Morphological analysis of leucocyte transmigration in the pleural cavity.

The role that pleural mesothelial cells play in leucocyte transmigration into the pleural cavity was investigated in lipopolysaccharide-stimulated mice. Changes in mesothelial cell morphology and changes in expression of adhesion molecules on mesothelial cells and leucocytes were analysed by light microscopy, immunohistochemistry, transmission electron microscopy (TEM) and immuno-scanning electron microscopy (immuno-SEM). After stimulation, the mesothelial cells separated completely from one another before leucocyte penetration across the mesothelial layer occurred. These changes occurred primarily in the immediate vicinity of ribs, where a large number of leucocytes accumulated. Immuno-SEM showed that the expression of intercellular adhesion molecule-1 (ICAM-1) on the parietal pleural mesothelial cells was significantly up-regulated by lipopolysaccharide stimulation, and that of vascular cell adhesion molecule-1 (VCAM-1) was induced. Both were restricted to the microvilli of the mesothelial cells. By contrast, expression of intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1), mucosal addressin cell adhesion molecule-1 (MAdCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1), peripheral node addressin (PNAd) and fibronectin were not detected. Lymphocyte function associated antigen-1 (LFA-1), macrophage-1 molecule (Mac-1) and very late appearing antigen-4 (VLA-4), all ligands of ICAM-1 and VCAM-1, were present on the transmigrated neutrophils and macrophages. These findings demonstrate that the immediate vicinity of ribs is a source of leucocyte migration into the pleural space.

Three-dimensional morphological analysis of antigen-antibody reaction in hepatic sinusoids preserved in hypothermic UW solution.

ICAM-1 antigen-antibody reaction was visualized by three-dimensional immunoscanning electron microscopy of hepatic sinusoids in rat liver treated with hypothermic University of Wisconsin (UW) organ preservation solution. The results were compared with similar antigen-antibody reactions carried out with immunoliposomes injected in vivo. Morphologically, the hepatic sinusoids were preserved well during the hypothermic procedure. Endothelial cells had a large number of fenestrations, which partly aggregated and formed sieve plates. ICAM-1 expression was induced by injection of LPS and detected by monoclonal antibody in the UW solution followed by gold-labeled secondary antibody. ICAM-1 was restricted mostly to the unique areas of sieve plates with immature, small fenestrations. A similar distribution of ICAM-1 was present when detected by in vivo injection of immunoliposomes containing the monoclonal ICAM-1 antibody. The results showed that antigen-antibody reactions can take place in livers preserved in hypothermic UW solution. Further, the reaction is similar to that which could occur in vivo during transplantation. This suggests that it may be possible to block potentially harmful antigen-antibody reactions by addition of appropriate antibodies to hypothermic UW solution prior to transplantation.

Retinal manifestations of HIV-1 and HIV-2 infections among hospital patients in The Gambia, west Africa.

BACKGROUND: In developed countries, 50-75% of AIDS patients develop retinal complications and about 20-40% acquire cytomegalavirus (CMV) retinitis. We conducted a cross-sectional survey to determine prevalence of these in The Gambia where both HIV-1 and HIV-2 infection are present and the prevalence of HIV-1 is rising. METHOD: All patients attending hospital whose percentage CD4+ cells (CD4%) was below 14, the level associated typically with an AIDS diagnosis, and one half of those whose CD4% was 14 or above were asked to join the study. Fifty-six HIV-1, 52 HIV-2 and 12 dually infected patients were recruited. Photographs of the fundi were taken and interpreted independently. The findings were related to the patients' percentage CD4+ cells. RESULTS: The CD4% was < 14 in 40 patients and < 7 in 17 patients. Thirty-six patients were male. No cases of CMV retinitis were found. Four patients whose CD4% were 4, 5, 11 and 23 had cotton wool spots ranging in number from 1 to 14 for any one patient. The prevalence of cotton wool spots was 8% (95% CI, 0-16%) among patients with CD4% below 14 and 12% (95% CI, 0-27) among patients with CD4% below 7. One of the 4 patients had associated microaneurysm and blot haemorrhages typical of more advanced HIV microvasculopathy. CONCLUSION: CMV retinitis is less common in The Gambia than in developed countries. Non-infectious retinopathy may also be less common.

Three-dimensional distribution of intercellular adhesion molecule-1 on lymphocytes in the high endothelial venule analyzed by backscatter electron imaging.

The intercellular adhesion molecule-1 (ICAM-1) is a ligand for the beta2 integrin. It is present on the vascular endothelium and plays an important role in cell to cell interaction in lymphocyte recirculation. ICAM-1 is expressed not only on the endothelium but also on other cells, especially lymphocytes adhering to the endothelium, B cells and mitogen-stimulated T cells. To define the functional role of ICAM-1 in lymphocytes, the three-dimensional distribution of ICAM-1 on the surface of lymphocytes in the rat mesenteric lymph node HEV was investigated by immunoscanning electron microscopy. ICAM-1 expression was closely associated with variations in surface structures of lymphocytes, which showed a cap-like structure, membrane ruffles and slender microvilli. ICAM-1 was preferentially localized on the cell surface including membrane ruffles (about 90% of all counted gold particles), but rarely on the microvilli (10%). The results suggest that activated lymphocytes with variations in surface structures demonstrate moderate expression and preferential localization of ICAM-1. This seems to enable a three-dimensional interaction of the microvilli of one lymphocyte with the surface membrane of another cell and might be a prerequisite for cluster formation between them, as seen in antigen presentation.

Ultrastructural localization of the intercellular adhesion molecule (ICAM-1) on the cell surface of high endothelial venules in lymph nodes.

BACKGROUND: The high endothelial venules (HEV) in the lymph nodes are essential for lymphocyte recirculation. As a first step, the HEV surface interacts with lymphocytes through adhesion molecules. It is important to know where adhesion molecules are expressed on the surface ultrastructure and how these structures interact with lymphocytes. METHODS: To demonstrate the ultrastructural mechanism of interaction between the HEV surface and lymphocytes through the intercellular adhesion molecule (ICAM-1), rat mesenteric lymph nodes were perfused through the superior mesenteric artery with the primary antibody (antirat ICAM-1 antibody) and secondary antibody (antimouse IgG coupled to 15 nm gold particles), which were diluted with hypothermic University of Wisconsin (UW) solution. After the immunoreaction, we analyzed the HEV three-dimensionally and quantitatively using immunoscanning electron microscopy (ISEM) combined with transmission electron microscopy (TEM). RESULTS: HEV expressed ICAM-1 in a 5-30-fold higher concentration than other vessels. Its distribution was extensive over the luminal surface of the cell down to the junctional area. The endothelial surface of HEV undulated to form branched microfolds, along which ICAM-1 was expressed. Cytoplasmic processes of lymphocytes were seen in microfurrows between microfolds and adhered to the sides of the folds. CONCLUSIONS: These observations imply that the microfolds expressing ICAM-1 and microfurrows are specific ultrastructural features for trapping lymphocytes, thus initiating lymphocyte emigration into the lymph node parenchyma.

Astrocytic contribution to functioning synapse formation estimated by spontaneous neuronal intracellular Ca2+ oscillations.

Glial contribution to in vitro synaptic function was investigated in a neuron-glia co-culture system by monitoring spontaneous oscillations of intracellular Ca2+ in neurons. Rat cortical neurons, grown stably on a cortical astrocyte monolayer, extended neurites resulting in marked functional synapse formation. Little synapse formation was observed in neuronal co-culture with meningeal fibroblasts or endothelial cells. Aged astrocytes in vitro (C35) were found to attenuate synaptic development, while young astrocytes (C5) markedly promoted synaptic function. C5 and C35 astrocyte media conditioned yielded no significant synaptogenic effect, indicating diffusible factor(s) are not responsible for our observation. Modulation of astrocytic proliferation and differentiation by gliostatin, a glial growth inhibitor, or dibutyryl cAMP affected neuronal synaptic function on the co-cultures. Site-specific analysis in homologous and heterologous neuron-astrocyte co-cultures among cortex, hippocampus, septum, and striatum revealed that homologous combinations of neurons and astrocytes derived from identical brain regions elicited the largest number of synchronizing neurons. These results suggest that in vivo neuronal synaptic function essentially requires the participation of adjacent astrocytes, which is site-specific and age-dependent.

Ultrastructural changes in hepatocytes, sinusoidal endothelial cells and macrophages in hypothermic preservation of the rat liver with University of Wisconsin solution.

To identify subtle changes which might lead to liver failure after liver transplantation, rat livers stored at 4 degrees C in University of Wisconsin solution for 8, 16, 24, and 32 h were examined by transmission electron microscopy, scanning electron microscopy, cellular matrix maceration and freeze fracture for ultrastructural analysis. Endothelial cells exhibited aggregation of intramembrane particles (IMPs) at 8 h and produced tiny blebs accompanied by marked development of pits. As deterioration advanced, endothelial cells exposed the perisinusoidal faces of hepatocytes directly to the lumen with destruction of sieve plates. They then degraded with loss of IMPs. Macrophages followed a similar deterioration process to endothelial cells. Membranes of hepatocytes did not demonstrate aggregations of IMPs for 32 h. Rough endoplasmic reticulum (rER) lost ribosomes and smooth ER (sER) increased in amount and dilated in an irregular form. Autophagosomes appeared in the cytoplasm, engulfed cytoplasmic matrix containing intracellular organelles and became autophagic vacuoles. At 32 h bile canaliculi were filled with detached vesicles. This may be one of the causes of preservation related bile duct complications after liver transplantation.

Neurotrophic action of gliostatin on cocultured neurons with glial cells.

Gliostatin is a polypeptide factor (apparent M(r) = 100 k with a homodimeric structure comprising two 50 kDa subunits) acting on cortical neurons (neurotrophic action) as well as astrocytic cells (growth inhibition). Under the coculture system of cerebral cortical neurons and astrocytes from fetal rats (E15 or E16), the neurotrophic action of gliostatin was examined immunocytochemically. Immunostaining by an anti-neurofilament (NF) monoclonal antibody visualized a marked neurite-outgrowth and interconnecting bundles of neuritic processes induced by gliostatin in the coculture system. Neurons stimulated by gliostatin formed dense aggregates in clumps, while neurons in control coculture spread out. Gliostatin has also shown survival-promoting effects on neurons. Furthermore, it was shown that gliostatin induced the differentiation of protoplasmic astrocytes to fibrous astrocytes. These results further support our previous contention that gliostatin plays physiological roles on neuronal and glial development.

Improvement of left ventricular function after renal transplantation in a patient with uremic cardiomyopathy: report of a case.

The improvement of heart function in a patient on hemodialysis with dilated cardiomyopathy by renal transplantation is herein reported. The patient was a 35-year-old woman. Hemodialysis had been initiated 3 months before, but she experienced difficulty with hemodialysis maintenance and exhibited congestive heart failure. The ejection fraction (EF) was decreased to 36.6% in the echocardiogram, and an intracardiac biopsy of the right ventricle showed myofiber degeneration and interstitial edema upon examination by light microscopy. She then underwent renal transplantation, and the postoperative recovery was almost uneventful. The cardiothoracic ratio decreased rapidly to around 40% after 1 month, although her body weight increased. The ejection fraction increased to 50% in the echocardiogram. An intracardiac biopsy of the right ventricle revealed disoriented myofibers, but myofiber degeneration improved, and no interstitial edema was present upon examination by light microscope. The electron microscopy showed that the intracellular edema had disappeared and other degenerative changes had also improved. The patient was discharged on the 44th postoperative day, with a serum creatinine of 1.3 mg/dl.

Effectiveness of modified University of Wisconsin solution for heart preservation as assessed in heterotopic rat heart transplant model.

A solution developed at the University of Wisconsin is an excellent preservation solution for the kidney, pancreas, and liver. We studied the effectiveness of a modified University of Wisconsin solution, which was made by reversing the sodium and potassium concentrations, in heart preservation. We first examined the relationship between total ischemic time and 1-week graft survival rates using a saline solution as a control preservation solution by transplanting a heart to the neck of test animals. To determine preserved heart viability, we palpated the heart 1 week postoperatively. The 1-week graft survival rates for the various total ischemic times were as follows: less than 1 hour, six of six; 4 hours, five of six; 8 hours, two of six, 12 hours, one of six; and 16 hours, one of six. These results demonstrate a correlation between total ischemic time and survival rate. Then we compared three preservation solutions--glucose-insulin-potassium, Bretschneider's solution, and the modified University of Wisconsin solution--for 16 hours of simple preservation. The 1-week graft survival rates were one of six for glucose-insulin-potassium, two of six for Bretschneider's solution, and five of six for the modified University of Wisconsin solution. The high survival rate for the modified University of Wisconsin solution under adverse conditions indicates its effectiveness in heart preservation.

[Clinical analysis on side effects of long-term oral administration of futraful to out-patients].

Ninety-six cases of cancer patients were treated with long-term oral administration of Futraful ranging more than one to five years. Group A consisted of 57 cases receiving the drug for 1-2 years and Group B of 39 cases receiving the drug for 3-5 years. In Group A abnormality of TTT was observed in 49% and also abnormality of LHD in 32% of the patients, abnormalities of hemoglobin, leucocyte count, lymphocyte per cent, GOT, GPT, ALP, Bilirubin and ZST were respectively reported in 10-20%. On the other hand, in Group B abnormalities of TTT (64%), hemoglobin (26%), LDH (21%) were observed as well as abnormalities 10-20% of leucocyte count, lymphocyte, GOT, and ALP in each approximately 10-20% of the patients. But all of the disorders were not severe and clinically not serious. These results suggested and safety of long-term oral administration of Futraful to out-patients.