A semithin light microscopic study of peritoneal cells of the mouse embryo

Using semithin plastic sections, sequential changes in the mouse peritoneal cells during their intrauterine life were observed by light microscopy. At 11-13 days of gestation, the peritoneal cavity contained a small number of free mononuclear cells, singly scattered. Most of them were 9 to 12 micron in cell diameter, and the N-C (nucleocytoplasmic) ratio was smaller than 0.7. The cytoplasm contained large phagocytic vacuoles, and long processes extended from the cell surface. These cells were considered mature macrophages. After 15 days, the peritoneal cavity contained small-sized mononuclear cells in addition to mature macrophages. The small mononuclear cells were 5-10 micron in cell diameter, and formed the major cell type at this time. The cytoplasm contained occasional small vesicles but no phagocytic vacuoles. The small mononuclear cells showed a larger N-C ratio, 0.8-1.7. In 18-day-old fetuses, the peritoneal cells consisted of mononuclear cells, 79.5%, and mast cells, 20.5%. During the initial 18 days of embryonic life, small lymphocytes, neutrophils and eosinophils were not contained in the mouse peritoneal space.     

The ultrastructure of megakaryopoietic cells of the yolk sac and liver in mouse embryo

Megakaryopoietic cells in the yolk sac and liver of mouse embryos were examined by electron microscopy. At 10 days' gestation, yolk sac vitelline vessels contained a few free megakaryopoietic cells. On the basis of the development of demarcation membranes and granules, yolk sac megakaryopoietic cells were classified into three types: YM1, YM2, and YM3. The YM1 cells, which comprised 75% of the yolk sac megakaryopoietic cells, had poorly developed demarcation membranes and few granules in the cytoplasm. The YM2 cells had a developed demarcation membrane system around the nucleus and comprised 24% of the yolk sac megakaryopoietic cells. The YM3 cells, the rarest type, had an eccentrically located nucleus and a cluster of demarcation membrane structures. In the liver of 11-day embryos, immature megakaryopoietic cells were present in both the sinusoidal lumen and the hepatic cords. Most of the hepatic megakaryopoietic cells of 11-day embryos had ultrastructural features similar to those of YM1 cells or intermediate between YM1 and YM2 cells.     

Ultrastructural studies of hepatoblast junctions and liver hematopoiesis of the mouse embryo]

To clarify the morphological changes in hepatoblast connections during the development of fetal liver hematopoiesis, ICR mouse livers of 11 to 19 days of gestation were studied by means of three-dimensional reconstruction, immunohistochemistry, electron microscopy and freeze fracture replica method. Embryonic liver weight showed rapid increase until 19 days of gestation, and an initial steep increase, due to hematopoietic development, was observed at 13 to 15 days of gestation. Hepatoblast volume appeared to be constant until 13 days of gestation, and, thereafter, showed a gradual increase. An 11-day primitive hepatic cord contained a few immature hematopoietic cells among hepatoblasts, and the hepatoblasts made contact with one another by short cytoplasmic projections. The area of the contact surface had a diameter of 4-5 microns, where E-cadherin-mediated adherens junctions were found. At 12-13 days of gestation, hepatoblasts surrounded large ellipsoidal hematopoietic foci, with long cytoplasmic projections. In addition to the adherens junctions, small desmosomes appeared to bind hepatoblasts together, and biliary canaliculi could be recognized between hepatoblasts. At peak stage of liver hematopoiesis at 14 days of gestation, both tight junctions and gap junctions appeared around the biliary canaliculi, and four types of specialized junctions, i.e., adherens junctions, desmosomes, tight junctions and gap junctions, appeared to be fully developed. After 15 days of gestation, hepatocyte volume showed rapid increase, and the surface areas between adjacent hepatocytes were markedly enlarged. As a result, the involuted hematopoietic foci were forced to move from interhepatocytic spaces to perisinusoidal space at the end of the intrauterine life.     

The endothelium in early experimental contact hypersensitivity of the oral mucosa. Leukocyte-binding properties and ultrastructure

Contact hypersensitivity is characterized by an early and specific diapedesis of mononuclear cells into the site of antigenic challenge. In order to study the functional and ultrastructural properties of the endothelium involved in the recruitment of leukocytes, Sprague-Dawley rats were skin sensitized to DNFB; and this was followed by challenge of the oral mucosa. In vitro binding of peripheral blood mononuclear cells to high endothelial venules in lymph nodes was highly specific but no affinity of peripheral blood mononuclear cells to the vessels was observed in normal oral mucosa or in early contact hypersensitivity. However, 10 days after repeated DNFB challenge, occasional vessels bound overlaid peripheral blood mononuclear cells. Ultrastructurally, we identified migration of mononuclear cells through small venules three h after challenge. The vessels involved, however, did not display morphological signs of activation reminiscent of high endothelial venules in lymph nodes. Mast cell degranulation was evident as early as 30 min after challenge, and a possible mechanism for mast cell-mediated leukocyte recruitment is discussed.     

Ultrastructural localization and identification of adrenergic and cholinergic nerve terminals in the olfactory mucosa

Pharmacological and ultrastructural methods were used to demonstrate alpha-adrenergic regulation of secretory granule content of acinar cells of Bowman's glands and to localize and identify adrenergic and cholinergic axonal varicosities and terminals in the olfactory mucosa of the tiger salamander. The alpha-adrenergic agonist phenylephrine caused secretory granule depletion from Bowman's glands; the alpha-adrenergic antagonist phentolamine partially blocked this effect. These observations were quantified using light microscopic computer-assisted morphometric techniques. Both drugs caused morphological signs of electrolye/water transport. Adrenergic axonal varicosities were identified by the presence of small granular vesicles (SGVs, 45-60 nm in diameter) containing electron-dense material that was enhanced by 5-hydroxydopamine loading and chromaffin reaction fixation techniques. Throughout the lamina propria, small fascicles with axons containing SGVs as well as varicosities and terminals with SGVs were located adjacent to blood vessels, Bowman's gland acini, and melanocytes. Mean vesicle diameters at these sites were 54 +/- 7 nm, 50 +/- 9 nm, and 56 +/- 8 nm, respectively; varicosities were located approximately 0.1-1.0 microns from their presumed cellular targets. Axonal varicosities containing small agranular vesicles (AGVs, 65 +/- 8 nm in diameter), identified as cholinergic by their size and by the absence of electron-dense material after 5-hydroxydopamine loading and chromaffin reaction fixation, were located between adjacent acinar cells. In addition, adrenergic varicosities containing SGVs (56 +/- 6 nm in diameter) were found within 1 micron of blood vessels associated with Bowman's gland ducts and sustentacular cells near the base of the olfactory epithelium. These results characterize the ultrastructural basis for adrenergic and cholinergic regulation of vasomotor tone and secretion within the olfactory mucosa.     

Structure of the endodermal epithelium of the chick yolk sac during early stages of development

The structure of the areas pellucida and vasculosa of the early chick embryo (stages 11–29) was examined by light, transmission and scanning electron microscopy. The most striking feature of the endodermal cells of these areas is the presence of large intracellular yolk drops which are characteristic of the regions in which they are found: lipid-like homogeneous drops in the area pellucida, heterogeneously composed pleomorphic drops in the mid-region of the area vasculosa and granular drops at the periphery of the area vasculosa in the region of the sinus terminalis. On morphological criteria it is postulated that granular drops may arise by direct engulfment of extracellular yolk, but this does not appear to be true for pleomorphic or homogeneous drops. Since the apical junctions between endodermal cells across the yolk sac are tight, they seal off the extraembryonic compartment from the vitelline circulation and presumably prevent intercellular passage of the yolk constituents. Thus the endodermal epithelium must mediate the transport of nutrients from the yolk mass to the developing embryo. Endodermal cells exhibit a variation across the yolk sac in the presence and number of structures associated with uptake of extracellular materials. The mid-region of the area vasculosa appears to be the most endocytotically active region with an abundance of microvilli, bristle-coated pits and vesicles and apical canaliculi and vacuoles. There is a close association between the endoderm and vitelline blood vessels and this association is maintained, as the yolk sac develops, by the formation of small vessels juxtaposed between the vascular surface of the endoderm and the walls of the large vitelline vessels.     

Ultrastructural differentiation of stromal and vascular components in early macaque placental villi

In this study, closely staged placental villi from rhesus monkeys between 19 and 60 days of gestation were used to examine (1) the origin of endothelial cells and the mechanism of angiogenesis in villi; (2) the origin of placental macrophages (Hofbauer cells), and (3) the origin of the reticular cells that compartmentalize the stroma. The results did not support the concept that early stromal cells in the villi were derived by in situ delamination from cytotrophoblast. The extraembryonic mesodermal (mesenchymal) cells at the earliest of ages examined contained considerable granular ER. These cells organized into groups and formed primitive intercellular junctions, thus giving rise to the early angioblastic masses. The angioblastic masses were cellular, not syncytial; and lumen formation was not the result of intracellular vacuolization, but rather was the result of the acquisition of junctionally defined spaces. The earliest capillaries lacked intravascular blood cells and a basal lamina. Later, blood cells were evident in the lumina. At about 45–50 days of gestation, fetal capillaries began to indent the basal surface of the trophoblast. The basal lamina of the fetal capillaries still had not developed by 60 days of gestation. Between 35 and 40 days of gestation, significant morphological changes took place in the villous stroma. Evidence was obtained that the mesenchymal cells differentiated into the reticular cells that subdivided the stroma into fibrilrich and fibril-free compartments. At the same time, Hofbauer cells were observed for the first time; they occupied the fibril-free regions of the stroma. We did not observe any clear indications of intermediate stages of differentiation between other stromal cell types and Hofbauer cells. It is suggested that placental macrophages may have an origin independent of other stromal types; one possibility is that they are derived from blood monocytes as in other tissues. It is further suggested that the activities of the macrophages and reticular cells may be important in remodeling the extracellular matrix and may be related to the process of branching morphogenesis in the villi.     

Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts.

Cocultures of dispersed human fetal liver cells with murine Swiss 3T3 fibroblasts resulted in the development of human mast cells after 1 to 4 weeks of culture. Mast cells were detected by immunohistochemistry using a murine monoclonal anti-tryptase antibody, before metachromasia appeared with toluidine blue. When subjected to double immunohistochemistry using murine monoclonal anti-chymase and anti-tryptase antibodies, 94% +/- 10% (SD) of the mast cells seen at day 30 of culture were of the MCT type. These results contrast with those obtained with human mast cells derived from cord blood mononuclear cells cocultured with murine 3T3 fibroblasts which are comprised of substantially greater numbers of MCTC cells, averaging 48% +/- 31% (SD) at day 30 of culture. Mast cells developed in vitro from fetal liver cells or cord blood mononuclear cells contained similar amounts (+/- SD) of histamine (0.9 +/- 0.5 pg/cell and 1.1 +/- 1 pg/cell, respectively) and tryptase (1.7 +/- 0.4 pg/cell and 1.9 +/- 1.2 pg/cell, respectively) on day 30 of culture. Fetal-liver-derived mast cells from a 30-day-old culture were identified by immunoelectron microscopy using gold-labelled antitryptase antibody. Typically, these mast cells appeared immature as they had large nuclear to cytoplasmic ratio and a small number of ill-formed cytoplasmic granules. For both fetal-liver- and cord-blood-derived mast cells, there was no evidence of conversion of the MCT type into the MCTC type provided by this study. These results suggest that commitment to develop as an MCT or MCTC type of mast cell may have occurred in mast cell precursors present in fetal liver and cord blood mononuclear cells, prior to granulation.     

Immunolocalization of CD34 in nasal polyposis. Effect of topical corticosteroids

Airway inflammation in patients with nasal polyps is characterized by the increased presence of eosinophils, the numbers of which are reduced after treatment with topical corticosteroids. Because eosinophilic responses in the airways are in part due to eosinophil progenitor differentiation, we hypothesized that CD34, a cell surface, sialomucinlike glycoprotein that specifically marks hemopoietic progenitors and endothelium, would be expressed in nasal polyp tissue. We sought to identify CD34(+) leukocytes or endothelial cells in nasal polyps. We also investigated the effect of the topical corticosteroid budesonide on the numbers of CD34(+) cells and vessels in nasal polyps. To accomplish this, we performed immunostaining for CD34 protein in tissue sections of nasal polyps from topical steroid-treated and -untreated groups of patients, as well as from one patient before and after systemic corticosteroid therapy. We also examined myeloid colony formation by isolated polyp mononuclear cells, and performed flow cytometry to detect the presence of CD34(+)/CD45(+) cells within these isolated populations. We also examined the in vitro effects of steroids on human umbilical vein endothelial cell (HUVEC) expression of CD34. We detected CD34-immunoreactive mononuclear cells and blood vessels in the lamina propria of all nasal polyps. CD34(+) mononuclear cells resembled immature hemopoietic cells morphologically. Mononuclear cell fractions from polyps contained myeloid colony-forming cells and cells bearing CD45, a pan-leukocyte marker, as well as CD34, and gated as true hemopoietic blast cells. The numbers of CD34(+) cells and CD34(+) vessels in steroid-treated nasal polyps were significantly higher than in steroid-untreated nasal polyps (15.67 +/- 2.08 cells/10 hpf, versus 5.33 +/- 1.36 cells/10 hpf, P = 0.002; 101.25 +/- 6.24 vessels/0.5 mm2 of lamina propria, versus 57.22 +/- 8.00 vessels/0.5 mm2 of lamina propria, P = 0.0008, respectively). A similar upregulation of CD34 immunostaining, especially for mononuclear cells, was observed in one patient after systemic corticosteroid therapy. Steroid treatment in vitro of HUVECs did not result in enhanced CD34 expression. Both CD34(+)/CD45(+) mononuclear cells and CD34(+) endothelial cells are present within nasal polyps, with higher numbers in patients who have received topical corticosteroid treatment. Because enhancement of CD34 expression was not seen in cultured umbilical vein endothelial cells treated in vitro with corticosteroid, the findings of the study suggest that in nasal polyp tissue, steroids enhance numbers of CD34(+) progenitors and endothelial cells via indirect mechanisms.     

Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration

Rat gestation sites were obtained on days 10 through 16 of normal pregnancy. Light and electron microscopic examination of day-10 sites revealed a consistent complex pattern of stromal cell morphologies. Six distinct regions were identified: an antimesometrial region of epithelioid decidual cells that form the gestation chamber containing the embryo and extraembryonic membranes; an abembryonic antimesometrial decidual region, the decidual crypt, where the cells are separated by large extracellular spaces; a mesometrial region with granule-containing cells and mesometrial decidual cells; a region of spiny cells that are lateral to the antimesometrial decidual cells and continuous with the mesometrial decidual cells; and a region of undifferentiated stromal cells adjacent to the myometrium. Between days 12 and 16, the antimesometrial decidua becomes thinner and is eventually sloughed into the newly formed uterine lumen. The role of the antimesometrial decidual cells is discussed with reference to trophoblast invasiveness, protein synthesis, and especially remodeling of the gestation chamber. Differences between decidua and deciduoma are considered.     

In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection

Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. We investigated NK cell homeostasis by labelling dividing cells in vivo using deuterium-enriched glucose in young and elderly healthy subjects and patients with viral infection. Following a 24-hr intravenous infusion of 6,6-D(2)-glucose, CD3(-) CD16(+) NK cells sorted from peripheral blood mononuclear cells (PBMC) by fluorescence-activated cell sorter (FACS) were analysed for DNA deuterium content by gas chromatography mass spectrometry to yield minimum estimates for proliferation rate (p). In healthy young adults (n=5), deuterium enrichment was maximal approximately 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (+/- standard deviation) proliferation rate was 4 x 3 +/- 2 x 4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 +/- (7 x 6) x 10(6) cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6 x 9 +/- 4 x 0%/day; half-life (T((1/2))) < 10 days]. Healthy elderly subjects (n=8) had lower proliferation and production rates (P=2 x 5 +/- 1 x 0%/day and 7 x 3 +/- (3 x 7) x 10(6) cells/l/day, respectively; P=0 x 04). Similar rates were seen in patients chronically infected with human T-cell lymphotropic virus type I (HTLV-I) (P=3 x 2 +/- 1 x 9%/day). In acute infectious mononucleosis (n=5), NK cell numbers were increased but kinetics were unaffected (P=2 x 8 +/- 1 x 0%/day) a mean of 12 days after symptom onset. Human NK cells have a turnover time in blood of about 2 weeks. Proliferation rates appear to fall with ageing, remain unperturbed by chronic HTLV-I infection and normalize rapidly following acute Epstein-Barr virus infection.     

Platinum in blood mononuclear cells from patients after cisplatin therapy

The feasibility of monitoring cisplatin chemotherapy by measuring platinum (Pt) concentrations in blood mononuclear cells was tested in six patients (four with squamous cell carcinomas of the head and neck, one with non-Hodgkin's lymphoma, and one with lung carcinoma). Blood samples (20 to 40 mL) were collected at intervals from 6 min to 21 days after an iv infusion of cisplatin (80 or 100 mg per m2). Blood mononuclear cells were harvested on a Ficoll-Hypaque gradient, washed repeatedly, counted, and homogenized by sonication in 0.5 mL of saline solution. Pt was analyzed in duplicate 40 microL samples of plasma (N = 26) or cell homogenates (N = 23) by electrothermal atomic absorption spectrophotometry with Zeeman background correction. Immediately after the cisplatin infusion, plasma Pt concentrations averaged 2.6 (SD +/- 0.2) mg per L and mononuclear cell Pt concentrations averaged 2.5 +/- 0.5 ng per 10(6) cells. At 24 to 26 hr post-infusion, plasma Pt concentrations averaged 1.6 +/- 0.2 mg per L and mononuclear cell Pt concentrations averaged 2.3 +/- 0.6 ng per 10(6) cells. Plasma Pt disappearance followed two-compartment kinetics in all patients; the plasma Pt disappearance half-time (T1/2, mean +/- SD) was 148 +/- 41 hours. The Pt concentrations in blood mononuclear cells diminished gradually during the period of observation; the T1/2 could not be reliably determined, but was estimated to be longer than two weeks. This study shows that Pt can be measured in mononuclear cells of patients after cisplatin treatment and that Pt disappears more slowly from blood mononuclear cells than from plasma of these patients.     

Live but not heat-killed mycobacteria cause rapid chemotaxis of large numbers of eosinophils in vivo and are ingested by the attracted granulocytes.

We studied leukocyte chemotaxis triggered by a local injection of mycobacteria (Mycobacterium avium and M. smegmatis) in BALB/c and C57BL/6 mice. Our experimental model consisted of the induction of a subcutaneous air pouch in the dorsal area of mice and inoculation 6 days later of 10(8) CFU of myocobacteria. Inflammatory exudates were harvested from the air pouch cavities 15, 30, and 45 min after the injection of the inocula. Injection of the microorganisms resulted in the migration of an elevated number of eosinophilic granulocytes into the inflammatory cavities. At 30 min after the inoculation of the mycobacteria, the air pouches contained between (3.9 +/- 0.3) x 10(5) (M. avium) and (3.3 +/- 0.3) x 10(5) (M. smegmatis) eosinophils, corresponding to more than one-third (41.4 to 38.3%) of the leukocytes present in the inflammatory cavities. Less than one-half of the eosinophils were attracted to the air pouches when the same number of heat-killed mycobacteria were inoculated [(1.3 +/- 0.2) x 10(5) cells for M. avium and (1.5 +/- 0.2) x 10(5) cells for M. smegmatis]. Injection of gram-negative bacteria (Escherichia coli), of latex beads, or of casein resulted in the attraction of inflammatory eosinophils in numbers that were comparable to those attracted by the heat-killed mycobacteria. Our data document the fact that live mycobacteria exert a rapid chemotactic effect on eosinophils. We therefore postulate that mycobacteria either contain or induce the production of an eosinophilotactic factor. Because this chemotactic effect occurs during the acute inflammatory response to mycobacteria, it cannot be due to the formation of immune complexes (a major infection-associated chemotactic factor for eosinophils). The attracted eosinophils had an important role in the local phagocytosis of mycobacteria, as indicated by our finding, derived from thin-section electron microscopy quantifications, that at 30 min after M. avium inoculation the inflammatory exudates contained (2.2 +/- 0.5) x 10(5) mycobacterium-bearing eosinophils (corresponding to 57% of the total eosinophils), as compared with (2.1 +/- 0.1) x 10(5) neutrophils and (1.5 +/- 0.2) x 10(5) macrophages with ingested bacilli. We conclude that mycobacteria induce the attraction of eosinophils to inflammatory sites and that these granulocytes have the capacity to phagocytize these bacilli in situ.     

Microanatomy of enkephalin-containing neurones in the developing rat spinal cordin vitro

Enkephalin has been demonstrated in neurones in organotypic cultures of embryonic rat lumbar spinal cord using the indirect immunofluorescence technique. The peptide was demonstrated in neurone precursors in cultures of spinal cord at stage 16 (10 days' gestation). The subsequent development of the cells was studied by culturing spinal cord tissue from stage 30–34 (14–17 days' gestation) embryos and applying sequential immunocytochemistry, silver impregnation histology and autoradiography to the preparations. Acetylcholinesterase and γ-aminobutyrate transaminase activity in neurones and fibres in stage 30–34 cultures was at a maximum three weeks after explantation. Following a 24 h pulse of [³H]thymidine, about one third of enkephalin-immunoreactive neurones were labelled in cultures 7 days old. The proportion fell to nearly zero in cultures 14 and 21 days old. At this time axonal and dendritic growth began in most immunoreactive cells. The disappearance of neuritic growth cones indicated that differentiation of the cells was not complete until 5–7 weeks after explanation. Fully-differentiated neurones had cell bodies of 10–15 μm diameter and processes spanning 400 μm or less. During development and at maturity, three groups of immunoreactive neurone were identified by morphological criteria. Immunocytochemical demonstration of leucine enkephalin-containing neurones in smear preparations of spinal cord from young animals suggested that their cytodifferentiation was also completed late in the process of development in vivo (after the second week of life).The results show that lumbar spinal neurones containing the putative transmitter, leucine enkephalin, have a prolonged but otherwise normal pattern of development when grown in tissue culture. When mature they appear to be small interneurones of several morphological types.     

Development of esophageal epithelium in the fetal and neonatal mouse

Development of the fetal mouse esophageal epithelium was followed using light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and radioautography. At 15 days of gestation in the cervical (C), mediastinal (M), and abdominal (A) segments of the esophagus, the epithelium was two or three cells thick, and only cells located in the basal (germinal) layer incorporated tritiated thymidine. Ciliated cells were sparse in all three segments. At 17 days of gestation, longitudinal mesenchymal ridges became more differentiated in the distal segment. Labeling indices were lower than at preceding stages in each segment. Ciliated cells had increased in number and appeared to be evenly distributed along the whole esophagus. In periodic acid-Schiff (PAS)-stained sections, an increasing proximodistal distribution of glycogen stores was observed, with greatest concentrations found in segment A. At 18 days of gestation, labeling indices were comparable in segments C and M (11.7% +/- 2.9% and 12.8% +/- 1.9%, respectively) but remained higher in segment A (17.9% +/- 2.0%). Ciliated cells were still present. At this stage, transverse circular furrows and ridges started to appear. They increased in number at 4 days after birth and were very closely distributed in the adult. In longitudinal sections, these ridges corresponded to projections of stratum granulosum and of the overlying stratum corneum. After birth, ciliated cells desquamated rapidly but some patches were still present at 4 days. At 8 days, the esophageal epithelium was not yet keratinized.     

Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography

The role of micrometer and submicrometer surface roughness on the interaction of cells with titanium model surfaces of well-defined topography was investigated using human bone-derived cells (MG63 cells). The early phase of interactions was studied using a kinetic morphological analysis of adhesion, spreading and proliferation of the cells. By SEM and double immunofluorescent labeling of vinculin and actin, it was found that the cells responded to nanoscale roughness by a higher cell thickness and a delayed apparition of the focal contacts. A singular behavior was observed on nanoporous oxide surfaces, where the cells were more spread and displayed longer and more numerous filopods. On electrochemically microstructured surfaces with hemispherical cavities, arranged in a hexagonal pattern, the MG63 cells were able to go inside, adhere and proliferate in cavities of 30 or 100 microm in diameter, whereas they did not recognize the 10 microm diameter cavities. Cells adopted a 3D shape when attaching inside the 30 microm diameter cavities. Condensation of actin cytoskeleton correlated with vinculin-positive focal contacts on cavity edges were observed on all microstructured surfaces. Nanotopography on surfaces with 30 microm diameter cavities had little effect on cell morphology compared to flat surfaces with same nanostructure, but cell proliferation exhibited a marked synergistic effect of microscale and nanoscale topography.     

Isolation, phenotyping, and functional analysis of lymphocytes from human liver.

The isolation of mononuclear cells from human liver tissues was achieved by a simple method consisting of enzymatic and mechanical dissociation, density gradient centrifugation, and adherence to plastic. The method was optimized to obtain a nearly complete recovery of different lymphoid subpopulations. The mononuclear cells recovered from "normal" liver tissues consisted of 33 +/- 9% (mean +/- SD) small lymphocytes, 44 +/- 6% large granular lymphocytes, 9 +/- 2% monocytes/macrophages, 9 +/- 3% granulocytes, and 5 +/- 2% other cells as determined by microscopic analysis of May-Grünwald-Giemsa-stained cytocentrifuge smears. Phenotypic analysis of the liver-infiltrating lymphocytes (LIL) by two-color flow cytometry showed that CD3-CD56+ NK cells represented 43 +/- 6% (mean +/- SD), CD3+CD56- T cells, 30 +/- 9%, and CD19+ B cells 3 +/- 1%. The predominant phenotype of the liver-infiltrating NK cells was CD3-CD56+CD16- in contrast to that of circulating NK cells, which are largely CD3-CD56+CD16+. The alpha/beta TCR+ T cells were 42 +/- 14% and gamma/delta T cells were infrequent (4 +/- 4%). The proportions of the three major lymphoid populations (T, NK, and B cells) recovered from liver tissues of 10 patients with different liver diseases were altered. Tissue sections from the same specimens were stained by the immunoperoxidase method to compare the in situ cellular composition with that determined by flow cytometry. LIL recovered from normal (control) and virally infected (non-A, non-B hepatitis) liver tissues had high NK activity (up to 1,000 LU/10(7) cells) as measured against K-562 targets in 4-hr 51Cr-release assays. NK activity was significantly lower (P less than 0.05) in LIL recovered from other liver diseases. LIL had spontaneous cytotoxicity against NK-resistant Daudi targets which was significantly higher (P less than 0.05) in control and virally infected than in other liver tissues. Our results indicated that human LIL recovered from normal and diseased liver tissues contained a high proportion of functionally active NK cells and that NK and lymphokine-activated killer activities but not the percentages of CD56+ cells were decreased in end-stage primary biliary cirrhosis and primary sclerosing cholangitis. In contrast, the proportions of NK cells and NK activity remain high in non-A, non-B hepatitis.     

Effect of in vitro colchicine and oral theophylline on suppressor cell function of asthmatic patients.

Extrinsic asthmatic patients have been reported to have a deficiency of concanavalin A (Con A)-induced suppressor cell function. We tested whether in vitro colchicine and oral theophylline can correct this immunoregulatory abnormality. Asthmatic patients' mononuclear cells were incubated with Con A and/or colchicine and then suppression of proliferation was measured by coculture of these cells with healthy volunteers' mononuclear cells and phytohaemagglutinin. The Con A induced suppressor cell function of 29 theophylline treated patients (26 +/- 16%, mean +/- s.d.) was significantly (P less than 0.002) increased as compared to 21 untreated patients (12 +/- 10%) but significantly (P less than 0.01) decreased as compared to 45 healthy volunteers (39 +/- 17%). A pharmacological concentration (10(-8) M) of colchicine had no significant effect on Con A-induced suppressor cell function of 19 untreated patients (from 12 +/- 9% to 9 +/- 22%) but significantly (P less than 0.05) increased Con A-induced suppressor cell function of 20 theophylline treated patients (from 26 +/- 17% to 36 +/- 19%). Thus asthmatic patients have decreased Con A-induced suppressor cell function which is partially corrected by oral theophylline and almost completely corrected by oral theophylline plus in vitro colchicine. This synergistic effect raises the possibility that oral colchicine together with theophylline may be useful in treating patients with extrinsic asthma.     

Quantitation of the intrarenal uptake of immunoglobulin aggregates by macrophages in diffuse proliferative glomerulonephritis.

The intrarenal processing of circulating immune-complex-like-material is traditionally attributed to resident glomerular mesangial cells. To assess the contribution of infiltrating mononuclear cells to macromolecule processing by diseased glomeruli we have utilized an isolated perfused kidney system (IPK) to quantify the specific glomerular uptake of heat-aggregated immunoglobulin (AIgG) (as micrograms of aggregated IgG per 10(4) glomeruli (microgram 10(4)glom)). Mononuclear cell infiltrated glomeruli from animals with diffuse proliferative glomerulonephritis had a significantly augmented uptake of AIgG (48.9 +/- 3.8 micrograms/10(4)glom; normal 11.8 +/- 0.6 micrograms/10(4)glom; P less than 0.01). Specific blockade of mononuclear cell function by perfusion with anti-macrophage-serum (AMS) prevented increased AIgG uptake (12.8 +/- 1.2 micrograms/10(4)gloms; P less than 0.01), but had no effect on the AIgG uptake of normal kidneys (13.1 +/- 1.2 micrograms/10(4)glom). Thus, in diffuse proliferative glomerulonephritis the observed increase in the glomerular clearance of AIgG was due to phagocytosis by mononuclear cells. This study suggests that infiltrating, bone-marrow derived mononuclear cells may significantly contribute to the glomerular handling of circulating immune complexes by nephritic glomeruli.     

Characterization by scanning and transmission electron microscopy of avian peripheral blood mononuclear cells exhibiting natural killer-like (NK) activity

Peripheral blood mononuclear cells (MN) were isolated from 10-week-old Single Comb White Leghorn female chickens (Cornell K strain) and incubated with either LSCC-RP9 or P815 tumor cells at a ratio of 200:1. Samples were filtered through a 2 micron nucleopore filter at 15 min, 30 min, 1 hr, 2 h, 3 h, and 4 h following incubation to isolate conjugates for scanning electron microscopy (SEM). For transmission electron microscopy (TEM), MN were incubated with LSCC-RP9 tumor cells at a ratio of 200:1 for 1 h. Conjugates in the suspension were fixed, stained, and embedded for thin sectioning. Samples were viewed to determine morphological characteristics of cells having NK-like ability and tumor cell destruction process. The MN possessing NK-like activity appear to be small lymphocytes (5-6 microns in diameter) with a small cytoplasm to nuclear ratio and many surface projections. In addition, it was shown that conjugate formation occurred in both tumor cell lines and the lymphocyte surface projections appear to play a role in the destruction process. Tumor cell destruction did occur with both cell lines, however, the LSCC-RP9 tumors cells were destroyed more rapidly.