and 12 (24.5%) of the 49 AML patients were anti-HCV positive whereas all of the ALL patients were seronegative. The total number of blood units transfused to 134 patients (data on two dialysis patients were not available) was 18,148, out of which 17,575 units had been transfused prior to the initiation of anti- HCV screening of blood donors. On the basis of the anti-HCV prevalence among blood donors and the incidence of post-transfusion hepatitis, the predicted number of seropositive patients was 11 and 18, respectively. Five of the 65 dialysis patients were anti-HBc positive, compared with only one of the 40 leukemia patients. It is concluded that the anti-HCV prevalence among dialysis and leukemia patients is concordant with the risk of receiving contaminated blood products, whereas hepatitis B infection may have other routes of transmission in dialysis patients. © 1993 Wiley-Liss, Inc.     

Immunogenic Deposition and Destruction of Glomerulus in IgA Nephritis

The correlation between glomerular immune deposition and histological alteration was studied in serial paraffin sections of kidney biopsy specimens from patients with IgA nephritis using the peroxidase antiperoxidase (PAP) method. IgA deposition was detected in preserved glomerular extracellular matrix or in newly formed interposed matrix along the capillary loops (mesangial interposition). On the other hand, deposition of IgA was scanty or absent in the distorted extracellular matrix where severe exudative inflammation or extracapillary escape of exudates was occurring segmentally. Ultrastructurally such extracellular matricial destruction was expressed as splitting or thinning of the lamina densa, as well as mesangial edema, reticulation or mesangiolysis of the axial matrix. Therefore it appears that at least two types of change in the extracellular matrix are induced by IgA immune complexes in IgA nephritis; immunogenic deposition and destruction.     

The Origin and Evolution of Human T-Cell Lymphotropic Virus Types I and II

Studies on human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) are briefly reviewed from the viewpoint of molecular evolution, with special reference to the evolutionary rate and evolutionary relationships among these viruses. In particular, it appears that, in contrast to the low level of variability of HTLV-I among different isolates, individual isolates form quasispecies structures. Elucidating the mechanisms connecting these two phenomena will be one of the future problems in the study of the molecular evolution of HTLV-I and HTLV-II.     

Localization of a tumor suppressor gene associated with progression of human prostate cancer within a 1.2 Mb region of 8p22-p21.3

Human prostate cancers frequently show loss of heterozygosity at loci on the short arm of chromosome 8. In order to take a step toward isolation of the putative tumor suppressor gene(s) on 8p via positional cloning, we performed high-resolution deletion mapping in 46 prostate cancers (stage B, 20 cases; stage C, 8 cases; endocrine therapy-resistant cancer death, 18 cases) using new 12 restriction fragment length polymorphism markers for this chromosomal region. Allelic losses were observed in 25 of the 44 cases (57%) that were informative with at least one locus. Detailed deletion mapping defined a 1.2 Mb commonly deleted region at 8p22-p2 1.3 flanked by markers cMSR-32 and C18- 105 1. A second region of common deletion was identified between C18-1312 and C18-494 at 8p21-8p11.22, suggesting that at least two tumor suppressor genes associated with prostate cancer are present on chromosome arm 8p. Allelic losses on 8p were observed more frequently in the cancer death cases (14/17, 82%) than in early-stage tumors (11/27, 40%; P < 0.01, Fisher's exact test). In two out of 7 patients for whom DNA was available from metastatic cancers as well as from normal tissues and primary tumors, the primary cancer foci had no detectable abnormality of 8p, but the metastatic tumors showed loss of heterozygosity. These results suggest that inactivation of tumor suppressor genes on 8p plays an important role in the progression of prostate cancer. © 1995 Wiley-Liss, Inc.