Selection of stereotyped VH81X-{micro}H chains via pre-B cell receptor early in ontogeny and their conservation in adults by marginal zone B cells

The pre-B cell receptor (preBCR) plays critical roles in early B cell differentiation. It has been shown that not all muH chains are capable of pairing with surrogate light (SL) chains to form preBCR. Here, we established a novel system to differentially identify two types of early pre-B cell populations in bone marrow and fetal liver of mice, one producing SL-pairing muH chains and the other producing SL-non-pairing muH chains. The former population accounted for 80% of all the early pre-B cells in adult bone marrow, while it accounted for only 20% of those in fetal liver. Comparison of the two types of pre-B cell populations in fetal liver revealed the structural difference between SL-pairing and -non-pairing muH chains encoded by the V(H)81X segment that was most frequently utilized in fetal liver pre-B cells but rarely expressed by B cells generated in adults. PreBCR played an important role in the positive selection of V(H)81X-muH chains carrying the characteristic sequences of the complementarity-determining region 3 with little or no nibbling or N nucleotide addition, leading to their predominance in neonatal splenic B cells. These fetal-type V(H)81X-muH chains were also detected in adult spleen, but almost exclusively in marginal zone (MZ) B cells in contrast to the adult-type V(H)81X-muH chains. This strongly suggests that neonatally generated and selected B cells expressing the stereotyped V(H)81X-muH chains are maintained in the adult MZ and could function as innate-like lymphocytes.     

IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-gamma increase in normal pregnancy but decrease in spontaneous abortion

Recent data demonstrated that CD4+CD25+ regulatory T (Treg) cells and an enzyme called indoleamine 2,3-dioxygenase (IDO) mediate maternal tolerance to the fetus. Interestingly, Treg cells express the CTLA-4 molecule on their surface, and B7 (CD80/86) ligation by CTLA-4 enhanced IDO activity of dendritic cells (DCs) and monocytes by the induction of interferon gamma (IFN-gamma) production. In this study, we studied the IDO expression on peripheral blood monocytes and decidual monocytes or DCs after treatment with CTLA-4/Fc fusion protein or IFN-gamma using flow cytometry. IDO expressions on both peripheral blood DC and decidual DC and monocytes were up-regulated during normal pregnancy. On the other hand, both IDO expression on DC and monocytes after IFN-gamma treatment or CTLA-4 treatment were decreased in spontaneous abortion cases. The expression of CD86 on peripheral blood and decidual monocytes and DC in spontaneous abortion cases was lower compared with those in normal pregnancy subjects. Also, IFN-gamma production by decidual and peripheral blood mononuclear cells after CTLA-4/Fc treatment in spontaneous abortion cases was significantly lower than those in normal pregnancy subjects. These data suggest that CTLA-4 on Treg cells up-regulates IDO expression on decidual and peripheral blood DC and monocytes by the induction of IFN-gamma production.     

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.     

Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.     

Mutational screening of APP gene in patients with early-onset Alzheimer disease utilizing mismatched PCR-RFLP

To elucidate the frequency of mutations of the β/A4 amyloid protein precursor (APP) gene in early-onset Alzheimer disease, we designed a mismatched PCR-RFLP that can identify all kinds of missense mutations at codon 717 in addition to the seven kinds of known mutations at exon 17. When we screened mutations at exon 17 utilizing this method and the double missense mutations at exon 16 of the APP gene by PCR-RFLP, no cases revealed mutations of the APP gene among 13 familial and 54 sporadic cases, except one family (OS-1) that had previously been reported and used as a positive control of APP717(Val → Ile). Our results support the hypothesis that mutations in the APP gene are not major causes in early-onset Alzheimer disease.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

Surface properties of LDL-binding lymphocytes in human peripheral blood.

Surface properties of low density lipoprotein (LDL)-binding lymphocytes were evaluated to determine whether LDL binds with a subpopulation of human peripheral blood lymphocytes (PBL). B- and T-cell rich fractions were prepared from PBL using E-rosette formation or nylon reticulum columns. Binding of FITC-labelled LDL with these cell fractions was determined with a fluorescent microscope and a fluorescence-activated cell sorter (FACS II). The specificity of the binding was evaluated by a dose-dependent inhibition of LDL binding with the addition of unlabelled lipoproteins. In parallel studies, surface properties including E-rosette formation, surface immunoglobulins, and receptors for IgG-Fc, as well as human and mouse C3 were examined. LDL binding lymphocytes were enriched in the B-cell rich fraction, and depleted in the T-cell rich fraction. In addition, FITC-LDL binding lymphocytes were selectively collected by the FACS II. These LDL binding cells restored surface immunoglobulins after incubation in serum-free medium following trypsinization. The majority of lymphocytes stimulated by PHA and PWM in vitro bound with LDL. It is concluded that LDL binds with B cells in fresh human PBL, while it binds with B and T cells in mitogen-stimulated lymphocytes. It is suggested that the selective collection of LDL binding lymphocytes by the FACS II can be applied to the evaluation of cellular interaction of these cells in various immunological reactions.     

Case report of de novo dup(18p)/del(18q) and r(18) mosaicism

This is a report of a 27-year-old woman with an unusual de novo chromosomal abnormality. Mosaicism was identified in peripheral blood cells examined by standard G-bands by trypsin using Giemsa (GTG) analysis and fluorescence in situ hybridization (FISH) analysis with chromosome-18 region-specific probes, 46,XX,del(18)(pter → q21.33:)[41], 46,XX,r(18)(::p11.21 → q21.33::)[8], and 46,XX,der(18)(pter → q21.33::p11.21 → pter)[1]. On the other hand, the karyotype of periodontal ligament fibroblasts was nonmosaic, 46,XX, der(18)(pter → q21.33::p11.21 → pter)[50]. All cell lines appeared to be missing a portion of 18q (q21.33 → qter). The pattern of the dup(18p)/del(18q) in the rod configuration raises the possibility of an inversion in chromosome 18 in one of the parents. However, no chromosomal anomaly was detected in either parent. The most probable explanation is that de novo rod and ring configurations arose simultaneously from an intrachromosomal exchange. The unique phenotype of this patient, which included primary hypothyroidism and primary hypogonadism, is discussed in relation to her karyotype.     

Mesenteric lymphadenitis of swine caused by Rhodococcus sputi.

Rhodococcus sputi caused tuberculosislike lymphadenitis of mesenteric lymph nodes in swine. This is the first study reporting that R. sputi can be a pathogen in swine.     

Interferon-alpha and dexamethasone inhibit adhesion of T cells to endothelial cells and synovial cells

We investigated whether interferon-gamma (IFN-gamma), interferon-alpha (IFN-alpha) and glucocorticoids affected the adhesion of T cells to human umbilical endothelial cells or human synovial cells. About 30% of peripheral blood T cells could bind to unstimulated endothelial cells, but only a few T cells could bind to unstimulated synovial cells. When both endothelial cells and synovial cells were cultured with recombinant IFN-gamma (rIFN-gamma), the percentage of T cell binding to both types of cells increased in a dose-dependent manner. rIFN-alpha and dexamethasone blocked the T cell binding to unstimulated endothelial cells. Furthermore, rIFN-alpha and dexamethasone suppressed T cell binding to both endothelial cells and synovial cells stimulated by IFN-gamma, and also inhibited intercellular adhesion molecule-1 (ICAM-1) expression on both endothelial cells and synovial cells stimulated by IFN-gamma. These results suggest that IFN-alpha and glucocorticoids may inhibit T cell binding to endothelial cells or synovial cells by modulating adhesion molecule expression on these cells.