In situ fluorescence imaging of organs through compact scanning head for confocal laser microscopy

We develop a compact scanning head for use in laser confocal fluorescence microscopy for in situ fluorescence imaging of organs. The head, cylindrical in shape, has 3.5 mm diameter and 30 mm length, and is thus small enough to operate in a living rat heart. The lateral and axial resolutions, defined as full widths at half maximum (FWHM) of a point spread function (PSF), measures 1.0 and 5.0 microm, respectively, for 488-nm excitation and 1.0 and 5.4 microm, respectively, for 543-nm excitation. The chromatic aberration between 488- and 543-nm laser beams is well suppressed. We perform Ca2+ imaging in cardiomyocytes through the right ventricular chamber of a perfused rat heart in line-scan mode with 2.9-ms time resolution. We also carried out two-color imaging of a fixed mouse heart and liver with subcellular resolution. The compact head of the microscope equipped with a line-scan imaging mode and two-color imaging mode is useful for in situ imaging in living organs with subcellular resolution and can advantageously be applied to in vivo research.     

"Homing to Niche," a new criterion for hematopoietic stem cells?

By combining cell surface staining with fluorochrome-conjugated monoclonal antibodies and Hoechst 33342 dye supravital staining, Matsuzaki et al. have succeeded in enriching hematopoietic stem cells (HSCs) essentially to homogeneity. When single-cell transplantation analysis was performed using the isolated cells, over 95% of the recipient mice showed long-term multilineage engraftment. The work demonstrates unexpectedly high marrow seeding efficiency of HSCs and proposes high marrow homing capacity as a new criterion for HSCs.     

The Natural History of Neuroblastic Cells in the Fetal Adrenal Gland

A study was made of neuroblastic nodules of the human adrenal gland in fetal life to determine their embryonic morphology and derivation. One hundred sixty-nine glands from 92 fetuses selected from 106 consecutively submitted fetuses formed the basis of this study. All adrenal glands examined contained neuroblastic nodules. They increased in number and maximum size with increasing age, peaked at 17 to 20 weeks gestational age, and regressed in the oldest fetuses. It is concluded that such neuroblastic nodules are an integral part of the normal morphogenesis of the adrenal gland. Their presence at birth and early infancy may represent a normal variation, and they do not necessarily constitute microscopic incipient malignancy.     

Microanatomical localization of PD-1 in human tonsils

PD-1 is an immunoinhibitory receptor, which belongs structurally to the CD28 family. PD-1-deficient mice show breakdown of peripheral tolerance and manifest multiple autoimmune symptoms. We previously described expression of PD-1 on activated T and B lymphocytes and myeloid cells. However, little is known about the microanatomical distribution of PD-1 in lymphoid organs. In this study, we performed immunohistochemistry using monoclonal antibodies against human PD-1. In human tonsils, PD-1 was expressed on most of T cells and a small subset of centrocytes in the light zone of germinal centers (GCs), where clonal selection of centrocytes takes place. These results suggest that PD-1 may play an important role in GC reaction.     

Histamine downregulates CD14 expression via H2 receptors on human monocytes

Lipopolysaccharide (LPS) binds to LPS-binding protein (LBP) in plasma and is delivered to the cell surface receptor CD14 on human monocyte. LPS is transferred to the transmembrane signaling receptor toll-like receptor (TLR) 4. In the present study, the effect of histamine on the expression of CD14 on human monocytes was investigated. Histamine concentration- and time-dependently decreased the expression of cell surface CD14, whereas histamine did not decrease mRNA for CD14 nor increase soluble CD14 (sCD14). The inhibitory effects of histamine on CD14 expression were antagonized by H2-receptor antagonist, but not by H1 and H3/H4 antagonist. The effects of selective H2-receptor agonists, 4-methylhistamine and dimaprit, on CD14 expression mimicked that of histamine indicating that histamine regulated CD14 expression through the stimulation of H2-receptors. The pretreatment with histamine partially inhibited the LPS-induced TNF-alpha production in human peripheral blood mononuclear cells (PBMC). Such inhibition might be due to the down-regulation of CD14 expression on monocytes by histamine.     

Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes

A mAb J43 has been produced against the product of the mousePD-1 gene, a member of the Ig gene superfamily, which was previouslyisolated from an apoptosis-induced T cell hybridoma (2B4.11)by using subtractive hybridization. Analyses by flow cytometryand immunoprecipitation using the J43 mAb revealed that thePD-1 gene product is a 50–55 kDa membrane protein expressedon the cell surface of several PD-1 cDNA transfectants and 2B4.11cells. Since the molecular weight calculated from the aminoacid sequence is 29,310, the PD-1 protein appears to be heavilyglycosylated. Normal murine lymphoid tissues such as thymus,spleen, lymph node and bone marrow contained very small numbersof PD-1⁺ cells. However, a significant PD-1⁺ population appearedin the thymocytes as well as T cells in spleen and lymph nodesby the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1antigen expression was strongly induced in distinct subsetsof thymocytes and spleen T cells by in vitro stimulation witheither anti-CD3 mAb or concanavalin A (Con A) which could leadT cells to both activation and cell death. Similarly, PD-1 expressionwas induced on spleen B cells by in vitro stimulation with anti-IgMantibody. By contrast, PD-1 was not significantly expressedon lymphocytes by treatment with growth factor deprivation,dexamethasone or lipopolysaccharide. These results suggest thatthe expression of the PD-1 antigen is tightly regulated andinduced by signal transduction through the antigen receptorand do not exclude the possibility that the PD-1 antigen mayplay a role in clonal selection of lymphocytes although PD-1expression is not required for the common pathway of apoptosis.     

Prevention of endotoxin shock by an antibody against leukocyte integrin {beta}2 through inhibiting production and action of TNF

Septic shock remains a serious disorder associated with highmortality. Accumulating evidence indicates that TNF is a majorand essential mediator of endotoxin shock. We report here thatadministration of an antibody against CD18 dramatically reducedendotoxin-induced shock inrabbits as revealed by preventionof severe hypotension, metabolic acidosis and a pathologicalchange suggestive of disseminated intravascular coagulationwith concomitant inhibition of elevation of plasma TNF activity.The anti-CD18 antibody also inhibited the hypotension inducedby administering recombinant TNF. Furthermore, an antibody againsta ligand for CD18 complexes, intercellular adhesion molecule-1,also prevented TNF-induced shock as well as endotoxin shockinrabbits. These observations suggest that adhesion of leukocytesto endothelium may be of primary importance in the action ofTNF as well as in the production of TNF in vivo and that theantibody against adhesion molecules could be of therapeuticbenefit in life-threatening septic shock in humans.     

Haplotype analysis in gelsoiin-related amyloidosis reveals independent origin of identical mutation (G654A) of gelsolin in Finland and Japan

Familial amyloidosis, Finnish type (FAF) (gelsolin-related amyloidosis) is an autosomal dominant form of systemic amyloidosis characterized by corneal lattice dystrophy and peripheral polyneuropathy. The accumulating protein in FAF consists of fragments of gelsolin, an actin-modulating protein. The gelsolin mutation G654A has been found in both Finnish and Japanese patients. To study the origin of the gelsolin mutation in these patients we performed haplotype analysis in 10 Finnish and 2 Japanese FAF families. Poymorphic DNA markers GSN, D9S103, AFMa061xd9, and AFMa139xb9 revealed a uniform disease haplotype in all the disease-associated chromosomes of the Finnish FAF families, which was different from the one observed in the Japanese families. The present results and the previously detected gelsolin mutation G654T in Czech and Danish FAF patients suggest that nucle otide 654 may represent a mutation hot spot in the gelsolin gene. The DNA markers studied here will be useful in future genealogical analyses of FAF. © 1995 Wiley-Liss, Inc.