Morphological, phenotypic and karyotypic characterization of a novel natural-killer-cell target - evidence of involvement of MHC class-I molecules in NK cell recognition.

A new cell line from a neoplastic ascites was established. This strain, designated PAT-206, was characterized by plastic adherence and a high proliferative potential without any specific growth factor requirement. Karyotype analysis showed that the line was of human chromosomal constitution and aneuploid. Surface marker analysis showed that CD45, CD33 and CD15 were positive. In addition, the presence of human cytokeratins was detected by cytoplasmic immunofluorescence. Interestingly, the cell line did not express major hystocompatibility complex (MHC) class I and II, and was more sensitive than the 'classic' K562 cell line, to killing mediated by fresh uncultured peripheral blood lymphocytes. Following differentiation with interferon-gamma, the cell line expressed MHC class I antigens and resulted resistant to natural killing mediated lysis. This novel NK cell target seems to be suitable for further studies on NK cell specificities.

Ultrastructural study of the vascular response in small early gastric cancer.

The microvasculature of the stroma of four cases of small early gastric cancer (EGC) was investigated by conventional electron microscopy. Severe damage to small and large fenestrated capillaries was observed around endothelium-adherent, partially degranulated neutrophils. The findings suggest the existence of neutrophil-mediated injury of endothelial cells during the development of inflammatory responses in small EGC. The severely injured microvessels exhibited increase in vasopermeability, microhaemorrhage, and platelet aggregates. Other microvascular changes included endothelial cell and pericyte activation as well as basal lamina replications, indicative of repeated episodes of endothelial injury, necrosis and regeneration. This new capillary growth within the old basal laminas shared morphological features with a peculiar angiogenic process described in man and animals.

Localization of vinculin and talin at perineurial cells of human sural nerve.

Immunolocalization of spectrin, vinculin, talin, desmin and titin was investigated in human sural nerve. No binding for spectrin and titin was seen in any structure of the nerve. Antibody against desmin immunostained sporadic epineurial vessels only. Endoneurial and epineurial vessels were intensely positive for vinculin and talin. We found expression of vinculin and talin at the perineurial cells, using immunocytochemistry and gold immunoelectron microscopy. Since vinculin and talin are known to be involved in cell-cell and cell-extracellular matrix transmembrane connections, we propose that they, possibly together with other cytoskeletal proteins, may be implicated in the permeability barrier property of the perineurium. In pathological conditions, perineurium plays an as yet unknown role. Future studies are needed to investigate expression of vinculin and talin in neuropathies.

Regional changes in spinal cord glucose metabolism in a rat model of painful neuropathy.

Spinal cord patterns of metabolic activity in a model of neuropathic pain were assessed in unanesthetized rats by the [14C]-2-deoxyglucose (2-DG) technique. Rats used in this procedure had demonstrable thermal hyperalgesia ipsilateral to sciatic nerve ligation and ipsilateral hindpaws that were lifted in a guarded position. The latter indicated possible spontaneous pain. Sciatic nerve ligation produced significant increases in glucose utilization in the dorsal and ventral horns of both sides, with greater activity present on the ipsilateral as compared to the contralateral side. Peak activity was in laminae V-VI, a region involved in nociceptive processing. Thus, a chronic increase in neuronal activity in these regions may reflect spontaneous neuropathic pain.

Effect of intracarotid injection of iopamidol on local cerebral glucose utilization in rat brain.

We assessed, by means of the [14C]-2-deoxy-D-glucose autoradiography method, the effect of intracarotid injection of a nonionic, low-osmolar contrast medium (iopamidol) on local cerebral glucose utilization in the rat brain. Contrast medium was injected at 20 degrees C and at 37 degrees C, and the relative changes in local cerebral glucose utilization were measured. At 20 degrees C the viscosity of the contrast agent was about twice that of the same solution at 37 degrees C, and resulted in a statistically significant increase in local cerebral glucose utilization in the hemisphere ipsilateral to the side of intracarotid infusion. Saline control studies showed that the metabolic change was not related to either the solution temperature or the osmolality. These findings suggest that increased viscosity of a contrast medium may contribute to its neurotoxic effects during cerebral angiography, hence emphasizing the importance of preheating contrast material to avoid adverse reactions.

Blood-brain barrier permeability changes after experimental subarachnoid hemorrhage.

Basic mechanisms underlying cerebrovascular permeability responses to subarachnoid hemorrhage (SAH) are still to be defined in detail. Previous investigations examining the occurrence of blood-brain barrier (BBB) breakdown after SAH in the experimental setting have yielded conflicting results. In a rat model of SAH, we assessed BBB changes by means of the quantitative [14C]-alpha-aminoisobutyric acid technique. Experiments were carried out on the second day post-SAH. In blood-injected rats [14C]-alpha-aminoisobutyric acid transport across the BBB increased significantly in cerebral cortices and cerebellar gray matter, averaging 1.3 to 1.5 times control values. The present data indicate that SAH induces well-defined changes in BBB function, possibly involved in the pathogenesis of post-SAH cerebral dysfunction in humans. Results reported here have also potential clinical implications for the management of aneurysm patients.

Effect of whole brain radiation on local cerebral glucose utilization in the rat.

We assessed, by means of the [14C]-2-deoxy-D-glucose autoradiography method, the effect of whole-brain x-radiation on local cerebral glucose utilization in the rat brain. Animals were exposed to conventional fractionation (200 +/- 4 cGy/day, 5 days/week; total dose, 4000 cGy). Metabolic experiments were made 2 to 3 weeks after completion of the radiation exposure. In comparison with control and sham-irradiated animals, cerebral metabolic activity was diffusely decreased after irradiation. Statistically significant decreases in metabolic activity were observed in 13 of 27 brain regions studied. In general, the brain areas with the highest basal metabolic rates showed the greatest percentage of decrease in glucose utilization. The concept that radiation suppresses glucose utilization before any morphological change takes place in the cell structures was the basis of this study. Metabolic alterations after irradiation may explain the syndrome of early delayed deterioration observed in humans after whole-brain radiotherapy. These studies have applications to observations made with the [18F]-fluorodeoxyglucose method in conjunction with positron emission tomographic scans in patients receiving radiation therapy for intracranial malignancies. The data reported here also have potential clinical implications for the evaluation of a risk/benefit ratio for radiotherapy in patients with benign neurosurgical diseases or children undergoing prophylactic treatment of the central nervous system.

Time-related ultrastructural changes in an experimental model of whole brain irradiation.

To stimulate therapeutic irradiation, we exposed rats to conventional fractionation (200 +/- 4 cGy/d, 5 d/wk; total dose, 4000 cGy). The effects of this regimen were assessed by electron microscopic examinations of brain microvascular and parenchymal cells 15 and 90 days after irradiation. Studies of the transendothelial passage of horseradish peroxidase provided information about the functional status of the blood-brain barrier. At 15 days after irradiation, there was an increased vesicular transport of horseradish peroxidase across the intact endothelium without opening of the tight junctions, and without evidence of structural alterations of neuropil, neuronal bodies, and astrocytes. Ninety days after irradiation, well-defined ultrastructural alterations were observed, involving the microvasculature, the neuropil, the neuronal bodies, and astrocytes. The main ultrastructural feature of cortical microvessels was their collapsed aspect, associated with perivascular edema containing cell debris. Altered neurons and reactive activated astrocytes were also noticeable. These data suggest a possible association, not necessarily causal, between damage of the microvascular/glial unit of tissue injury and development of radiation-induced brain toxicity.

Quantitative study of blood-brain barrier permeability changes after experimental whole-brain radiation.

Basic mechanisms underlying the tolerance and reaction of the central nervous system to ionizing radiation are not known precisely. We investigated the possibility of a change in blood-brain barrier (BBB) function as a causative factor for early delayed whole-brain radiation-induced cerebral dysfunction. Rats were exposed to conventional fractionation (200 cGy/d, 5 d/wk; total dose, 4000 cGy). BBB changes were assessed by means of the quantitative 14C-alpha-aminoisobutyric acid technique and electron microscopy. Studies of the passage of horseradish peroxidase across the BBB permitted comparative quantitative isotopical and qualitative morphological data. Experiments were carried out 2 to 3 weeks after the completion of the radiation exposure. The transport of 14C-alpha-aminoisobutyric acid across the BBB increased significantly in cerebral cortex and cerebellar gray matter, averaging 1.3 to 1.5 times over the normal values. Electron microscopy disclosed an intense vesicular response of the cortical microvascular endothelium that occurred without the opening of the tight junctions and resulted in an intense transport of HRP across the intact endothelium. The present data indicate that moderate doses of whole-brain radiation induce well-defined changes in BBB function, which possibly are involved in the pathogenesis of radiation-induced cerebral dysfunction in humans.

Scanning electron microscope study of human arachnoid villi.

The functional morphology of human arachnoid villi obtained from surgical biopsy specimens has been studied by scanning electron microscopy (SEM). On SEM examination, the villi appeared to be distended, as if functioning normally. The endothelial cells constituting the cerebrospinal fluid (CSF)-blood interface were covered by numerous microvilli, uniformly oriented along the major axis of the villus. Examination for cell-to-cell contact revealed only occasional areas of tight adherence between adjacent endothelial cells, while widened intercellular spaces were frequently observed. Generally corresponding to the apex of the villus, points of emergence of endothelium-lined hollow structures were identified; these may represent apical openings of open pathways from the subarachnoid space to the venous system. Ultrastructural arrangements consistent with a closed system of CSF reabsorption were also observed. Large cells maximally distended and protruding into the sinus lumen were commonly seen; these were interpreted as the result of the formation of giant vacuoles within the endothelium covering the villus. This study has provided ultrastructural evidence for both closed and open systems of CSF reabsorption. Ultrastructural findings, such as gaps between endothelial cells and tubule-like endothelium-lined structures as previously identified in animals and observed in man by transmission electron microscopy, were demonstrated in human biopsy specimens by SEM.

Progressive perturbations in cerebral energy metabolism after experimental whole-brain radiation in the therapeutic range.

Basic mechanisms underlying the tolerance and reaction of the central nervous system to ionizing radiation have not been fully elucidated in the literature. The authors employed the [14C]-2-deoxy-D-glucose autoradiography method to investigate the effect of whole-brain x-irradiation on local cerebral glucose utilization in the rat brain. The animals were exposed to conventional fractionation (200 +/- 4 cGy/day, 5 days/week for a total dose of 4000 cGy), and the effects of this regimen were assessed at 2 weeks and 3 months postirradiation. In rats evaluated 2 weeks after treatment, statistically significant decreases in cerebral metabolic activity were found in 13 of the 27 regions studied, compared to control animals. In rats studied 3 months after treatment, additional metabolic suppression and statistically significant decreases in cerebral metabolic activity were found in 11 of the 27 regions, compared to rats studied 2 weeks after treatment. A weighted-average rate for the brain as a whole was approximately 15% and approximately 25% below that of control animals 2 weeks and 3 months after exposure, respectively. Although the difference in species is significant enough so that direct extrapolation to humans may not be appropriate, the data reported here may have potential clinical implications for the evaluation of the risk-benefit ratio for radiotherapy. This model can be used reproducibly for further investigations, including evaluation of therapies that may reduce irradiation-induced brain injury.

The effect of the calcium antagonist nimodipine upon local cerebral glucose utilization in the rat brain.

The new calcium antagonist Nimodipine has been shown to have more powerful dilator action on cerebral than peripheral vessels. The effect of the drug on cerebral metabolism was studied in conscious rats using the /14C/-2-deoxyglucose quantitative autoradiographic technique. Intravenous injection of Nimodipine, 2 mcg/Kg, determined significant increases in local cerebral glucose utilization that appeared to be homogeneous in magnitude and anatomic distribution throughout the brain. This study raises the question whether Nimodipine affects brain functions by other mechanisms than an increase in cerebral blood flow.

Primary adenocarcinoma of the arytenoid: an ultrastructural study.

Primary adenocarcinoma of the larynx is a rare neoplasm that tends to spread to both regional lymph nodes and distant sites. A case of primary adenocarcinoma of the arytenoid in a 74-year-old man is presented. The tumor was evaluated by light and electron microscopy. A high percentage of intranuclear pseudoinclusions (more than 20% of the neoplastic cells) was a peculiar characteristic of the tumor. To the best of our knowledge, such a feature has not been reported previously and should be considered a hallmark of more aggressive behavior.

Effect of x-radiation to brain on cerebral glucose utilization in the rat.

We assessed, by means of the [14C]-2-deoxy-D-glucose autoradiography method, the effect of whole-brain x-radiation on local cerebral glucose utilization in the rat brain. Animals were exposed to conventional fractionation (200 +/- cGy/day given 5 days a week) to a total dose of 4000 cGy. Metabolic experiments were made 2 weeks after completion of the radiation exposure. In comparison with control and sham-irradiated animals, cerebral metabolic activity was diffusely decreased following irradiation. Statistically significant decreases in metabolic activity were observed in 13 of 27 brain regions studied. In general, brain areas with the highest basal metabolic rates showed the greatest percentage drop of glucose utilization. Post-irradiation metabolic alterations possibly provide an explanation for the syndrome of early delayed deterioration observed in humans after whole-brain radiotherapy.

Radiation-induced blood-brain barrier changes: pathophysiological mechanisms and clinical implications.

The pathophysiology of whole-brain radiation (WBR) toxicity remains incompletely understood. The possibility of a primary change in blood-brain barrier (BBB) associated with microvascular damage was investigated. Rats were exposed to conventional fractionation in radiation (200 +/- cGy/d, 5d/wk; total dose, 4,000 cGy). BBB changes were assessed by means of the quantitative 14C-alpha-aminoisobutyric acid (AIB) technique coupled with standard electron microscopy (EM) and morphometric techniques as well as studies of the transcapillary passage of horseradish peroxidase (HRP). At 15 days after WBR, AIB transport across BBB increased significantly in cerebral cortex. EM disclosed vesicular transport of HRP across the intact endothelium without opening of the tight junctions. Ninety days after WBR, well-defined alterations of the microvasculature were observed. The main feature of cortical microvessels was their collapsed aspect, associated with perivascular edema containing cell debris. Data suggest a possible association between damage of the microvascular/glial unit of tissue injury and development of radiation-induced brain cerebral dysfunction. We hypothesize the following sequence of pathophysiological events: WBR causes an early increase in BBB permeability, which produces perivascular edema and microvascular collapse. The interference with microcirculation affects blood flow and energy supply to the tissue, resulting in structural damage on an ischemic/dysmetabolic basis.

Response of satellite cells and muscle fibers to long-term compensatory hypertrophy.

The ultrastructural changes of the plantaris muscle of the rat have been investigated during long-term compensatory hypertrophy. The latter was induced by the removal of the synergistic gastrocnemius muscle. Transmission electron microscope (TEM) observations were performed on the 20th, 40th and 60th days after surgery on overloaded and control muscles. The plantaris muscle had hypertrophied 54.8% after 20 days, 74.4% after 40 days and 79.8% after 60 days. It was observed that activation of the satellite cell developed as shown by an increase in cytoplasmic volume, together with diffuse presence of polyribosomes and a rough endoplasmic reticulum. On the 40th day masses of swollen mitochondria grouped at the edge of muscle fibers were observed. On the 60th day structures completely covered with basal lamina, whose cytoplasm was filled with mitochondria and polyribosomes, were partially detached from the fibers. The above ultrastructural changes were absent in control contralateral muscles. These findings suggest that surgically induced overload produces a relevant ultrastructural re-arrangement in muscle tissue.

Development of the pigeon bursa of Fabricius. A scanning and transmission electron microscope study.

The development of the pigeon bursa of Fabricius was investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Particularly SEM emphasized that the bursa during development progressively undergoes hypertrophia and hyperplasia of lobules constituting the plicae. Moreover modifications of surface epithelium were observed. During early days of development after hatching, epithelium showed evenly distributed microvilli which become shorter and shorter and also unevenly distributed with aging. In addition TEM allowed us to state that the bursa of Fabricius in pigeons, during development, undergoes morphological modifications among which one of the most remarkable is the gradual and continuous increasing of lymphocytes. Another one, as it was already observed in chickens (Frazier 1974), consists in the contemporaneous but independent development of cortex and medulla, even if we observed undifferentiated epithelial cells moving from medulla to cortex and a migration of lymphocytes and mesenchymal cells from cortex to medulla (Tar et al. 1958; Ackerman et al. 1964; Naukkarinen et al. 1978).

Chronic heat-induced skin lesions (erythema ab Igne): ultrastructural studies.

Erythema ab igne (EI) is an uncommon skin lesion caused by mild and repeated exposure to infrared sources. The aim of this study was to investigate the ultrastructural alterations in this condition. The ultrastructural study was carried out on 5-outpatients who presented typical EI of their exposed sites. Skin punch biopsies were processed for standard electron microscopy. The epidermis was hyperpigmented, with focal regressive changes of basal keratinocytes. An apparent functional activation of melanocytes with numerical increase of dendritic processes was also observed. The dermis showed abundant melanophages and occasional elastic fiber alterations similar to actinic elastosis. No alterations consistent with preneoplastic skin conditions were observed. The ultrastructural findings associated with EI seem to be nonspecific and consistent with moderate regressive changes of keratinocytes as well as a consensual melanocytic activation and elastic fiber modifications. Similar alterations can be observed in chronic actinic skin damage. This condition is presumably more benign than the ultraviolet exposure. The association of EI and premalignant skin lesions, though occasionally described, seems relatively infrequent.

Eruptive syringomas with calcium deposits in a young woman with Down's syndrome.

Eruptive syringomas are uncommon in the general population. We describe here an 18-year-old female, affected by Down's syndrome, who presented with an abrupt eruption of small skin-colored or reddish papules on the face, neck and limbs. Light microscopy allowed us to diagnose syringomas, whereas the study of the ultrastructural features revealed calcium deposits in many lumina and also in the mitochondria. This observation confirms the hypothesis that the syringeal structure plays a role in the pathogenesis of calcinosis cutis.

Primary macular amyloidosis: an ultrastructural approach to diagnosis.

Seven cases of primary macular amyloidosis were studied on skin biopsies. The Congo red stain was positive only in three cases, whereas the ultrastructural observation allowed for the detection of amyloid deposits in all biopsies. Fibrillary degeneration of basal keratynocytes was occasionally observed, and regressive changes of keratynocytes and dermal nerve bundles presumably related to the intensity of the scratch trauma were detected in one case. In six biopsies mast cell profiles exhibiting various degrees of degranulation were detected in the dermis. Melanosome aggregates were also observed consistently in dermal macrophages and occasionally in Schwann cells. A variable degree of structural alteration was observed in dermal unmyelinated nerve fibers. Even if the intimate mechanism of amyloid deposition was not explained by the ultrastructural study, this approach is a useful instrument in the differential diagnosis of cutaneous macular hyperpigmented lesions.