Association of hypercalcemia with tumors producing colony-stimulating factor(s).

Two human malignant tumors, which we previously reported to produce colony-stimulating factors (CSFs), were found to be accompanied by remarkable hypercalcemia. A patient with a CSF-producing lower jaw cancer (squamous cell carcinoma) developed a marked granulocytosis (150,000/microliters) and hypercalcemia (more than 215 mg/dl). The tumor was successfully transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and hypercalcemia (20 mg/dl). White blood cell and serum calcium concentrations of these mice decreased promptly to normal levels when the tumor was excised. Treatment with prednisolone (1.5 mg/kg) or indomethacin (5 mg/kg) had no effect on the serum calcium level of these mice. Parathyroid hormone or prostaglandin E was not increased in the serum of the mice or in the tumor tissue. However, the mice bearing the tumor excreted extremely large amounts of calcium in their urine, and their bony tissues contained less calcium and phosphorus than controls. Moreover, histology of bony tissues of these nude mice clearly demonstrated the decrease in trabecular tissues and cortical thickness as well as remarkable activation of osteoclasts. Another patient with a CSF-producing bronchogenic squamous cell carcinoma showed mild granulocytosis and hypercalcemia. The biopsied tumor tissue was transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and also severe hypercalcemia (18 mg/dl). These results suggest the presence of a new syndrome of granulocytosis and hypercalcemia associated with CSF-producing tumors. The causal mechanism of the hypercalcemia was shown to be some humoral factor which activates osteoclasts other than parathyroid hormone. Neither prostaglandins nor osteoclast-activating factor seemed to be the cause of the hypercalcemia.

Lignoceroyl-coenzyme A synthetase from developing rat brain: partial purification, characterization and comparison with palmitoyl-coenzyme A synthetase activity and liver enzyme.

As part of a long-term study of sphingolipid metabolism in brain, we have purified and partially characterized a long-chain acyl-CoA synthetase from microsomes of developing rat brain and compared it with the hepatic microsomal enzyme from the same animals. Both enzymes were solubilized from microsomes by treatment with Triton X-100 and then chromatographed successively on Blue-Sepharose and DEAE-Sepharose. Blue-Sepharose chromatography yielded a single peak with acyl-CoA synthetase activity, whereas DEAE-Sepharose chromatography of both brain and liver preparations yielded two peaks. Elution patterns of lignoceroyl-CoA synthetase and palmitoyl-CoA synthetase activities were identical throughout these steps and were similar in brain and liver. Gel filtration of each DEAE-Sepharose fraction on Sephadex G-200 also yielded two peaks of activity. The more rapidly eluted material contained much more lignoceroyl-CoA synthetase activity, while the activity for palmitoyl-CoA synthetase was higher in slower eluting peaks. In all preparations the ratio of lignoceroyl-CoA synthetase activity to palmitoyl-CoA synthetase activity was much higher in brain than in liver. These results suggest that although the brain acyl-CoA synthetase is chromatographically similar to the liver enzyme, there are differences in substrate specificity.

Development and characterization of cationic liposomes conjugated with HVJ (Sendai virus): reciprocal effect of cationic lipid for in vitro and in vivo gene transfer.

Today, nonviral gene transfer vectors attract more attention as a therapeutic strategy for human diseases, because viral vectors such as adenoviral and herpes viral vectors have been proven to have problems, especially in immunogenicity and cytotoxicity. However, the main limitation of nonviral vectors has been low efficiency of gene expression. To overcome this defect, we have developed a new class of transfection vehicles, HVJ-cationic liposomes. The use of the cationic lipid DC-cholesterol facilitates efficient entrapment of negatively charged macromolecules (plasmid DNA, oligodeoxynucleotides, and proteins) and efficient interaction with negatively charged plasma membranes of cultured cells in vitro. Moreover, the fusogenic envelope proteins of hemagglutinating virus of Japan (HVJ) enhance delivery of the enclosed materials into cells. Using firefly luciferase as a marker, we optimized the liposome formula. As a result, we have succeeded in obtaining 100-800 times higher gene expression in vitro than with the conventional HVJ-anionic liposomes. However, in vivo gene transfer experiments have revealed that the use of cationic lipid instead of anionic lipid reduced the transgene expression dramatically in organs such as muscle and liver. We further discovered that the use of anionic liposomes with a viral-mimic king lipid composition increased transfection efficiency by several times in vivo. We conclude that the alternative usage of transfer vectors, for example, HVJ-anionic liposomes for in vivo delivery to extended areas of organs and HVJ-cationic liposomes for in vitro delivery (and possibly for in vivo delivery to a restricted area of organs), is of significance.

Visualization of the retinal microvasculature by targeted dye delivery.

Although fluorescein angiography has proven to be an important tool in the diagnosis and management of retinal vascular diseases, it is subject to certain limitations, namely the presence of the choroidal background, which usually precludes a detailed examination of the retinal microvasculature. Moreover, the inability to repeat the bolus reduces the chance of obtaining high-quality photographs of early phases, and does not allow for a complete binocular examination or for testing the response to induced physiologic changes. We have developed a method of targeted dye delivery that consists of encapsulating the dye in lipid vesicles, injecting them intravenously, and causing them to release their contents locally when a short heat pulse is induced in a retinal artery by a laser. This method was applied in the rhesus monkey in order to visualize the retinal microvasculature. A well-defined bolus and absence of background fluorescence permitted both following of the dye front through the vasculature and clear imaging of the capillary network over the whole posterior pole. The bolus delivery could be repeated as many as 100 times in 45 min without significant loss of contrast. The comparison of these results with conventional fluorescein angiography illustrated the advantage of the new method. The examination of the safety of the delivery system indicates that there is no major obstacle to the eventual application to humans.

Quantitative analysis of retinal hemodynamics using targeted dye delivery.

A new method designed to allow repeated mapping of retinal hemodynamics on a macro- and microcirculatory level was evaluated in the primate eye. The method, called "targeted dye delivery," consists of encapsulating a fluorescent dye in temperature-sensitive liposomes, injecting the liposomes systemically, and using a light pulse from an argon laser to release a bolus of dye in a targeted retinal vessel. The follow-up of the well-defined dye front thus generated allows calculation of the blood flow and capillary transit time. Evaluation of targeted dye delivery in a monkey indicated that centerline blood velocity and the vessel diameter can be measured with a reproducibility of 10% and 4%, respectively, in vessels that are 40 microns and larger. These measurements yielded flow values that had a reproducibility of 10% on the same day and 13% on different days. The normalization of flow rate by the vessel diameter was consistent with theoretic estimates and promises to be a circulation indicator independent of variations between individual and species. The transit time across capillary beds at different locations was found to be similar, thus indicating that the method could be used to evaluate the local viability of the microcirculation.

Noninvasive visualization of the choriocapillaris and its dynamic filling.

PURPOSE: The choroidal microvasculature and its circulation are inadequately assessed by presently available techniques. Laser-targeted delivery was applied to generate local, repetitive angiograms of the choriocapillaris in primates. METHODS: Carboxyfluorescein was encapsulated in heat-sensitive liposomes and injected intravenously in monkeys. The liposome contents were then released locally in the choroid by application of a short heat pulse provided by an infrared laser. The bolus of dye spread rapidly downstream from the underlying arterioles into clusters of lobules. Video angiograms were generated with excitation illumination provided by an argon laser. RESULTS: Laser-targeted delivery choroidal angiography performed on three monkeys indicated that the fluorescence was emitted mainly from the choriocapillaris. Clusters of irregular shape with well-defined margins were observed. Adjacent arteries typically supplied separate clusters that fit together like a jigsaw puzzle. The dynamic filling and emptying patterns, recorded at video rate, revealed that macular lobules were filled by a central arteriole and drained by a venous annulus. The average dye transit time through a lobule (n = 10) was 118 +/- 26 msec (mean +/- SD), and the dye transit velocity was 2.53 +/- 0.55 mm/sec. CONCLUSIONS: This study clearly documents the segmental nature of the primate choroidal microvasculature. It also illustrates that choroidal angiography by laser-targeted dye delivery provides information useful for studying the response of the choriocapillaris to physiological and pathologic changes.

Feasibility of targeted drug delivery to selective areas of the retina.

A new method was developed to deliver locally a bolus dose of a drug to the retinal vasculature. The targeted delivery system was based on encapsulating the drug in heat-sensitive liposomes, which are injected intravenously and lysed in the retinal vessels by a heat pulse generated by a laser. To test if substances delivered in the vessels could also penetrate into the surrounding tissue, 6-carboxyfluorescein was encapsulated in liposomes and used as a marker for drug penetration. Moderate argon laser pulses were applied to the retinal vessels of Dutch pigmented rabbits to induce breakdown of the blood-retinal barrier (BRB). A suspension of liposomes at a dose of 2 ml/kg body weight, corresponding to a carboxyfluorescein dose of 12 mg/kg, was injected into the ear vein. The dye was released from the liposomes proximal to the damaged portion of the vessel. Fundus fluorescein angiograms were recorded with a video camera and digitized for subsequent image analysis. The penetration of carboxyfluorescein into the retinal tissue was evaluated by comparing the fluorescence intensity of the area around the damaged vessel with that of an adjacent control area. The dye penetration increased with the numbers of laser applications (P less than 0.001). The leakage was localized distally to the released site and was restricted to areas with a disrupted BRB. The mass of carboxyfluorescein that penetrated gradually spread with time. Both veins and arteries could be used for the targeted delivery. These results indicated that this delivery system, which is fully controllable by laser through the pupil, can deliver drugs inside the vasculature and into the retinal tissue wherever the BRB is disrupted.

Asian hereditary neuropathy patients with peripheral myelin protein-22 gene aneuploidy.

Japanese hereditary neuropathy with liability to pressure palsy (HNPP) patients have a deletion of one peripheral myelin protein-22 (PMP22) gene region in distal chromosome band 17p11.2 as do Caucasian patients. Japanese and Asiatic Indian CMT1A patients have a PMP22 gene duplication that results in Charcot-Marie-Tooth disease type IA (CMT1A; HMSNIA) in patients of European and Middle Eastern ancestry. About 70% of Japanese CMT1 patients have a PMP22 duplication as do Caucasians, while Japanese CMT1B, CMT2 and Dejerine-Sottas patients to not have PMP22 gene region aneuploidy. Although HNPP and CMT1A genotypes are generated simultaneously by unequal recombination that results in PMP22 gene aneuploidy in each daughter cell, only 3 Japanese HNPP probands with PMP22 deletion from a large patient population were referred to a single center compared to 18 referred CMT1A probands with PMP22 duplication. This lower HNPP frequency more likely reflects lower HNPP reproductive fitness than patient ascertainment bias because disease severity and variation in severity is about the same in CMT1A and HNPP patients and because all patients of both types were referred regardless of disease severity. These results, along with an apparently high de novo CMT1A mutation rate, suggest that common ancestors of Japanese, Asian Indians, and Caucasians carried PMP22 geneflanking sequences that enhance unequal crossing over.

Effect of streptozotocin diabetes on gingivitis in plaque-susceptible rats.

The periodontal pocket probing depths of mandibular incisors of plaque-susceptible (Sus) rats, which spontaneously exhibit gingivitis with accumulation of plaque, were increased 20 days after injection of streptozotocin (70 mg/kg, i.v.). The accumulated plaque weights were also increased in Sus rats with streptozotocin diabetes, and a positive correlation was found between the plaque weights and the pocket depths. Histological findings showed that this inflammatory reaction in gingival tissue was higher and more extensive in diabetic Sus rats than in control Sus rats. These findings suggest that the accumulated plaque is the important factor for the severe breakdown of gingival tissue in this experimental model.

Malignant mixed mesodermal tumour of the peritoneum with a complete response to cyclophosphamide.

We treated a 66-year-old Japanese woman with malignant mixed mesodermal tumour of the heterologous type arising from the paracolic peritoneum. The tumour contained heterologous elements in the form of cartilage in addition to carcinosarcomatous areas. This is the third case of malignant mixed mesodermal tumours originating from the peritoneum to be documented in the literature. A complete resection of the tumour was carried out but multiple lymph nodes metastases in the left neck occurred 5 months later. Cyclophosphamide in doses of 150 mg daily by mouth was initiated, with the result of a complete regression of the malignant lesions. She died of myocardial infarction 21 months after surgery.

Dentinogenic ghost cell tumor: histologic aspects, immunohistochemistry, lectin binding profiles, and biophysical studies.

Dentinogenic ghost cell tumor accompanied with calcifying odontogenic cyst (COC) was described in terms of its clinical, histological, immunohistochemical, lectin binding and biophysical properties. The case was a 38-year-old Japanese female, in whom the tumor had arisen in the right mandibular premolar and molar region. Material obtained by partial mandibulectomy was used. Decalcified paraffin sections were used to detect keratins, involucrin, and lectin binding; and non-decalcified thin sections were used for biophysical analysis. The lesion comprising dentinogenic ghost cell tumor and COC contained odontogenic epithelium with ghost cells, eosinophilic amorphous materials and osteodentin. Some of the eosinophilic material had undergone transformation into osteodentin. Keratins in odontogenic epithelia showed positive PKK1 staining in peripheral tumor cells, and stainings with KL1 and involucrin were positive in centrally located cells. Lectin binding in the amorphous materials was comparatively strong for PNA, and SBA, moderate for WGA, RCA-1, and UEA-1, and slight for DBA and ConA. Lectin binding affinities were higher in the amorphous materials than in the osteodentin. Elemental analysis with an electron probe X-ray microanalysis of the amorphous materials and osteodentin showed a pattern similar to that found in the normal dentin. The biologic properties of the eosinophilic amorphous materials suggested the material to be poorly calcified osteodentin, which gradually transformed into the well-calcified type.

Metallothionein immunoreactivity in head and neck carcinomas; special reference to clinical behaviors and chemotherapy responses.

Metallothionein (MT), has selectively binding affinity for heavy metal ions and over expression of MT has a potential against resistance for CDDP anticancer agents and radiation treatment. The role of MT immunoreactivity of squamous cell carcinoma in oral and pharyngeal regions (n = 28) and in the maxillary sinus region (n = 3) was evaluated for distribution patterns of MT and clinicopathologic behaviors. All the sections were examined in 400x and counted for MT positive cells over 5 fields of tumor growing foci. MT immunoreactivity was expressed in both tumor cell cytoplasm and nuclei, and showed heterogeneous localization in tumor epithelial cells and in the stroma. Immunohistochemical localizations showed mosaic patterns as the highest MT staining tumor cells intermingled with negative or low staining cells in neoplastic foci, and in stromal cells. Histiocytic and fibrocytic cells in both peripheral and interstitial stromas were also not stained homogeneously. In oral and pharyngeal carcinomas (n = 28), MT positive cell index in treated cases (n = 11) was 17.85% and that in non treated tumors (n = 17) was 25.19%. In maxillary sinus carcinomas (n = 3), MT positive index was 4.56% and showed lowers levels as compacted to other SCC sites. Among histological grading in oral and pharyngeal SCCs, MT index of well differentiated SCC (n = 9) was 17.04%, of moderately differentiated SCC (n = 13) 21.92% and poorly differentiated SCC (n = 6) was 31.06%. There is no significant correlation of positive index of metallothionein between treated and untreated samples taken in oral and pharyngeal SCCs.

Effects of in vivo soluble selectin gene introduction of lps-induced leukocyte accumulation in the murine lung.

The selectin family adhesion molecules exert a crucial role in accumulation of leukocytes at the site of inflammation. To test the biological effects of soluble selectin on lung inflammation, we introduced an expression plasmid vector of soluble selectin gene via HVJ-liposome into a murine model of LPS-induced lung injury. The myeloperoxidase activity in LPS-injected mice was suppressed by the in vivo injection of soluble P-selectin gene relative to control mice. On the contrary, soluble E- or L-selectin genes did not exert suppressive effects. Our observations suggest that P-selectin plays a crucial role in the initial steps of lung inflammation, and exogenous introduction of soluble P-selectin by in vivo gene transfer method may be a useful strategy for regulating inflammation of the lung.

Experimental gingivitis in ODU plaque-susceptible rats. V. The presence of bradykinin in the gingival tissue and the bradykinin inactivating factor in rat dental plaque.

Plaque formation and gingival inflammation were found in ODU plaque-susceptible rats. A study was performed to determine the relationship between the presence of bradykinin in gingival tissue and the inflammatory capacity of rat plaque. Bradykinin content in gingival tissue and the bradykinin inactivating activity of dental plaque of 12-month-old ODU rats were measured. The presence of bradykinin was observed in the gingival tissue of the ODU rat and a bradykinin inactivating factor was found in the rat plaque. The factor was destroyed at 60 degrees C for 30 minutes, at pH 1.5 or by treatment with perchloric acid. The optimum pH of the factor was approximately 8.0. Chelate reagents, such as O-phenanthroline and EDTA . 4Na, strongly inhibited the bradykinin inactivating factor. The protease inhibitor, Trasylol, also inhibited this factor. The SH reagent, L-cysteine, inhibited weakly and Zn++ inhibited less. The investigation suggested that the rat plaque posseses the ability to inactivate bradykinin and plays an important role in modifying the inflammatory response caused by bradykinin in gingival tissue.

Experimental gingivitis in ODU plaque-susceptible rats. VI. Histopathological studies.

The purpose of this study was to document the histological development of gingivitis in the plaque-susceptible ODU rat. Specimens were taken every 3 months over a 12 month period. In the 1st month a mile lymphocytic infiltrate was seen near the junctional epithelium. This infiltrate spread to the deeper connective tissue by the 3rd month. In the 6 to 9 month animals the cells of the junctional epithelium were partially destroyed resulting in beginning of sulcus ulceration. At this time osteoclasts were noted. By 12 months the cellular infiltrate was diffuse and large macrophages were evident. There were numerous enlarged capillaries.

Experimental gingivitis in ODU plaque-susceptible rats. III. Toxic activity of the rat dental plaque.

The toxicity tests of the dental plaque from ODU plaque-susceptible rats showed strong lethal effect on mice, and abscess forming effect on guinea-pigs. Bacterial cells isolated from the rat dental plaque also showed strong toxicity on both animals and capillary permeable activity on rabbits. Among these bacterial cells, Corynebacterium showed the strongest toxic effects on these animals. These facts suggested an important role of the dental plaque on initiation and development of gingivitis and that especially Corynebacterium may play an important role on gingivitis in ODU plaque-susceptible rats.

Experimental gingivitis in ODU plaque-susceptible rats. I. Changes of plaque formation and body weight.

New strains of ODU plaque-susceptible and resistant rats have been developed starting from litter mating at our laboratory, in 1972. Those strains were selected and mated for further generations. ODU plaque-susceptible and resistant rats are those that show the presence or absence of plaque formation in their gingiva of lower incisors after they are fed a commercially available powder diet. Experiments were started when rats weighing 75 to 85 gm were about 5 weeks old being fed with 20 gm of a powder diet a day. Almost every 4 days, plaque formation was observed in terms of 5 degrees from zero to 4 degrees and body weight. As a result, plaque formation in plaque-susceptible and resistant rats clearly showed their quality in their 3rd generation. Those qualities will be further emphasized as the generation advances. In the 3rd generation of plaque-susceptible rats, no difference was found in growth between those ingested with a powder diet and those with a solid diet of the same ingredients. But in the first 2 months, male plaque-resistant rats of the 3rd generation grew more quickly than susceptible rats.

Experimental gingivitis in ODU plaque-susceptible rats. II. Biochemical nature of the rat plaque.

The characterization of the dental plaque from ODU plaque-susceptible rats is reported. The comparison between the rat dental plaque and a human dental plaque indicates that content of extractable hexose and inorganic salts and amino acid composition are different from each other. Electron microscopic study indicates that bacterial flora of the rat dental plaque seems to be little different from a human dental plaque. In spite spite of obvious differences in biochemical nature between the rat dental plaque and the human dental plaque, there are enough similarities to warrant intensive investigation regarding the relationship between the dental plaque and gingivitis.

Experimental gingivitis in ODU plaque-susceptible rats. V. On the vascular permeability factor of dental plaque.

Plaque-induced inflammation has been documented from various aspects, but is still not well understood. In an effort to clarify the physicochemical properties of dental plaque and to isolate the active factor which give rise to inflammation, we studied a factor in plaque which may be a direct and local cause of gingival inflammation in ODU rats. Rat dental plaque was dissolved in physiologic saline. After being homogenized, the solution was centrifuged and the supernatant (RP) was used as a sample. Protein component (pRP) of PR was isolated by ammonium sulfate fractionated procedure and then deproteinized RP (dpRP) was obtained by trichloroacetic acid treatment. The vascular permeability factor existed in RP and it was affected by heating and pH. Changes in vascular permeability were directly proportionate to the logarithm of the concentration of protein fraction in dental plaque, and were of a delayed type. It was surmised that pRP contained in RP was the main vascular permeability factor, and it was purified by gel filtration on Sephadex G-100 and G-200.

Inhibitory effect of tea polyphenols on histamine and leukotriene B4 release from rat peritoneal exudate cells.

The effect of tea polyphenols on the release of chemical mediators, histamine and leukotriene B4 (LTB4), from rat peritoneal exudate cells (PEC) was studied. Among polyphenols, (-)-epigallocatechin gallate (EGCG) most strongly inhibited the histamine release from the cells stimulated with a calcium ionophore, A23187 or compound 48/80. Though (+)-catechin (C) and (-)-epicatechin (EC) had no effect, (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECG) moderately inhibited the histamine release. Similarly, EGCG, ECG, and EGC inhibited LTB4 release from PEC, whereas C and EC were not effective. The magnitude of the inhibitory effect on the release of these mediators of tea polyphenols was in the order of EGCG > ECG > EGC. These results indicated an important role of the triphenol structure in the inhibitory activity. Therefore, the possible antiallergic effect of tea polyphenols can be expected.