Experimental arthritis induced by continuous infusion of IL-8 into rabbit knee joints.

To determine the roles of IL-8 in inflammatory synovitis, examination was made of the results of continuously injecting human recombinant IL-8 into the knee joints of New Zealand while rabbits. Recombinant human IL-8 was infused continuously into the joint cavity at 75 ng/h for 14 days by a polypropylene catheter connected to a mini-osmotic pump implanted in each rabbit. Infiltration of inflammatory cells into joint cavity and histopathological changes in synovial tissue were examined at 7 and 14 days following the start of infusion. The continuous infusion of IL-8 for 14 days led to severe arthritis characterized by apparent erythema and joint pain, the accumulation of leucocytes, infiltration of mononuclear cells in synovial tissue, and marked hypervascularization in the synovial lining layer. IL-8 may be a factor which can contribute to the inflammatory process of chronic arthritis by mediating leucocyte recruitment and hypervascularization in inflamed joints.     

Suppression of development of diabetes in NOD mice by lactate dehydrogenase virus infection

It has been reported that lactate dehydrogenase virus (LDV) selectively infects a subpopulation of macrophages, thereby affecting the immune system. We studied the effects of LDV infection on the development of diabetes in non-obese diabetic (NOD) mice. Five-week-old female NOD mice were infected with LDV (10(8) ID50/mouse) and observed until 23 weeks of age. None of the 21-LDV-infected mice developed diabetes, whereas 10/14 (71.4%) uninfected mice did. Although the subpopulations of T cells and the percentage of Mac1-positive cells in the NOD murine spleen and the number of harvested peritoneal macrophages were unaffected by LDV infection, the proportions of Ia-positive peritoneal macrophages were significantly decreased in LDV-infected compared with uninfected mice (1.1 +/- 0.2%, 6.5 +/- 2.9%; P < 0.01). In LDV-infected NOD mice, insulitis of the same grade as that seen in uninfected NOD mice was observed. In another experiment, 3, 5, 10 or 16-week-old female NOD mice were infected with LDV. None of the mice infected with LDV at 3, 5 or 10 weeks of age developed diabetes and only one of six infected at 16 weeks of age did. These findings indicate that LDV infection suppresses the development of diabetes in female NOD mice by reducing the capacity of Ia-positive macrophages, and suggest that the development of human type 1 diabetes may be suppressed by certain viral infections.     

Analysis of mRNA with microsomal fractionation using a SAGE-based DNA microarray system facilitates identification of the genes encoding secretory proteins

In the regulation of host defense responses such as inflammation and immunity, the secretory proteins, including membrane proteins, play central roles. Although many secretory proteins have been identified by using methods such as differential display, random screening, or the signal sequence trap method, each method suffers from poor reproducibility, low sensitivity, or time-consuming or laborious work. Therefore, the strategy for facilitating the selection of the genes encoding the secretory proteins is desired. In this paper, we describe a system for isolating the genes encoding secretory proteins by analyzing mRNAs with microsomal fractionation on serial analysis of gene expression (SAGE)-based DNA microarray system. This system succeeded in discriminating the genes encoding secretory proteins from ones encoding nonsecretory proteins with 80% accuracy. We applied this system to human T lymphocytes. As a result, we were able to identify the genes that are not only encoding secretory proteins but also expressing selectively in a specific subset of T lymphocytes. The SAGE-based DNA microarray system is a promising system to identify the genes encoding specific secretory proteins.     

Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus

B1 cells have different origin and function from conventional B (B2) cells and are considered to be involved in autoantibody production in the development of autoimmune disease. We found that B1 cells preferentially accumulated in the target organs including thymus in aged BWF1 mice, a murine model for systemic lupus erythematosus, and that B lymphocyte chemoattractant (BLC/CXCL13) expression was increased in the thymus before the onset of lupus nephritis, while stromal cell-derived factor-1 (SDF-1/CXCL12) and secondary lymphoid tissue chemokine (SLC/CCL21) expression remained unchanged. Adhesion molecules such as peripheral node addressin (PNAd), ICAM-1, and VCAM-1 were also expressed on endothelial cells in the enlarged thymic perivascular space (PVS) in aged BWF1 mice. BLC protein and PNAd were co-localized on these high-endothelial-venules-like vessels in enlarged PVS. B1 cells expressed higher level of costimulatory molecules and showed a potent antigen-presenting activity in allogeneic mixed lymphocyte reaction comparable to splenic dendritic cells. Interestingly, B1 cells stimulated proliferation of autologous thymic CD4 T cells in the presence of IL-2. These results indicate that aberrant B1 cell trafficking into the thymus due to ectopic high expression of BLC may result in an activation of self-reactive T cells in the development of murine lupus.     

Z-line structural diversity in frog single muscle fiber in the passive state

The structural changes of the Z-line between small square net (ss) and basket weave (bw) cross-sectional patterns were examined using intact single fibers and mechanically skinned fibers in the passive state to determine if the pattern is related to the sarcomere length (SL) and if the pattern undergoes a reversible transition in low- and high-osmotic medium.Frog single fibers were isolated from the anterior tibial muscle in Ringer's solution. Entirely or partially skinned single fibers were prepared in relaxing solution (also called low-osmotic medium).The high osmotic medium contained 10% polyvinylpyrrolidone (PVP) in relaxing solution.The sarcomere length (SL) of each fiber was measured directly by use of a laser beam or indirectly from electron micrographs with use of a correction factor. The ss and bw forms in cross sections were quantified by analysis of electron micrographs. The results show that the structural change of Z-line occurs around bw 2.3–2.4m ss (n = 25) and bw 3.1–3.2m ss (n = 13) in intact single fibers and skinned fibers, respectively. With the quick freeze-freeze substitution method, an intact single fiber with a SL of 2.35m showed almost 100% of ss form. The structural transition in cross section was also confirmed in four partially skinned fibers, where patterns went from mostly ss form (intact portion) to mostly bw form (skinned portion) at the SL between 2.40 to 3.20m.The reversibility of the change between ss and bw was proved by using low- and high-osmotic medium. The transition and reversion of cross-sectional patterns both occur in the passive state.     

PRQFVamide,a novel pentapeptide identified from the CNS and gut of Aplysia

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.     

Establishment of a human B cell line that proliferates in response to B cell growth factor

A human B cell line which shows a marked dose dependence on B cell growth factor (BCGF) when cultured in less than or equal to 2% serum has been established. Human B lymphocytes were obtained from peripheral blood of normal donors and cultured in the presence of anti-IgM (mu chain specific) and BCGF. Frequent refeedings with fresh medium containing BCGF and anti-IgM led to the establishment of a long term cultured human B cell line, HAB-40. Phenotyping of HAB-40 revealed that the cell population consisted predominantly of IgM-bearing (72%) and B1 (100%) positive cells. This B cell line consistently secreted IgM and IgG when co-cultured in the presence of PMA, anti-IgM and beta or gamma interferon (IFN). Also, it was Epstein-Barr virus nuclear antigen (EBNA) positive (100%). HAB-40 cells have been successfully maintained in the presence of BCGF without anti-IgM for over a year. Removal of BCGF led to the rapid loss of viable cells in cultures containing less than 2% serum. HAB-40 cells in microassays exhibited a marked dose-dependent incorporation of [3H]thymidine in response to BCGF in the absence of any exogenous stimulants such as anti-IgM or Staphylococcus aureus Cowan I (SAC). Recombinant interleukin 2 (IL-2) failed to augment the [3H]thymidine uptake by these B cells despite the low density expression of Tac antigen (IL-2 receptor) on their cell surface, or even when the cells were stimulated with phorbol myristate acetate (PMA) to express higher density of Tac antigen (48%). HAB-40 cells could be maintained in BCGF which was partially purified to deplete it of other contaminating proteins. None of the seven well established EBNA-positive human B cell lines nor two chronic B lymphocytic leukemia (B-CLL) cell lines that were tested showed BCGF dependence. The same BCGF-active chromatographic fractions that were active on HAB-40 cells also stimulated BCL1 and normal human B cells stimulated with anti-IgM. In the presence of less than or equal to 2% serum proteins this cell line provides a simple, reproducible assay for BCGF even in the presence of contaminant IL-2.     

Orientational behavior of side-chain liquid-crystalline polysiloxanes deduced from measurements of second harmonic generation

Side-chain functionalized polysiloxanes were prepared via polymer-analogous esterification of poly[(3-chloroformylpropyl)methylsiloxane] with 4-(4-hydroxyphenylazo)nitrobenzene ( P1 ), 4-[4-(ω-hydroxyalkyloxy)phenylazo]nitrobenzene ( P2 – P4 ), 4-{4-[N-(2-hydroxyethyl)-N-methyl]anilinoazo}nitrobenzene ( P5 ), 4-(4-hydroxypiperidino)nitrobenzene ( P6 ), or 4-[4-(2-hydroxyethyl)piperidino]nitrobenzene ( P7 )., P1 , P3 , P4 and P5 exhibit liquid crystallinity, as deduced from differential scanning calorimetry, polarized microscopic observations and X-ray diffraction measurements. The liquid-crystalline phase of P1 and P5 is a nematic phase, and that of P3 and P4 is a smectic one. The second harmonic generation (SHG) measurement of a spin-coated film of P1 was carried out by the Maker fringe method using a Q-switched Nd:YAG laser (1064 nm). The SHG profile after the heat treatment of a spin-coated film suggests a perpendicular orientation of the mesogenic molecules to the glass substrate. The SH light intensity of a corona-poled film was 20-fold higher than that of a film which was only heated, though no differences were observed in their UV-vis absorption spectra. These findings suggest that the mesogenic-molecular dipole moments are aligned to the same direction in the crystalline or liquid-crystalline phase by a poling treatment.