Detection of polymeric IgA in glomeruli from patients with IgA nephropathy.

A study on the detection of polymeric IgA in glomeruli from renal biopsy specimens in patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These specimens were stained with FITC-labelled anti-human J chain antisera and then examined with a fluorescent microscope. The J chain was observed in the glomerular mesangium by immunofluorescent staining. In parallel studies, renal biopsy specimens were treated with citrate buffer (pH 3.2) and the 'eluate' was neutralized by sodium hydroxide. The eluate was labelled with iodine-125, and the radiolabelled 'eluate' was fractionated by sucrose density-gradient ultracentrifugation. Polymerized IgA in the 'eluate' obtained from patients with IgA nephropathy was found to sediment predominantly as 9S to 11S using a sucrose density gradient analysis. Polymeric IgA in the fractions of the density gradient analysis was determined by anti-human IgA and anti-human J chain antisera. It was demonstrated that IgA and J chain were eluted from the glomeruli in some patients with IgA nephropathy. It is concluded that IgA deposited in the glomeruli is composed of dimers and/or larger polymers of circulating IgA in some patients with IgA nephropathy.     

Ontogeny of macrophage-mediated protection against Listeria monocytogenes.

We investigated the ontogenic development of macrophage functions which are important in the expression of host defense against infection by Listeria monocytogenes. Macrophage functions, including accumulation in response to local stimuli, chemotaxis in vitro, and intracellular killing, as well as number of macrophages, were examined by using mice 1, 2, 3, 4, and 8 weeks old. The number of peritoneal macrophages was extremely low in younger mice even when their body weights were taken into consideration. Macrophage accumulation in response to infectious stimulus with viable listeria was poor in younger mice and showed an age-dependent development. In younger groups, chemotaxis in vitro was as immature as chemotaxis in vivo. In 1- and 2-week-old mice, macrophages did not show any intracellular killing activity against L. monocytogenes, but killing was observed in mice over 3 weeks of age. These functions developed in an age-dependent manner and reached the 8-week-old adult level after the mice were 4 weeks of age. In adult mice, these macrophage functions were shown to be enhanced after immunization with viable listeria; however, such an immunization-induced enhancement was very poor in the younger groups of mice. Protection judged by mortality and in vivo bacterial growth was weaker in the younger groups against both primary and secondary challenges. In vivo protection against L. monocytogenes seemed to develop in the same age-dependent manner as the development of macrophage functions. These results indicate that age-dependent immaturity of macrophage functions mainly comprises the age-dependent immaturity of protection against L. monocytogenes.     

Molecular cloning and complete nucleotide sequence of the genome of Japanese encephalitis virus Beijing-1 strain

The genomic RNA of the Japanese encephalitis virus (JEV) Beijing-1 strain was reversely transcribed and the synthesized cDNA was molecularly cloned. Six continuous cDNA clones that cover the entire virus genome were established and sequenced to determine the complete nucleotide sequence of the JEV RNA. The precise genomic size was estimated as 10,965 bases long. With flanking 95 bases at the 5 and 583 bases at the 3 non-coding regions, one long open reading frame (ORF) was revealed encoding a virus polyprotein with 3,429 amino acid residues. Because of sequence homologies observed between JEV and other flaviviruses, the genome organization of JEV appears to be identical with other flaviviruses. Genetic variation detected among flavivirus genomes is consistent with the established serological relatedness between JEV and other members of flaviviruses. The secondary structure of the JEV genome is deduced and discussed concerning its involvement in genome replication.     

Characterization of the poliovirus 147S particle: new insights into poliovirus uncoating

A Sabin 1 strain poliovirus (PV) mutant, S1(2Y-1I), carrying a Tyr at amino acid position VP2(142) and an Ile at position VP1(160), can establish persistent infections in HEp-2c cells. This mutant forms atypical 147S particles upon interaction at 0 degrees C with either cells expressing PV receptor (PVR) CD155, or PVR-IgG2a, a chimeric molecule consisting of an extracellular moiety of PVR and the hinge and Fc portion of a mouse IgG2a. Upon interaction with PVR at 37 degrees C, S1(2Y-1I), similar to the parental strain, forms both 135S A particles and 80S empty capsids. At 0 degrees C, surprisingly, at a concentration equal to or greater than 5 nM, PVR-IgG2a induced both the extrusion of VP4 from the capsid of S1(2Y-1I) and the formation of 80S particles. The same transitions were observed at 0 degrees C with the parental strain Sabin 1 at 40 nM PVR-IgG2a. Thus, the formation of 80S particles and VP4 extrusion, considered as one of the steps of PV uncoating, can be temperature-independent at high PVR concentration. This implies that structural changes of the PV capsid occurred following adsorption at low temperature.     

Isolation of vaccine-derived type 1 polioviruses displaying similar properties to virulent wild strain Mahoney from sewage in Japan

Type 1, 2, and 3 vaccine-derived polioviruses were isolated from a sewage disposal plant located downstream of the Oyabe River in Toyama Prefecture, Japan, between October 1993 and September 1995. Neurovirulence was analyzed in 13 type 1 vaccine-derived strains, using mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC). Nine strains (69%) were estimated to have marked neurovirulence. Some of the neutralizing antigenic sites, temperature sensitivity, and plaque-forming ability of two virulent vaccine-derived poliovirus strains were similar to Mahoney strain. The neutralizing activity of human sera obtained after oral poliomyelitis vaccine (OPV) administration against one of the virulent vaccine-derived polioviruses was examined. Although all human sera showed sufficient neutralizing activity for the prevention of poliomyelitis by vaccine-derived poliovirus strains, a lower titer than that against Sabin type 1 strain was observed. Vaccination against virulent vaccine-derived poliovirus will be effective. However, the environmental presence of viruses that have properties similar to those Mahoney strain is a threat. The introduction of inactivated poliovirus vaccine (IPV), and well-maintained herd immunity, together with reinforced environmental surveillance is important for the final phase of the polio eradication program by the World Health Organization (WHO).     

Infection of Mycobacterium bovis bacillus Calmette-Guérin in antibody-mediated gamma delta T-cell-depleted mice.

To evaluate the hypothesis that gamma delta T cells participate in protective immunity against mycobacterial infection, we depleted gamma delta T cells from mice by administration of anti-T-cell receptor (TCR)gamma delta monoclonal antibody (mAb) and analysed protection against Mycobacterium bovis bacillus Calmette-Guérin (BCG). The gamma delta T-cell-depleted mice did not show any exaggerated bacterial multiplication compared with control mice. In contrast, alpha beta T-cell-depleted mice, which were administrated anti-TCR alpha beta mAb before BCG infection, showed a depressed protective immunity. These results suggest that gamma delta T cells are not essential for coping with a primary BCG infection.     

Inhibition of skin xenograft rejection by depleting T-cell receptor alpha beta-bearing cells without T-cell receptor gamma delta-bearing cells or natural killer cells by monoclonal antibody.

We compared the effects of in vivo administration of the anti-T-cell receptor (TCR) alpha beta monoclonal antibody (mAb) (H57-597) to those of the anti-CD3 mAb (145-2C11), with or without anti-NK1.1 mAb (PK136), on xenogeneic skin graft survival in mice. In anti-TCR alpha beta mAb-treated B6 mice, F344 rat skin grafts survived for about 54 days, whereas in anti-CD3 mAb-treated B6 mice with or without anti-NK1.1 mAb treatment grafts survived about 25 days. In anti-TCR alpha beta mAb-treated B6 mice, TCR alpha beta-bearing T-lymphocyte function was completely abrogated, although TCR gamma delta-bearing T-lymphocyte function was still intact on day 9. In the anti-CD3 mAb-treated mice, the functions of both types of T lymphocytes were completely abrogated. On day 32, when most of the skin xenografts had been rejected in the anti-CD3 mAb-treated mice, the functions of both T lymphocytes had recovered considerably, and could actually respond to F344 antigens. In contrast, the function of TCR alpha beta-bearing cells had only partially recovered in the anti-TCR alpha beta mAb-treated mice. Finally, natural killer (NK) activity in the anti-TCR alpha beta mAb-treated mice was intact on day 32, when rat skin grafts still survived. In contrast, NK activity in the anti-CD3 mAb plus anti-NK1.1 mAb-treated mice did not recover on day 32, when skin xenografts had already been rejected. These results suggest that TCR gamma delta-bearing T cells and NK cells by themselves, at least in the absence of TCR alpha beta-bearing T cells, do not mediate xenogeneic skin graft rejection in mouse/rat combinations.     

Poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing

The replication of type 1 poliovirus in 13 established human blood cell lines differing in the differentiation stage and cell lineage was investigated. Three T (CCRF-CEM, CCRF-HSB-2, and Molt-3) and three B (Raji, CCRF-SB, and RPMI 8226) cell lines showed no cytopathic effects (CPE) or virus production. CPE associated with virus production were detected in the other seven cell lines: HL-60, ML-1, and KG-1 (granulocytic lineage), U-937 and THP-1 (monocytic lineage), K-562 (erythroid lineage), and Molt-4 (T cell lineage). These susceptible cell lines greatly differed in the speed at which the CPE progressed. The progression of CPE was faster in relatively well-differentiated cell lines such as HL-60 and U-937, independently of the multiplicity of infection, than in less differentiated cell lines such as K-562, KG-1, and THP-1. Thus, for the same lineage, the speed at which CPE progressed became proportionally higher with subsequent differentiation stages. In the K-562 cell culture, CPE were not observed until at least 5 days postinfection (p.i.), while more than 80% of HL-60 cells were killed within 3 days p.i. There were no significant differences between infected HL-60 and K-562 cells in the efficiency of infection determined at 8 hr p.i. by the indirect immunofluorescent technique, the rate of virus growth, or the amount of viral capsid protein synthesized. This indicated that there were similar viral replication cycles in the two cell lines. These observations suggest that the killing function of the virus is expressed more slowly in K-562 cells than in HL-60 cells.     

Different expression of T-cell receptor beta-chain variable region genes in lymph nodes of lpr mice with different alleles of the major histocompatibility complex.

In order to search for a possible role of abnormally proliferating T cells in developing autoimmune disease in lpr mice, and to define the difference of the T cells among various lpr-congeneic mice with different clinicopathological findings, the T-cell receptor (TcR) V beta gene expression in the enlarged lymph nodes (LN) of C3H/HeJ-lpr/lpr (C3H-lpr), C57BL/6-lpr/lpr (B6-lpr) and MRL/Mp-lpr/lpr (MRL-lpr) mice was analysed. A RNA blot analysis using several V beta-specific probes showed that the V beta 3 gene, whose products are important for recognizing Mlsb/a, was used in B6-lpr and MRL-lpr with the Mlsb/b but not in C3H-lpr with the Mlsb/a. The V beta 5 gene, which is selectively related to I-E molecules, was predominantly used in B6-lpr(I-E-) but not in C3H-lpr(I-E+) nor MRL-lpr(I-E+). Similarly, the V12 gene was also expressed in B6-lpr but not in C3H-lpr. To compare in detail in V beta repertoire among lpr mice with different major histocompatibility complex (MHC) backgrounds, the V beta gene sequences in the cDNA libraries from LN cells of C3H-lpr were analysed, following the recent investigation of B6-lpr mice (Ohga et al., 1989). Eleven beta-chain cDNA out of 32 beta cDNA in B6-lpr and 24 beta-chain cDNA out of 55 beta cDNA in C3H-lpr were found to contain sequences with open reading frames that potentially encode functional TcR beta-chain. The frequencies of the messages in the cDNA libraries from these mice were consistent with the RNA blot analysis using V beta 3- and V beta 5-specific probes. It was notable that 36% of the functional beta-chain mRNA in B6-lpr and 50% of the beta mRNA in C3H-lpr expressed the V beta 8 gene family. When the TcR V beta gene expression was compared between the LN cells in C3H-lpr, B6-lpr and MRL-lpr, as reported by Singer et al. (1986), the usage of V beta genes other than the V beta 8 gene family in B6-lpr (H-2b) LN cells differed significantly from those in C3H-lpr (H-2k) and MRL-lpr (H-2k). The results presented here indicate that the usage of V beta genes is heavily influenced by the genetic background of lpr mice, similar to normal mice, but with preferential usage of the V beta 8 gene family as a common structural feature in lpr gene-induced cell populations.