Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy

Soluble human interleukin-6 receptor (sIL-6R) was measured in the serum of 30 healthy individuals, 32 individuals with monoclonal gammopathy of undetermined significance (MGUS), 20 patients with early multiple myeloma (MM) and 54 patients with overt MM. The serum activity recognized by an immunoradiometric assay was determined to be sIL-6R, because of its binding capacity to IL-6 and its molecular mass of 55 kDa. All sera of healthy individuals contained sIL-6R (mean value: 89 ng/ml, range 17-300 ng/ml). Serum sIL-6R levels were increased by 51% in patients with MGUS (mean value: 135 ng/ml, p<0.005), by 44% in patients with early myeloma (mean value: 128 ng/ml, p<0.001) and by 116 % in patients with overt MM (mean value: 193 ng/ml, p<0.001). In patients with MM, a complete lack of correlation (p>0.7) was found between serum sIL-6R levels and other previously recognized prognostic factors in this disease, particularly serum IL-6 levels and those factors related to tumor cell mass. The independence of serum sIL-6R levels on tumor cell mass was directly demonstrated by studying four patients with MM treated with autologous bone marrow transplantation for periods of between 320 and 760 days. These levels were found to be remarkably stable and constant, independent of whether patients relapsed or achieved complete remission. Finally, physiological concentrations of sIL-6R were found to increase by tenfold the sensitivity of human myeloma cell lines to IL-6. These observations suggest a high control of the sIL-6R level in vivo, and, possibly, an important functional role of this circulating protein in patients with monoclonal gammopathies.     

The role of basophils and mast cells in cutaneous basophil hypersensitivity reaction.

Basophils are the main cell component in cutaneous basophil hypersensitivity (CBH) reactions, but the role of basophils and the factors which gather them into CBH reaction sites are unknown. To investigate these problems, we induced CBH reactions in guinea pigs and observed basophils and mast cells in the skin reaction sites using light and electron microscopy. Basophils infiltrated into CBH reaction site appeared at 5 h after the challenge with antigen, increased till 48 h and decreased thereafter. On the other hand, the number of mast cells and their granules decreased after the challenge with antigen and reached a minimum at 48 h, but recovered at 96 h. The changes in the number of basophils and mast cells were complementary. This result suggested that the granules of mast cells may have a factor to gather basophils into the CBH skin reaction site. Furthermore, basophils infiltrated in the CBH reaction site were degranulated by the rechallenge with antigen, which was considered to be by an anaphylactic reaction.     

Altered mRNA splicing of the skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a CTG repeat expansion in the DMPK gene. Aberrant splicing of several genes has been reported to contribute to some symptoms of DM1, but the cause of muscle weakness in DM1 and elevated Ca2+ concentrations in cultured DM muscle cells is unknown. Here, we investigated the alternative splicing of mRNAs of two major proteins of the sarcoplasmic reticulum, the ryanodine receptor 1 (RyR1) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 1 or 2. The fetal variants, ASI(-) of RyR1 which lacks residue 3481-3485, and SERCA1b which differs at the C-terminal were significantly increased in skeletal muscles from DM1 patients and the transgenic mouse model of DM1 (HSA(LR)). In addition, a novel variant of SERCA2 was significantly decreased in DM1 patients. The total amount of mRNA for RyR1, SERCA1 and SERCA2 in DM1 and the expression levels of their proteins in HSA(LR) mice were not significantly different. However, heterologous expression of ASI(-) in cultured cells showed decreased affinity for [3H]ryanodine but similar Ca2+ dependency, and decreased channel activity in single-channel recording when compared with wild-type (WT) RyR1. In support of this, RyR1-knockout myotubes expressing ASI(-) exhibited a decreased incidence of Ca2+ oscillations during caffeine exposure compared with that observed for myotubes expressing WT-RyR1. We suggest that aberrant splicing of RyR1 and SERCA1 mRNAs might contribute to impaired Ca2+ homeostasis in DM1 muscle.     

New criteria for immunofluorescence assay for Q fever diagnosis in Japan

A study was made to evaluate the cutoff value of indirect immunofluorescent-antibody (IFA) test for Q fever diagnosis in Japan. We used 346 sera, including 16 from confirmed Q fever cases, 304 from Japanese pneumonia patients, and 26 from negative cases. Thirteen sera from the confirmed Q fever cases with an immunoglobulin M (IgM) titer of > or =1:128 and/or IgG titer of > or =1:256 by the IFA test were positive by both enzyme-linked immunosorbent assay (ELISA) and Western blotting assay (WBA), whereas 298 sera from pneumonia patients and 26 negative sera with an IgM titer of < or =1:16 and an IgG titer of < or =1:32 by the IFA test were negative by both ELISA and WBA. In the proposed "equivocal area," with an IgM titer of > or =1:32 and < or =1:64 and/or an IgG titer of > or =1:64 and < or =1:128, we found 9 sera, 3 from confirmed Q fever cases and 6 from Japanese pneumonia patients, by the IFA test. Three sera from the confirmed Q fever cases and one of the sera from pneumonia patients were IgM and/or IgG positive by both ELISA and WBA. These results suggest that a single cutoff value for the IFA test may cause false-positive and false-negative results. In conclusion, this study showed that an "equivocal area" should be used for the IFA test rather than a single cutoff value and that sera in the equivocal area should be tested by additional serological assays for confirmation.     

Immune responses in aged mice: changes of antibody-forming cell precursors in antigen-reactive cells with ageing

The depressed immune response to sheep erythrocytes in young syngeneic irradiated recipients, adoptively restored with splenic cells from old donor mice, was used as an index of the changes in the immunocompentent cells with ageing. The numbers of antibody-forming cell precursors in both spleen and bone marrow appeared to be diminished with ageing. Some reduction of antigen-reactive cells was also observed in old spleens.     

The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor

Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin(-)CD19(-)c-kit(+)IL-7Ralpha(+)AA4.1(+), the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1(-/-) embryos but not in IL-7(-/-) embryos. Lin(-) T cell progenitors appeared normal in SDF-1(-/-) embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.     

Characterization of Chlamydia pneumoniae species-specific proteins immunodominant in humans.

Proteins of Chlamydia pneumoniae immunodominant in humans were characterized with the sera of 13 patients who were not likely to have been exposed to C. trachomatis or C. psittaci. The serological responses among these patients were similar on a qualitative basis, but some differences were found quantitatively. However, the serological responses of the patients who were infected with C. pneumoniae differed markedly from those of two patients who were infected with C. trachomatis and two who were infected with C. psittaci and those of mice that were transtracheally infected with C. pneumoniae. Among proteins immunodominant in the patients who were infected with C. pneumoniae, a 40-kDa major outer membrane protein was genus specific and 53-, 46-, and 43-kDa proteins were species specific in their reactions with the majority of the human sera used. A few sera reacted strongly with a 73-kDa protein genus specifically. Some proteins with weak immunogenicity exhibited species specificity. An antigenic analysis with human sera and murine monoclonal antibodies against the 53-kDa protein showed that hte antigenicities were strictly conserved among the seven strains of C. pneumoniae tested. The genus-specific 73-kDa protein was solubilized with octylglucoside. All of the species-specific immunodominant proteins were solubilized with sodium dodecyl sulfate, but the genus-specific major outer membrane protein was not. These results suggest that a serological diagnosis of C. pneumoniae infection could be achieved species specifically by comparison of the serum responses to sodium dodecyl sulfate- and octylglucoside-soluble fractions.     

Mechanism of the formation of megamitochondria by copper-chelating agents. V. Further studies on isolated megamitochondria.

Effect of cuprizone has been studied on some biochemical properties of megamitochondria obtained from the mouse liver. (1) Contents of Ca2+, Mg2+ and Cu2+ in the blood or the liver homogenates were not altered by cuprizone intoxication, whereas those in liver mitochondria were significantly altered: after 3-4 days' intoxication, content of Ca2+ was decreased and was remarkably increased after 14-15 days' intoxication. Content of Mg2+ behaved contrarily. (2) Both cytochrome oxidase and ATPase activities were unchanged in the liver megamitochondria, but monoamine oxidase (MAO) activity was significantly decreased. Value of I50 (50% inhibition) for MAO was determined to be 0.33 mM using the control liver mitochondria. Cuprizone had almost no effect on MAO activity of kidney or heart mitochondria both in vivo and in vitro. (3) The amount of lysolecithin was increased in the liver megamitochondria. These results were discussed in the light of membrane fusion phenomenon which plays a key role in the mechanism of megamitochondrial formation.     

Zonation of the adrenal cortex. III. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex.

A microsomal fraction was obtained from the zona glomerulosa of the bovine adrenal cortex. Glucose-6-phosphate activity of the fraction was found to be much lower than that of the liver. Contents of RNA and phospholipids, besides electron microscopic findings, of the fraction also indicate that it is rich in smooth-surfaced endoplasmic reticulum. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including the microsomal fraction described above. The amount of cytochrome P-450 in mitochondria and that in microsomes were determined to be 0.73 and 0.32 nmoles/mg protein, respectively. The CO-difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40-50% of cytochrome P-450 in the samples were converted to cytochrome P-420 within 20-30 seconds of incubation with deoxycholate.     

Fat of Coxiella burnetti in macrophages from immune guinea pigs.

The interaction between Coxiella burnetii and peritoneal macrophages obtained from immune guinea pigs was studied by transmission electron microscopy. Phagocytosis and subsequent fate of ingested phase I and II rickettsiae were compared. Phase I rickettsiae were more resistant to phagocytosis than were phase II organisms. Macrophages from phase I- and II-immunized animals were equally capable of phagocytizing rickettsiae. Phase I and II rickettsiae previously treated with normal serum multiplied and destroyed macrophages from guinea pigs that had been immunized with phase II rickettsiae. Phase II organisms were initially suppressed in macrophages from phase I-immunized animals, but eventually multiplied in these cells. In contrast, only phase I organisms were destroyed by macrophages from phase I-immunized animals. Treatment of rickettsiae with immune serum enhanced ingestion by macrophages and potentiated the destruction of organisms by both types of macrophages. The macrophage migration inhibition assay was performed on peritoneal exudate cells from immune animals. Migration of peritoneal macrophages from phase I-immunized guinea pigs was inhibited, whereas macrophages from phase II-immunized animals migrated when cells were cultured in the presence of killed, intact phase I or II C. burnetii.