Different stromal cell lines support lineage-selective differentiation of the multipotential bone marrow stem cell clone LyD9.

An interleukin 3-dependent multipotential stem cell clone, LyD9, has been shown to generate mature B lymphocytes, macrophages, and neutrophils by coculture with primary bone marrow stromal cells. We report here that coculture with the cloned stromal cell lines PA6 and ST2 can support differentiation of LyD9 cells predominantly into granulocyte/macrophage colony-stimulating factor (GM-CSF)- and granulocyte (G)-CSF-responsive cells, respectively. However, these stromal cell lines were unable to support lymphopoiesis of LyD9 cells. The GM-CSF-dependent line, L-GM, which was derived from LyD9 cells cocultured with PA6 stromal cells, could differentiate into macrophages and granulocytes in the presence of GM-CSF. The L-GM line can further differentiate predominantly into neutrophils by coculture with ST2 stromal cells. The G-CSF-dependent line, L-G, which was derived from LyD9 cells cocultured with ST2 stromal cells, differentiated into neutrophils in response to G-CSF. Although the stromal cell-supported differentiation of LyD9 cells required the direct contact between LyD9 and stromal cells, a small fraction of LyD9 cells that were pretreated with 5-azacytidine could differentiate into neutrophils and macrophages without direct contact with stromal cells. These results indicate that different stromal cell lines support lineage-selective differentiation of the LyD9 stem cell and that 5-azacytidine treatment can bypass the requirement of direct contact with stromal cells, albeit with a lower frequency.

Evidence of different pharmacokinetics between cyclosporine and tacrolimus in renal transplant recipients: why cyclosporine is monitored by C2 level and tacrolimus by trough level.

The clinical efficacy of calcineurin inhibitors administered to renal transplant recipients is considered to be a strong function of the area under the concentration time curve (AUC). Monitoring of blood concentrations for two similar calcineurin inhibitors, cyclosporine (CyA) and tacrolimus (TAC) are different. Namely, CyA blood concentration is usually monitored at two hours after administration (C2), a surrogate for peak concentration (Cp), and TAC at trough concentration (Ct). We examined the behavior of blood concentration curves simultaneously for both CyA and TAC in renal transplant recipients with similar clinical backgrounds. Furthermore, we analyzed the correlation of Cp and Ct vs AUC implementing an area under the trough level, or area above the trough level as new pharmacokinetic parameters, so that C2 for CyA and Ct for TAC has validated using controlled clinical data. We observed differences in the pharmacokinetics between.

ABO-incompatible adult living donor liver transplantation for hepatocellular carcinoma.

Living donor liver transplantation (LDLT) offers timely transplantation for patients with hepatocellular carcinoma (HCC). If ABO-incompatible LDLT is feasible, the need for pretransplantation treatment may be eliminated, which may reduce overall morbidity. In this article, we have described 8 adult HCC patients who successfully underwent LDLT from ABO-incompatible donors. Antirejection therapy included multiple preoperative plasmaphereses, splenectomy, and an immunosuppressive regimen with tacrolimus, methylprednisolone, and mycophenolate mofetil. The maintenance dose of immunosuppression did not differ from that of the ABO-identical cases. In addition, we also performed intrahepatic arterial infusion of prostaglandin E1. In 5 patients, we administered a single dose of rituximab, a chimeric CD20 monoclonal antibody. As a result of this treatment, 6/8 patients are still alive. Our experience has shown that it is possible to control antibody-mediated humoral rejection and other complications in adult ABO-incompatible LDLT.

Biliary complications after 52 adult living donor liver transplantations: a single-center experience.

OBJECTIVE: The incidence of biliary complications after adult living donor liver transplantation (ALDLT) are still high even though various devices have been reported to overcome them. METHOD: From October 2000 to April 2007, we performed 52 ALDLTs which included 15 ABO-incompatible grafts. Median follow-up was 565 days. In 49 procedures, we used duct-to-duct anastmosis with a stent inserted in the recipient duct and out through the common bile duct wall as an external stent, and in 3 procedures, we used duct-to-jejunostomy anastomosis. We investigated postoperative biliary complications and their management. RESULTS: Forty-four patients received right lobe grafts and 8 received left lobe grafts. Among patients in whom duct-to-duct anastomosis was used, nine (20.5%) developed biliary complications including bile leakage in five and biliary strictures in four. All bile leakage was treated with reoperation. Three biliary strictures were treated with stent placement, and one biliary stricture was treated with magnetic compression anastomosis. Among the three patients in whom duct-to-jejunostomy was used, two (66.7%) had bile leakage and stricture, respectively. Two of four ABO-incompatible patients (50%) had hepatic artery thrombosis with biliary complications, a high incidence. CONCLUSION: In our series of ABO-incompatible patients undergoing ALDLT, those who developed hepatic artery thrombosis exhibited a high incidence of biliary complications.

How can we increase living related donor renal transplantations?

BACKGROUND: In Japan, living donor renal transplantation has gained momentum due to an increased number of patients with end-stage renal disease. Living donation not only provides better outcomes, but also the recipients usually need less medications, thereby increasing the quality of life and reducing the potential side effects of immunosuppression. MATERIALS AND METHODS: For the past 25 years, our center had performed 140 open donor nephrectomy (OPNx) renal transplantations. Since July 2003, we changed our procurement operation to living hand-assisted laparoscopic donor nephrectomy (HALNx) in 49 cases. Our operative technique consisted of two 12-mm ports placed in the midaxillary line at the superior and inferior levels of the umbilicus. Next, a 5-cm incision was made in the midline periumbilicus and the hand port system fitted through a midline abdominal incision. RESULTS: In 49 cases, HALNx was completed successfully; no patient required conversion to laparotomy. The estimated blood loss was 33.0 +/- 43.4 g and no patient required blood transfusion. In comparison, in OPNx the blood loss was 426.5 +/- 247.6 g (P < .001). The mean operative times were 167.4 +/- 39.7 minutes for HALNx and 228.4 +/- 35.7 minutes for OPNx (P < .001). The postoperative hospital stays were 9.1 +/- 3.8 days for HALNx and 13.0 +/- 1.9 days for OPNx (P < .001). For 3 years prior to introduction of HALNx, we had performed only 10 living donor renal transplantations. Since the introduction of HALNx in 2003, the number of living donors has tripled during the following 3 years. CONCLUSIONS: Herein we have reported that HALNx was superior in terms of less operative time and blood loss, postoperative pain and recovery, and shorter hospital stay. Overall donor patient satisfaction was also better in the HALNx group. HALNx is a safe procedure that makes kidney donation more appealing to potential live donors and has increased the living donor pool at our center.

The role of plasmapheresis therapy for perioperative management in ABO-incompatible adult living donor liver transplantation.

BACKGROUND: Although living donor liver transplantation (LDLT) was established as a treatment for end-stage liver disease in Japan, the indication for LDLT across an ABO-incompatible barrier remains controversial. The purpose of this study was to elucidate the role of plasmapheresis in incompatible LDLT. METHODS: Eleven adult patients (seven men and four women) who underwent incompatible LDLT were enrolled in this study. Of these three patients had hepatocellular carcinoma, three chronic hepatitis C, one Wilson's disease, one autoimmune hepatitis, one chronic hepatitis B, one hemochromatosis, and one fulminant hepatic failure. The immunosuppressive regimen consisted of tacrolimus, prednisolone, mycophenolate mofetil (or cyclophosphamide), and prostaglandin E1 in all patients. Multiple plasmapheresis was performed perioperatively to reduce the recipient's antibody titers against the donor's blood type. RESULTS: Plasmapheresis was useful for the reduction of the recipient's antibody titers to x 16 or lower before and after transplantation. There was no difference in transplant outcome between the 11 patients with incompatible blood group and 30 patients with identical or compatible blood groups. DISCUSSION: Major postoperative complications such as intrahepatic biliary complications and hepatic necrosis may occur in incompatible transplantation. Several investigators suggested that anti-immunoglobulin (Ig) M and anti-IgG antibody titers sustained these complications. The antibody titers must be decreased sufficiently with plasmapheresis. An elevation of anti-ABO titers after transplantation may be a predictive risk factor for increased mortality and morbidity. In order to perform LDLT in a safer manner, plasmapheresis is an indispensable treatment to improve the outcome of ABO-incompatible cases.

Radial flow bioreactor for the creation of bioartificial liver and kidney.

A radial flow bioreactor (RFB) is used for a three-dimensional perfusion culture of hepatocellular carcinoma (HCC) cells and renal cells, to create a bioartificial liver and kidney. The cylindrical reactor is filled with porous cellulose microcarrier. RFB can be characterized as a system in which the medium flows from the periphery toward the center of the reactor, thereby delivering an adequate supply of oxygen and nutrients to cells at the center as well as at the periphery. HCC cells incubated in the RFB system at high density maintained viability for long periods of time. Proximal tubular cells (LLC-PK1) as well as HCC cells, but not human immortalized mesangial cells (HMC) were cultured in the RFB for more than 14 days. The mRNA expression of some enzymes involved in the urea cycle, cytochrome P450s in HCC cells, and the 1-alpha-hydroxylase (CYP27B1) in LLC-PK1 cells was higher than that in monolayer cultures. These results suggest that the RFB system composed of HCC cells or renal cells may be useful for a bioartificial liver and kidney.

Evaluation of appropriate blood level in continuous intravenous infusion from trough concentrations after oral administration based on area under trough level in tacrolimus and cyclosporine therapy.

The target blood concentrations of tacrolimus (TAC) and cyclosporine (CYA) during continuous intravenous infusion (C(ss)) have been determined based on clinical experience. However, it is desirable that C(ss) should be set so that the AUC after intravenous infusion is equal to the AUC after oral administration (AUC(po)). Accordingly, we performed 12-hour monitoring of blood concentrations to calculate C(ss) from the blood trough levels (C(TL)) on 15 kidney recipients administered TAC and 12 recipients administered CYA (Neoral). We used an area under the trough level (AUTL) as a new pharmacokinetic parameter. The C(ss) was evaluated from C(TL), AUC(po), and AUTL was calculated to be C(ss) = C(TL) x (AUC(po)/AUTL). In addition, AUTL/AUC(po) ratio and blood peak/trough level ratio (C(max)/C(min)) were examined to compare pharmacokinetics of TAC and CYA. The formula for TAC was C(ss) = C(TL) x 1.40 and that for CYA, C(ss) = C(TL) x 2.55. The calculated target C(ss) of TAC was 1.40 times that of C(TL), which was similar to the present clinical C(TL). In contrast, the calculated target C(ss) of CYA was 2.55 times the C(TL), and therefore an extremely high C(ss) was necessary to obtain a sufficient AUC that will be available after oral administration. Consequently, intravenous administration of CYA twice a day was considered to be more appropriate to obtain sufficient CYA pharmacokinetics, rather than a continuous intravenous administration. We conclude that the formula, C(ss) = C(TL) x (AUC(po)/AUTL) was useful to calculate the target blood concentration of calcineurin inhibitors when changing from continuous intravenous infusion to oral administration of these drugs.

Pharmacokinetic differences between morning and evening administration of cyclosporine and tacrolimus therapy.

We performed 24-hour monitoring of cyclosporine (NEO) and tacrolimus (TAC) blood concentrations, evaluating pharmacokinetic parameters and characterizing circadian variations. The monitoring was performed in 10 instances on nine patients administered NEO and 12 out of 11 patients administered TAC. All cases were administered equally divided doses of drugs twice daily orally. Blood samples were taken before and 1, 2, 3, 4, 6, and 12 hours after NEO or TAC administration in the morning and evening. The pharmacokinetic parameters were compared between morning and evening administrations of both drugs. AUC0-12, AUC0-4, C(max), C2, and C(max)/C(min) of NEO and TAC were significantly lower during the evening compared with morning administrations. C(min) values were significantly higher in the evening. T(max) of NEO was longer in evening, although there was not a significant difference; T(max) of TAC was significantly longer in the evening. We found that NEO and TAC administrations in the evening resulted in reduced bioavailability and delayed absorption when compared with drug administrations in the morning. It was thought that the difference in bioavailability between morning and evening administrations was smaller with TAC, because TAC shows lower peak levels and a flatter blood concentration curve than NEO. C(min) was higher after evening administration than morning because of delayed absorption, though the bioavailability of both drugs decreased in the evening. These results suggest that we have to appreciate apparently high trough levels.

Optimal dose and target trough level in cyclosporine and tacrolimus conversion in renal transplantation as evaluated by lymphocyte drug sensitivity and pharmacokinetic parameters.

We evaluated the relative clinical potency of cyclosporine (CyA) and tacrolimus (Tac) using pharmacodynamic and pharmacokinetic parameters of the drug to obtain the most suitable converting dose and target trough level. The relative pharmacodynamic potency was examined by the mean ratio of drug concentrations giving 50% inhibition of blastogenesis of lymphocytes (IC50) in 66 chronic renal failure patients. The relative potency estimated from clinical pharmacokinetic parameters was examined by the mean ratio of each pharmacokinetic parameter value of CyA versus Tac. The pharmacokinetic parameters were estimated by 12-hour monitoring of drug blood concentrations in seven CyA patients and seven Tac patients. The mean IC50 ratio of CyA and Tac (CyA/Tac of IC50) was 25.1. The mean area under the concentration-time curve (AUC) ratio (CyA/Tac of AUC) was 25.5, the mean trough level (C(min)) ratio (CyA/Tac of C(min)) was 13.2, and the mean dose per body weight ratio was 25.2. The relative potency estimated from AUC that is the most reliable pharmacokinetic parameter for the estimation of clinical efficacy of calcineurin inhibitors appeared to agree with the relative pharmacodynamic potency estimated from IC50. The data suggest that TAC 25-fold more potent than CyA, which represents a suitable converting dose ratio, and that target trough level of CyA is about 13-fold greater than Tac based on CyA/Tac of C(min). We conclude that these relative values may be useful to estimate the suitable dose and target trough levels to convert between CyA and Tac.

Hyperglycemia is a factor for an increase in serum ceruloplasmin in type 2 diabetes.

OBJECTIVE: To examine if there is a correlation between high blood glucose and serum ceruloplasmin (Cp) levels. RESEARCH DESIGN AND METHODS: Serum Cp levels were measured in 637 patients with type 2 diabetes (all type 2 diabetes group). For the follow-up type 2 diabetes group, 161 patients who had not had any changes in their situation during the last year that are known to influence serum Cp levels were reexamined 1 year later. The control group was composed of 158 healthy individuals. Serum Cp and blood HbA1c levels were measured by radial immunodiffusion and high-performance liquid chromatography assays, respectively. RESULTS: Serum Cp levels in the all type 2 diabetes group were significantly higher than those in the control group (P < 0.0001), although the serum Cp levels did not correlate with the blood HbA1c levels in the all type 2 diabetes group (r = 0.055, P = 0.351). Then we evaluated those factors (delta-log Cp and delta-HbA1c) in the follow-up type 2 diabetes group to minimize changes from the genetic differences and to exclude any known factors influencing serum Cp levels. This indicated that the delta-HbA1c had a positive correlation to the delta-log Cp (r = 0.304, P < 0.0001). CONCLUSIONS: A persistent high blood glucose (namely HbA1c) is associated with an increase in serum Cp levels over 1 year.

Three-dimensional structure of astrocytes in the rat dentate gyrus.

Structural features of astrocytes in the rat dentate gyrus were studied by means of light and high-voltage electron microscopy of Golgi-impregnated materials, conventional electron microscopy, combined Golgi-electron microscopy, and immunohistochemistry for glial fibrillar acidic protein. Astrocytes in the dentate gyrus were of the protoplasmic type and were classified into six subtypes based on the location of their somata; i.e., astrocytes in the polymorph layer, in the subgranular zone, in the granular cell layer, at the border of the granular cell layer and the molecular layer, in the molecular layer, and subjacent to the pia surface. Stereoscopic observations of 5-micron-thick sections of Golgi-impregnated materials revealed three-dimensional structural details of astroglial processes not apparent in either the light microscope or in conventional thin-section electron microscopy. Most of them were basically thin sheets, varying in shape and size in accordance with their sites. In the granule cell layer, thin veillike sheets or lamellae, originating from three kinds of astrocytes (subtypes 2, 3, and 4), intervened between granule cell somata, whereas in the plexiform and molecular layers small leafletlike appendages originating from astrocytes (subtypes 1, 2, 4, and 5) intermingled with one another, making spongelike conglomerates. Thus astrocytes in the subgranular zone and those at the border of the granule cell layer and the molecular layer showed prominent regional differentiation of their processes among layers. In addition to these sheetlike processes, thin threadlike processes were also common.

Presence of the ruffed cell in the olfactory bulb of the catfish, Parasilurus asotus, and the sea eel, Conger myriaster.

The ruffed cell is present in the olfactory bulb of the catfish, Parasilurus asotus, and of the sea eel, Conger myriaster. Its morphological features have been studied by light microscopy, high-voltage electron microscopy, and conventional electron microscopy. The ruffed cell of the catfish is very similar to that of the goldfish in its location and in its structural features. It has a spheroidal or ovoid cell body about 15 to 30 micrometer in diameter. Several dendrites arise from the cell body to form a rounded dendritic field about 100 micrometer in diameter near the cell body. The initial unmyelinated portion of its axon (IP) consists of a shaft and many protrusions arising from it. The shaft, about 1 micrometers in diameter, located extends for about 100 to 200 micrometer, where it acquires a myelin sheath. The protrusions intermingle with one another in a complex manner to form a rather discrete spheroidal field about 20 to 50 micrometers in diameter, located in the vicinity of the cell body. In contrast, the ruffed cell of the sea eel differs rather significantly from that of the goldfish in its morphological features. The ruffed cell of the sea eel is of a bipolar type. One thick dendrite arises from the cell body and extends for about 50 to 100 micrometers, where it gives rise to many thread-like dendritic branches. The IP arises from the cell body as a smooth thin process. However, about 30 to 70 micrometers distant from its origin, many elaborate protrusions arise from the axonal shaft. These intermingle with one another to form a spheroidal or ovoid field about 20 to 40 micrometers in diameter. Distal to this protrusion-bearing part, the axon continues as a smooth, thin process. In spite of these differences in structural features, the ruffed cell of the catfish and that of the sea eel are very similar in their synaptic features in the nest (the special synaptic field around the ruffed cell IP, composed of its protrusions, of granule cell dendrites, and of other neuronal processes); that is, synapses between the ruffed cell IP (its shaft and protrusions) and granule cell dendrites and serial synapses made by the ruffed cell IP, granule cell dendrites, and perinest cell dendrites. These results suggest that the ruffed cell is generally present in the teleostean olfactory bulb, although its detailed structural features may vary from species to species. Moreover, the neuronal organization of the olfactory bulb seems to be fundamentally similar in various species of teleosts.

Ruffed cell: a new type of neuron with a distinctive initial unmyelinated portion of the axon in the olfactory bulb of the goldfish (Carassius auratus) I. Golgi impregnation and serial thin sectioning studies.

A new type of neuron was recognized in the olfactory bulb of the goldfish (Carassius auratus) by means of light and high voltage electron microscopy of Golgi-impregnated material, combined Golgi-electron microscopy, and electron microscopy of serial thin sections. The neuron is located in the layer between the olfactory nerve layer and the anterior olfactory nucleus. It has a spherical cell body, about 10--20 microns in diameter, and several dendrites which form a spherical dendritic field, about 70--100 microns in diameter, in the vicinity of the cell body. The most remarkable structural feature of this neuron is that its initial unmyelinated portion of the axon (IP) has elaborate protrusions with many synapses. The IP can be divided into three parts, parts 1, 2 and 3, based on its structural features. Part 1 is the initial part of the IP, about 20--40 microns in length. Many elaborate protrusions arise from the shaft and intermingle with one another to constitute a spherical field, about 20--40 microns in diameter, around the shaft. Part 2 is the middle part of the IP, about 10--20 microns in length. There are several collateral-like protrusions, which are scattered along the shaft and extended laterally about 5--15 microns. Part 3 is the last part of the IP, and is cylindrical without protrusions. The length of part 3 varies from 20 to more than 100 microns. The axon acquires a myelin sheath at distance of 70--250 microns from its origin. Protrusions make synaptic contacts mainly with granule cell dendrites. Some of them are of the reciprocal type. Protrusion are presynaptic in asymmetrical synapses, and postsynaptic in symmetrical synapses with granule cell dendrites. The shaft of the IP also has synapses similar to those on protrusions. The neuron described is a new type of neuron in the vertebrate central nervous system. We propose for it the name "ruffed cell."

Structure of the mitral cell in the olfactory bulb of the goldfish (Carassius auratus).

The mitral cell in the olfactory bulb of the goldfish was examined by means of light microscopy, high-voltage electron microscopy, and conventional electron microscopy. Mitral cells are located rather diffusely throughout the glomerular and plexiform layers. They do not make their own discrete layer. The cell bodies are rounded or triangular, and are about 10-25 micrometers in diameter. In Golgi-impregnated material, thick cylindrical dendrites can be seen arising from the cell bodies and branching in the glomerular layer. Dendritic branches of some cells make two or more rather compact tufts, while the dendrites of other cells intermingle loosely with one another. In semithin and thin sections, darkly stained nodules appear to be scattered diffusely in the glomerular layer without clustering into discrete spheres, which are characteristic of the mammalian glomerulus. Hence, instead of the glomerulus, the "glomerular area" is defined as an area consisting of darkly stained nodules with rather pale granular regions surrounding them. Branches of mitral cell dendrites in the glomerular area consist of cylindrical shafts and irregular appendages arising from them. The shafts appear in the pale granular region and the appendages are found in the darkly stained nodules. Synapses can be found on all parts of the mitral cell: the soma, axon hillock, axon initial segment, thick dendritic stems, and dendritic branches. The abundance of synapses seems to vary considerably from part to part, and is highest on the dendritic branches in the glomerular area. The mitral cell is postsynaptic to olfactory nerve terminals and granule cell dendrites, and presynaptic to granule cell dendrites and some processes of unknown origin. Olfactory nerve terminals make asymmetrical synapses specifically on the appendages of the dendritic branches. Of the synapses on the shafts of the mitral cell dendritic branches in the glomerular area, 90% are with granule cell dendrites. Of the synapses between two different kinds of processes 30% are mitral-to-granule asymmetrical synapses, 20% are granule-to-mitral symmetrical synapses, and 50% are reciprocal pairs. Gap junctions and mixed synapses are also seen on branches of mitral cell dendrites. Features of the goldfish mitral cell are compared with those of the mammal. The differences in neuronal organization between the olfactory bulbs of teleosts and mammals are discussed.

Ruffed cell: a new type of neuron with a distinctive initial unmyelinated portion of the axon in the olfactory bulb of the goldfish (Carassius auratus). III. Three-dimensional structure of the ruffed cell dendrite.

The three-dimensional structure of the ruffed cell dendrite in the olfactory bulb of the goldfish (Carassius auratus) was studied by means of light and high-voltage electron microscopy of Golgi-impregnated material, combined Golgi-electron microscopy, and electron microscopy of serial thin sections. Ruffed cell dendrites ramify in the glomerular area with tufted branching patterns. They give rise to many appendages of various shapes and sizes: sheetlike processes, fingerlike projections, or small tuftlike appendages. These appendages, singly or together, appear to entwine or cover other neuronal processes. In thin sections ruffed cell dendritic appendages frequently look like glial processes; that is, they appear to conform in shape to the contours of surrounding neuronal elements, such as mitral and granule cell dendrites. Ruffed cell dendrites display intimate relationships with mitral cell dendrites. The former appear to coil around and cover the latter. Nothwithstanding the intimate relationship between ruffed cell dendrites and mitral cell dendrites, there seem to be no synaptic connection between them. Ruffed cell dendrites bear a rather small number of presynaptic and postsynaptic sites, most of which area with granule cell dendrites. Ruffed cell dendrites seem to receive no synapses from olfactory nerve terminals.

Gap junctions between non-pyramidal cell dendrites in the rat hippocampus (CA1 and CA3 regions): a combined Golgi-electron microscopy study.

Non-pyramidal cells in the rat hippocampus were examined with a combined Golgi-electron microscopic method. The somata of non-pyramidal cells were ovoid, about 15 X 30 micron, and several smooth and/or varicose dendrites extended from them. With electron microscopy, Golgi-impregnated gold-toned non-pyramidal cells showed distinctive fine structural features. The somata displayed large nuclei and an extensive perikaryal cytoplasm. The nuclei showed extensive cytoplasmic invaginations, little heterochromatin, conspicuous nucleoli, and intranuclear rods composed of filamentous bundles. The perikaryal cytoplasm was rich in cell organelles such as well-developed cisternae of rough endoplasmic reticulum and Golgi apparatus, and numerous clusters of free ribosomes and mitochondria. Many synaptic boutons, most of which formed asymmetrical synapses, impinged upon the somata and dendrites. Gap junctions were seen on varicose dendrites of Golgi-impregnated non-pyramidal cells. These gap junctions were patch-like, about 0.1-0.6 micron in diameter, and situated in the stratum radiatum or stratum oriens of the CA1 and CA3 regions 70-230 micron from the soma. They displayed a characteristic cytoplasmic semidense material undercoating the junctional membranes. The gap junctions were usually formed between impregnated and unimpregnated varicose dendrites. Thirteen of a total of 22 gap junctions involving the impregnated dendrites were situated singly, whereas the remaining nine were on four impregnated dendrites in clusters of two or three side by side. In the latter cases, two pairs of junctions were formed between pairs of dendrites running parallel to each other, and each of the other two pairs was formed among three dendrites, appearing to make a dendritic network bridged by gap junctions. One gap junction was seen between two impregnated dendrites originating from two identified Golgi-impregnated non-pyramidal cells. These observations revealed unequivocally that non-pyramidal cells in the hippocampus form gap junctions with one another on their dendrites.

GABAergic neurons containing CCK-8-like and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus.

The coexistence of cholecystokinin-octapeptide-like (CCK-L) and/or vasoactive-intestinal-polypeptide-like immunoreactive (VIP-LI) materials and glutamate decarboxylase (GAD) was studied in the rat hippocampus and dentate gyrus by means of immunohistochemistry. Consecutive 40-micron-thick sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for these peptides and GAD in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell, incubated in two different antisera. Almost all of the CCK-LI neurons were also GAD immunoreactive, whereas only about 10% of the GAD-immunoreactive neurons were CCK-LI. The percentages of GAD-immunoreactive neurons which were also immunoreactive for CCK were dependent on the laminar area in which they were found: i.e., 15-20% in the stratum radiatum of the hippocampus, about 10% in the stratum pyramidale, and about 6% in the stratum oriens. In contrast to the CCK-LI neurons, only about 40% of the VIP-LI neurons were identified to be also GAD immunoreactive, which might correspond to only part of the GAD-immunoreactive neurons. Furthermore the coexistence of VIP-LI and CCK-LI materials was recognized in about 10% of the CCK-LI neurons or about 35% of the VIP-LI neurons, indicating that some GABAergic neurons (presumably about 1%) in the rat hippocampus and dentate gyrus may contain both CCK-LI and VIP-LI materials.

Use of high concentrations of glutaraldehyde for immunocytochemistry of transmitter-synthesizing enzymes in the central nervous system.

Aldehyde fixatives containing high concentrations of glutaraldehyde, usually used for conventional electron microscope studies, were successfully used for immunocytochemistry of transmitter synthesizing enzymes, glutamate decarboxylase and tyrosine hydroxylase, in the rat central nervous system. Although a high concentration of glutaraldehyde could cause tremendous non-specific staining, this was almost completely absent after treating sections with 1% sodium borohydride for 30 min. Furthermore, it was shown that a high concentration of glutaraldehyde might cause no appreciable reduction of the antigenicities of glutamate decarboxylase and tyrosine hydroxylase when compared with fixatives containing a low concentration of glutaraldehyde. It is suggested that fixatives containing high concentrations of glutaraldehyde are very useful, not only for conventional electron microscope studies, but also for light and electron microscope immunocytochemistry of some antigens, including glutamate decarboxylase and tyrosine hydroxylase.

Application of a human T cell line derived from a Sézary syndrome patient for human interleukin 4 assay.

Interleukin 4 (IL-4) is a multi-functional biological regulator molecule. We developed a simple assay system for human IL-4, using a IL-2-dependent T cell line designated Sez 627 cell line. Sez 627 cells were found to respond only to human IL-4 and human IL-2, but not to other available lymphokines. In combination with mouse IL-2-dependent CTLL-2 cell line, which responds to both human and murine IL-2 but not to human IL-4, human IL-4 activity can be discriminated from human IL-2 activity.