Phase I clinical trial of oral furtulon and combined hepatic arterial chemoembolization and radiotherapy in unresectable primary liver cancers, including clinicopathologic study.

Surgical resection has been accepted as the only curative therapy for primary liver cancer (PLC). Unfortunately, most patients are surgically unresectable when they seek treatment. An alternative therapeutic approach for some of these patients is transcatheter arterial chemoembolization. However, this is not curative by itself, and additional therapy is required to eradicate residual disease. This study investigates the approach of preoperative hepatic arterial chemoembolization followed by the combination of oral Furtulon (5'-deoxy-5-fluorouridine) as a radiosensitizer and external beam radiotherapy (RT). From July 1997 to December 1998, 25 patients with unresectable PLC were treated with hepatic arterial chemoembolization followed by limited-field radiotherapy plus oral Furtulon as a radiosensitizer. Hepatic arterial chemoembolization was performed with 5-fluorouracil 1 g, cisplatin 80 mg (DDP), mitomycin C (MMC) 10 mg, and arterial embolization with iodized oil-10 ml mixed with 10 mg MMC. Hepatic arterial chemoembolization was performed at regular intervals of 6 weeks, and the patients then received limited-field RT. Mean tumor dose was 4,600 cGy (range, 4,100-5,200 cGy) in daily 1.8-Gy fractions, 5 times a week. The toxicity and responses between RT and surgery were assessed. After surgical evaluation, resection was performed. The histopathologic study was also performed in the specimens of both normal and radiation-injured liver tissues from the patients who underwent resection. Seventeen of 25 patients (68%) showed an objective response. One patient with cholangiocarcinoma involving the portal lymph nodes attained a complete response. Eight patients (32%) underwent sequential resection. The most common toxicity was an increase in liver enzymes, which were less than twofold of the upper limit of normal. Follow-up computed tomography studies after treatment showed a low-attenuation area adjacent to the hepatic tumor in the target volume. On pathologic evaluation, the low-attenuation area revealed hyperemia, distended hepatic sinusoids packed with erythrocytes, and hepatic cell loss when examined with microscopy; "new-born" hepatocytes, hepatic cords in the process of forming, and endothelial cells have appeared on electronic microscopic examination. The combination of hepatic arterial chemoembolization and external radiotherapy is efficacious and a safe modality for unresectable primary liver cancers. Furtulon offers the potential for use as a clinical radiosensitizer. Radiation can significantly damage the liver tissue between 41 Gy and 52 Gy, but the new hepatocytes were forming within the radiation-injured liver after RT.

Spindle cell carcinoma of cornea and conjunctiva with pseudosarcomatous metaplasia. Light microscopic, electron microscopic and immunohistochemical findings in 2 patients.

We studied the electron microscopic and immunohistochemical findings in two elderly patients with spindle cell carcinoma of the cornea and conjunctiva. By light microscopy, correct diagnosis was made by finding the transition between the pleomorphic spindle shaped tumor cells and the overlying squamous epithelium. Ultrastructural findings indicated that squamous cell origin included tonofilament-associated desmosomes and keratohyaline granules in the tumor cells. Positive immunohistochemical staining with antikeratin antibodies provided further evidence to prove the origin of spindle cell carcinoma. Follow-up of the two patients showed the malignant tumor to be quite aggressive in terms of intraocular invasion in the first patient and local recurrence in the second.

Electronmicroscopic observation on chronic cardiopathy in spontaneous diabetic biobreeding rats.

The ultrastructural changes of chronic cardiopathy in diabetic biobreeding rats were observed electronmicroscopically. The thickness of the myocardial capillary basement membrane (CBM) and volume fraction of myofilaments in the myocardium were quantitatively measured with a video interactive display system III (VIDS-III). The effect of metabolic control on chronic diabetic cardiopathy was also investigated.

Streptozotocin induced cardiomyopathy in diabetic rats.

Microscopic quantitative cardiomyopathy was previously reported in spontaneous diabetic biobreeding (BB) rats, but the nature of the lesions remains obscure. To further study the specificity of cardiomyopathy in streptozotocin-diabetic rats and discuss the existence of diabetic heart disease, we compared 40 streptozotocin-diabetic rats (D group) with 40 normal control rats (C group) at various durations in plasma glucose, fructosamine, plasma lipid, left ventricular enzyme contents and alterations of myocardial cells and coronary artery tree under electron and light microscope respectively. In 4 weeks, D rats showed myocardial mitochondrial swelling and degeneration, whereas at 8 weeks, myocardial enzyme contents markedly decreased, and myocardial lesions were more conspicuous with disruption of myocardial cells, formation of myocardial contraction bands, dilatation of intercalated discs, deposition of glycogen granules, etc. At 11 weeks, microscopic changes were essentially similar, whereas the decrease of enzyme contents became more conspicuous. During the 11 weeks, persistent and marked hyperglycemia and elevation of fructosamine were observed, however, no abnormalities were found along the coronary artery tree in the D rats. The results indicated that cardiomyopathy is diabetes specific, and exists independently as one of the three important components of diabetic heart disease.

[Ultrastructural and clinical study on neuroblastoma].

Ultrastructures of 11 cases of neuroblastoma and ganglioneuroblastoma were observed. Ultrastructurally, the neuroblastoma could be classified into four types: immature, poorly differentiated, differentiated and ganglioneural cell. Neurosecretory granules (NSG) of round or oval shapes with various diameters in the tumor cells and neural processes were observed. The presence of NSG in the tumor tissue is one of the pathognomic features for neuroblastoma. Comparing and study of the relation between the ultrastructural findings and the excretory patterns of the urinary catecholamine as well as the prognosis, it was shown that the tumor of the differentiated type and the presence of abundant NSG lead to a good prognosis. Some slight differences in ultrastructure were found in neuroblastoma specimens from children of various ages. Infants under 18 months had a longer survival time, which is related to the differentiation and other features of the tumor cells.

[Relative changes between calcium content and number of atrial specific granules induced by water and salt loading].

Rats prepared by either deprivation of water intake for 5 d or drinking 2% NaCl solution for 4 d were sacrificed for dissection of auricle, and making ultrathin sections. The numerical density (Nv) of atrial specific granules (ASG) were counted by using electron microscopic morphometry, and Ca concentration in ASG and in terminal cisterna of sarcoplasmic reticulum (TSR) was determined by using electron microscopic X-ray microanalysis. In the drinking 2% NaCl solution group, the Nv of ASG decreased from 6.02 +/- 2.30 to 2.96 +/- 1.62/microns 3 (P < 0.01) and all companied by a decrease of Ca concentration from 64 +/- 16 to 38 +/- 21 mmol/kg (P < 0.01), while the secretion of atrial natriuretic peptide (ANP) showed some increase. Water deprivation induced all the contrary results. However, the Ca concentration in TSR was not affected by water-salt loading. It is suggested that when the atrial cardiocytes were stimulated by salt loading, Ca in ASG was released into cytosol and thus promoted fusion of ASG membrane with sarcolemma which resulted in release of ANP. Therefore, it is considered that ASG as an intracellular Ca store participates in the stimulus-secretion coupling of ANP through the release of Ca ions.

[Atrial specific granules are an intracellular calcium store].

In order to elucidate whether the secretion of atrial natriuretic peptide (ANP) is correlated to the release of calcium (Ca) from the atrial specific granules (ASG), it is necessary to explore whether the ASG contain highly concentrated Ca and how the high Ca is maintained in the ASG. The present study was designed to determine Ca in the ASG with the quantitative electron microscope X-ray microanalysis and Ca(2+)-ATPase with electron microscopic (EM) cytochemical technique. The ultrathin cryosections of rapid frozen fresh rat auricles were used for measuring Ca concentration with a JEM-1200EX electron microscope equipped with a Link AN 10,000 energy dispersive X-ray spectroscope. The measurement showed that the Ca concentration in the ASG was quite high, being 81 +/- 15 mmol/kg (n = 10), comparable with that found in the sarcoplasmic reticulum. With the Ca(2+)-ATPase EM cytochemical technique, the reaction products proved to be deposited on the membrane of the ASG. It was postulated that the Ca(2+)-ATPase on the membrane of ASG pumped Ca2+ out of the cytosol into the ASG and thus maintained a high Ca concentration inside the ASG. Therefore, the ASG might be considered to be a Ca store in atrial cardiocytes.

[The relationship between cytosolic Ca2+ and two kinds of granule number in activated platelets].

The numerical density (Nv) of the alpha-granule (alpha G) and dense granule (dG) measured by using electron microscopic morphometry and the cytosolic free calcium concentration ([Ca2+]i) by using Ca-fluorescent indicator were observed for studying the relationship of [Ca2+]i and Nv of alpha G and dG in platelets activated by A23187, thrombin or ADP. The results showed that the [Ca2+]i rose markedly by A23187. Thrombin or ADP could also induce an increase of [Ca2+]i concentration-dependently. Under 3 different degrees of activation induced by different agonists, the close relationship between Nv of both granules, alpha G and dG, and [Ca2+]i in platelets was found (Pr(alpha G) < 0.05, Pr(dG) < 0.01). These findings indicate that the increase in [Ca2+]i may enhance the secretion of alpha G and dG.

[The effects of stimulation of the brain osmoreceptor on the tubular reabsorption of sodium, chloride and potassium in rats].

The tubular reabsorption of sodium, chloride and potassium was studied with micropuncture technique and electron probe X-ray microanalysis before and following intracerebroventricular administration of hypertonic saline (icv. HS) in rats. At the late proximal convoluted tubules, the fractional delivery of sodium increased from 53.0 +/- 2.1% to 66.0 +/- 2.9% (P < 0.01), the fractional delivery of chloride increased from 65.4 +/- 3.4% to 78.2 +/- 3.9% (P < 0.05), but the fractional delivery of potassium and tubular fluid osmolarity were unaltered. At the early distal convoluted tubules, the fractional delivery of sodium increased from 8.2 +/- 0.9% to 13.6 +/- 1.8% (P < 0.05), the fractional delivery of chloride increased from 5.4 +/- 0.8% to 9.5 +/- 1.4% (P < 0.05), the tubular fluid osmolality increased from 139.8 +/- 6.9 mOsm/kg H2O to 181.3 +/- 15.6 mOsm/kg H2O2 whereas fractional delivery of potassium did not show significant change. Under the condition of diuresis provoked by the intravenous administration of furosemide the kaliuresis induced by icv. HS was abolished, while the icv. HS-elicited diuresis and natriuresis remained unaffected. These results indicate that stimulation of the brain osmoreceptor inhibits the proximal tubular reabsorption of sodium chloride which in turn enhances sodium-potassium exchange in the distal tubules and collecting ducts.

[The relationship between the chromaffin granule number and the granule calcium content in the adrenal medulla of restrained rats].

Morphometry of numerical density and electron probe X-ray quantitative microanalysis were used to measure the alterations in the number and calcium content of chromaffin granules in adrenal medulla cells during emergency reaction of restrained rats. It was found that both the alterations showed monotonic decrease during the period of restraint, but the rate of decrease of the calcium content was significantly faster than that of the granules. These data are consistent with the hypothesis that calcium release from the granules may increase the cytoplasmic calcium concentration, which again triggers off the granule exocytosis.

[Acetylcholine and A23187 stimulate epinephrine secretion of isolated rat adrenal medulla cells in vitro].

Acetylcholine and A23187, two acetylcholinergic agonists, were used to induce the stimulus-secretion coupling of the isolated rat adrenal medulla cells. Morphometry of numerical density and electron probe X-ray quantitative microanalysis were adopted to measure the alterations in the number and calcium content of chromaffin granules in adrenal medulla cells during the agonist treatment. Secretion of epinephrine was detected by the high performance liquid chromatography (HPLC). It was found that the Ca content of the isolated rat adrenal medulla chromaffin granules dropped significantly after a 10-min incubation with the agonists, whereas the number of chromaffin granule decreased slowly during the agonist treatment and the epinephrine concentration raised significantly after a 20-min incubation with the agonists. The decrease of granule Ca content occurred earlier than the increase of epinephrine concentration, suggesting that the calcium released from chromaffin granule may in part account for the induced cell secretion.

[Electron microscopy observation on pre- and post-treatment skin of dorsum pedis of chyluric patient treated with heat-clearing and hemostatic drugs--the lymphangial wall damage being the pathogenesis of chyluria].

Heat-clearing and hemostatic drugs were effective in treating chyluria. In order to explore the curative mechanism, a group of 10 in-patients with chyluria were chosen and treated with heat-clearing and hemostatic drugs for 45 days. Some pre- and post-treatment skin from dorsum pedis of the patients were taken off and observed under electron microscope. It showed that the walls of lymphatic capillaries were seriously damaged before treatment and rehabilitated wall after treatment. The pinocytotic vesicles were packed tightly in the endothelial cells of capillaries before treatment and their number was decreased remarkably after treatment; the mast cells were rare in number and abnormal as spindle shape before treatment and they increased in number and became round or oval in shape, the nucleus became large, the granules they contained became more in number and larger in size after treatment, lymph edema was noticed before treatment and it disappeared after treatment. The result showed that the extensive lesion of lymphangial walls is the pathogenesis of chyluria, while the rehabilitation of them is the curative mechanism. It seems that in the process of rehabilitation the mast cells act took the role of "soldier-engineer".

[Ultrastructural studies on effects of colchicine in treating hepatic fibrosis of schistosomiasis rabbits].

8 rabbits were infected percutaneously with the cercariae of Schistosoma japonicum. After hepatic fibrosis had developed in these rabbits 4 months after infection, 4 out of the 8 infected rabbits were given colchicine orally at a dosage of 40 micrograms/kg per day for 7 weeks. Another uninfected rabbits were used as controls. The therapeutic effects of colchicine on hepatic fibrosis were studied by transmission electron microscopy and morphometry, in which the area of collagen microfibrils in the space of disse and liver cells was measured. The results showed that colchicine relieved ultrastructural injury of liver cells and reduced the number of active fibroblasts and collagen microfibrils. The area of collagen microfibrils measured in the liver of the infected rabbits and colchicine-treated rabbits accounted for 42.5% and 0.2%, respectively (P less than 0.01), suggesting that colchicine has therapeutic effect against schistosomal liver fibrosis.

X-ray microanalysis of calcium in ischemic skeletal muscle cells.

Tissue sections of both transiently and persistently ischemic musculus latissimus dorsi of rabbits show degenerated and necrotic cells in varying degrees. Degenerated mitochondria appearing in the cells contain moderate electron-dense fluffy intramatrical deposits. However, only in the transient ischemia sample can other two kinds of electron-dense granules be seen between myofibrils. According to their ultrastructure, size and distribution, the larger granules are considered the degenerated mitochondria, and the smaller ones the degenerated sarcoplasmic reticulum. X-ray microanalysis proves that these two kinds of granules contain high concentration of calcium, while the fluffy deposits contain little calcium. The findings suggest that the calcium deposits in the degenerated and necrotic cell seen under light microscope occur in mitochondria and sarcoplasmic reticulum of the transiently ischemic muscle cell. The conditions of calcium accumulation in ischemic muscle cells are discussed.

Inhibitory effects of vinpocetine on sodium current in rat cardiomyocytes.

AIM: To study the effects of vinpocetine (Vin) on the sodium current (INa) in cardiomyocytes. METHODS: The sodium current in adult rat ventricular myocytes was measured by whole cell patch-clamp technique. RESULTS: The INa in cardiomyocytes was blocked reversibly by Vin, in concentration-dependent and voltage-dependent manner, but not rate- or use-dependent. The INa was attenuated by 13%-75% when the Vin concentration was raised from 10 to 80 mumol.L-1. The IC50 (95% confidence limits) was 36.4 (28.1-47.1) mumol.L-1. When the membrane potential depolarized over the range of -90 mV to +40 mV in 10-mV step, inhibitory effect of Vin on the INa was 39% at first, then maintained at a higher level, about 52% +/- 5%. The maximal depression (57%) reached at about 0 mV. Vin influenced both the activation and inactivation processes of sodium channel, and resulted in attenuation of the window currents (the slowly inactivating sodium currents). CONCLUSION: Vin inhibited sodium currents in rat ventricular myocytes.