Strong immunogenicity of a multicomponent peptide vaccine developed with the branched lysine oligopeptide method for human immunodeficiency virus infection

We synthesized one V3 peptide each from HTLV-III_B, Thai A and Thai B, conjugating then to the T cell epitope of the env region, and we also synthesized a p17 protein peptide of the gag region (HGP-30). These peptide were then coupled to 8-lysine copolymers using N-succinimidyl maleimido carboxylate (M_r = ca 60 000). We designated this the branched lysine oligopeptide method. The large peptide complexes constructed from these four macromolecular peptide were used with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay showed high titres of anti-peptide antibodies to each V3loop peptide and the HGP-30 peptide. Strong inhibition of CD4⁺ dependent cell fusion was obtained with these antisera when III_b, Thai A and Thai B strains of human immunodeficiency virus (HIV) were used. Strong anti-fusion inhibition was also observed with two other HIV strains. In addition, an increase of the anti-HIV effect was observed when we used sera obtained by multicomponent vaccine immunization. The same kind of inhibition was also observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in mice immunized with this vaccine. These results suggest that immunization with macromolecular peptide complexes can result in strong immunogenicity towards HIV-1.     

Bone marrow analysis of the myelodysplastic syndromes: histological and immunohistochemical features related to the evolution of overt leukemia

Bone marrow trephines from 31 patients with an initial diagnosis of myelodysplastic syndromes (MDS) were examined and analyzed histologically and immunohistochemically. In those cases terminating in overt leukemia (6/31, 19%), the number of bone marrow mast cells was significantly reduced, compared with those which did not evolve to overt leukemia. The bone marrow lymphoid cells that may participate in immunosurveillance against the proliferation of blast cells were also significantly reduced in cases terminating in overt leukemia. However, S-100 protein-positive cells, which include histiocytes and suppressor T-cells, were increased in cases terminating in overt leukemia. The results indicated that examination of the bone marrow to determine the proportions of mast cells and lymphoid cells which may be involved in host defense systems may be useful in predicting the evolution to overt leukemia in MDS. In the present series, patients with a hypocellular marrow (5/31, 16%) did not progress to overt leukemia and had a significantly lower bone marrow reticulin content, a significantly lower megakaryocyte count, a relatively higher mast cell count and a significantly higher lymphoid cell count than those with a normocellular or hypercellular marrow. These findings may reflect the initial features of MDS or, possibly, that hypocellular MDS is an independent entity with a low potential for blastic proliferation.     

Production and characterization of murine monoclonal anti-human DNase II antibodies,and their use for immunoaffinity purification of DNase II from human liver and urine

Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.     

Second analysis of mortality of nuclear industry workers in Japan, 1986-1997

IA cohort study of nuclear industry workers was initiated in 1990 to determine the possible health effects of low-level radiation. A total of 5,527 deaths were ascertained among 176,000 male workers who had been retrospectively and/or prospectively followed for an average of 7.9 years during the observation period 1986-1997. Statistical analyses were made mainly on the prospective follow-up outcome of 120,000 workers followed for an average of 4.5 years. The standardized mortality ratio (and its 95% confidence interval) was 0.94 (0.90, 0.97) for 2,934 cases of all causes combined and 0.86 (0.82, 0.91) for 1,305 cases of non-malignant diseases combined, which suggested a healthy worker effect. For 1,191 cases of all cancers combined, it was 0.98 (0.93, 1.04), indicating no difference in mortality from that of the general population. In tests for trend of death rate with increasing radiation dose, no significant correlation was found for all cancers combined. For site-specific cancers, most cancers including leukemia showed no positive correlation with dose, except for cancers of the esophagus, stomach and rectum and multiple myeloma. External causes showed a significant correlation with dose. A separate questionnaire study indicated that these positive findings could be ascribed in part to lifestyle characteristics of the workers. For leukemia only, we attempted to estimate the excess relative risk per unit dose of radiation, which, with reservations because of its wide confidence interval, was within the range of variation of the risks reported in other radiation epidemiological studies. This population must be studied for a longer time and with a consideration of the possible effects of confounding factors.     

Reducing sludge production and the domination of Comamonadaceae by reducing the oxygen supply in the wastewater treatment procedure of a food-processing factory

Sludge production was reduced remarkably by reducing the dissolved oxygen supply to less than 1 mg/l in the conventional wastewater treatment procedure of a food-processing factory that produced 180 m(3) of wastewater of biochemical oxygen demand (BOD) of about 1,000 mg/l daily. DNA was extracted from the sludge and subjected to PCR amplification. The PCR product was cloned into a plasmid and sequenced. Estimation of the resident bacterial distribution by 16S rDNA sequences before and after improvement of the system suggested a remarkable gradual change in the major bacterial population from Anaerolinaeceae (15.6%) to Comamonadaceae (52.3%), members of denitrifying bacteria of Proteobacteria. Although we did not directly confirm the ability of denitrification of the resulting sludge, a change in the major final electron acceptors from oxygen to nitrate might explain the reduction in sludge production in a conventional activated sludge process when the oxygen supply was limitted.     

Association of cerebrospinal fluid anti-Sm antibodies with acute confusional state in systemic lupus erythematosus

Introduction

Neuropsychiatric manifestation in systemic lupus erythematosus (NPSLE) is one of the most serious complications of the disease. Previous studies revealed the strong association between serum anti-Sm and organic brain syndrome, consisting mainly of acute confusional state (ACS) of diffuse psychiatric/neuropsychological syndromes (diffuse NPSLE). However, the precise mechanism by which anti-Sm causes diffuse NPSLE remains unclear. Of note, recent studies demonstrated that anti-U1 RNP antibodies (anti-RNP) in cerebrospinal fluid (CSF) are associated with NPSLE. The present study was designed to explore the association of anti-Sm antibodies in CSF with NPSLE.

Methods

Paired serum and CSF specimens were obtained from 72 patients with NPSLE (49 with diffuse NPSLE, 23 with neurological syndromes or peripheral neuropathy (focal NPSLE) and from 22 control patients with non-SLE neurological diseases. Sera were also obtained from 41 patients with active SLE without neuropsychiatric manifestations (non-NPSLE). Anti-Sm and anti-RNP were measured by enzyme-linked immunosorbent assay (ELISA). Blood-brain barrier (BBB) function and intrathecal anti-Sm production were evaluated by Q albumin and CSF anti-Sm index, respectively. Binding of anti-Sm to neuroblastoma cell lines SK-N-MC and Neuro2a was examined by flow cytometry and by cell ELISA.

Results

Anti-Sm and anti-RNP in CSF and sera were elevated in NPSLE compared with non-SLE control. CSF anti-Sm, but not CSF anti-RNP, was significantly elevated in ACS compared with non-ACS diffuse NPSLE or with focal NPSLE. By contrast, there were no significant differences in serum anti-Sm or anti-RNP among subsets of NPSLE and non-NPSLE. Whereas there were no significant differences in CSF anti-Sm index, Q albumin was elevated in ACS compared with non-ACS or with focal NPSLE. Notably, CSF anti-Sm was correlated with Q albumin (r = 0.2373, P = 0.0447) or with serum anti-Sm (r = 0.7185, P <0.0001) in 72 patients with NPSLE. Finally, monoclonal anti-Sm and purified human anti-Sm bound to the surface of SK-N-MC and Neuro2a.

Conclusions

These results demonstrate that the elevation of CSF anti-Sm through transudation from systemic circulation due to damaged BBB plays a critical role in the pathogenesis of ACS. More importantly, the data indicate that anti-Sm is yet another autoantibody with presumed neural toxicity, but might not be the last.     

Blood-brain barrier damages and intrathecal synthesis of anti-N-methyl-D-aspartate receptor NR2 antibodies in diffuse psychiatric/neuropsychological syndromes in systemic lupus erythematosus

Introduction

Although neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the recalcitrant complications of the disease, its pathogenesis still remains unclear. Previous studies revealed that antibodies reactive with NMDA (N-methyl-D-aspartate) receptor NR2 (anti-NR2) are elevated in cerebrospinal fluid (CSF) of patients with diffuse psychiatric/neuropsychological syndromes (diffuse NPSLE), which is usually more recalcitrant than neurologic syndromes of NPSLE (focal NPSLE). Two mechanisms have been implicated for the elevation of CSF IgG, including intrathecal synthesis and transudation through the damaged blood-brain barrier (BBB). The present study was designed in order to elucidate the roles of BBB function and intrathecal synthesis of anti-NR2 in the elevation of CSF anti-NR2 with regard to the severity in NPSLE.

Methods

Paired serum and CSF samples were obtained from 81 systemic lupus erythematosus (SLE) patients when they presented active neuropsychiatric manifestations, and from 22 non-SLE control patients with non-inflammatory neurological diseases. The 81 SLE patients consisted of 55 patients with diffuse NPSLE, including 23 patients with acute confusional state (ACS), the severest form of diffuse NPSLE, and 26 patients with neurologic syndromes or peripheral nervous system involvement (focal NPSLE). IgG anti-NR2 and albumin were measured by ELISA. BBB function and intrathecal synthesis of anti-NR2 were evaluated by Q albumin and by CSF anti-NR2 index, respectively.

Results

CSF anti-NR2 levels, Q albumin and CSF anti-NR2 index were significantly higher in NPSLE than in non-SLE control. CSF anti-NR2 levels and Q albumin were significantly higher in ACS than in non-ACS diffuse NPSLE (anxiety disorder, cognitive dysfunction, mood disorder and psychosis) or in focal NPSLE, whereas there was no significant difference in CSF anti-NR2 index among the 3 groups. CSF anti-NR2 levels were significantly correlated with Q albumin in diffuse NPSLE (r = 0.3754, P = 0.0053).

Conclusions

These results demonstrate that the severity of BBB damages plays a crucial role in the development of ACS, the severest form of diffuse NPSLE, through the accelerated entry of larger amounts of anti-NR2 into the central nervous system.     

Combinatorial Contextualization of Peptidic Epitopes for Enhanced Cellular Immunity

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our “motif-programming” approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund''s adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.     

The development of structure and function in chloroplasts of greening mutants of Scenedesmus III. Biosynthesis of {delta}-aminolevulinic acid

Cells of the mutant C-2A' of Scenedesmus obliquus which requirelight for chlorophyll formation were assayed for in vivo activityof ALA synthesis. In general, ALA and chlorophyll syntheseswere coupled during the greening process. The action spectrafor ALA and chlorophyll syntheses both show the highest activitiesin the blue region, but were different in details. Under certainconditions, ALA synthesis occurred without a corresponding synthesisof chlorophyll. Reasons for these variances were discussed. The controlling action of light on ALA synthesis may occur throughthree different, but related, mechanisms. The principle mechanismappeared to be linked to lightenhanced respiration since itsinhibition by cycloheximide blocks ALA synthesis. The Hill coefficientof this inhibition is 2. After the light-induced enhancementof respiration had ceased, the Hill-coefficient of inhibitionof ALA synthesis became 1. Thus, in addition to enhanced respiration,ALA formation depends on its sensitizing enzyme having a half-lifetime of less than 1 hr. Finally, the dependence of the synthesisof ALA precursors on light was evident. ¹ On leave from the Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. (Received November 11, 1974; )