Haemoglobin-vesicles as artificial oxygen carriers: present situation and future visions.

During the long history of development of haemoglobin (Hb)-based O2 carriers (HBOCs), many side effects of Hb molecules have become apparent. They imply the physiological importance of the cellular structure of red blood cells. Hb-vesicles (HbV) are artificial O2 carriers that encapsulate concentrated Hb solution with a thin lipid membrane. We have overcome the intrinsic issues of the suspension of HbV as a molecular assembly, such as stability for storage and in blood circulation, blood compatibility and prompt degradation in the reticuloendothelial system. Animal tests clarified the efficacy of HbV as a transfusion alternative and the possibility for other clinical applications. The results of ongoing HbV research make us confident in advancing further development of HbV, with the expectation of its eventual realization.

A histochemical study of the camel (Camelus bactrianus) duodenal glands.

The complex carbohydrates in the camel duodenal glands were examined histochemically at light and electron microscopic levels. The duodenal glands of the camel were distributed in the submucosa 2 m caudal from the pylorus. These were branched tubuloalveolar glands. The terminal portion of each lobule was formed by only one type of mucous cell. The duodenal gland cells contained acidic and neutral carbohydrates. The mucous cells mainly contained sulfate and carboxyl carbohydrate with sialic acid, and they also contained a few neutral carbohydrates with different saccharide residues such as mannose, glucose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. The results showed that the secretary granules of the duodenal glands in the camel contain mainly acidic carbohydrates. These findings seem to be the morphological characteristics of the duodenal glands in the camel.

Poly(ethylene glycol)-conjugation and deoxygenation enable long-term preservation of hemoglobin-vesicles as oxygen carriers in a liquid state.

The stability of hemoglobin vesicles (HbV) as an oxygen infusion was tested during the storage for 1 year at 4, 23, and 40 degrees C. The surface of the HbV was modified with poly(ethylene glycol) (PEG), and the suspension was deoxygenated with nitrogen bubbling. The samples stored at 4 and 23 degrees C showed a stable dispersion state for 1 year, though the sample stored at 40 degrees C showed the precipitation and decomposition of vesicular components, a decrease in pH, and 4% leakage of total Hb after 1 year. The PEG chains on the vesicular surface stabilize the dispersion state and prevent the aggregation and fusion due to their steric hindrance. The original metHb content (ca. 3%) before the preservation gradually decreased to less than 1% in all the samples after 1 month due to the presence of homocysteine inside the vesicles which consumed the residual oxygen and gradually reduced the trace amount of metHb. The rate of metHb formation was strongly dependent on the partial pressure of oxygen, and no increase in metHb formation was observed due to the intrinsic stability of the deoxygenated Hb. Preservation at 4 and 23 degrees C slightly reduced P(50) (increased the oxygen affinity) from 38 Torr to 32 and 31 Torr, respectively. These results indicate the possibility that HbV suspension can be stored at room temperature for at least 1 year.

Poly(ethylene glycol)-modification of the phospholipid vesicles by using the spontaneous incorporation of poly(ethylene glycol)-lipid into the vesicles.

The critical micelle concentrations of 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 microM, respectively, in buffer solutions ([Tris] = 20 mM, [NaCl] = 140 mM, pH 7.4) at 37 degrees C. When these PEG-lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEG-DSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEG-DPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.

[MR imaging of hepatocellular carcinoma following microwave coagulation therapy].

MRI was performed in 13 patients who had microwave coagulation therapy (MCT) for hepatocellular carcinoma. Six of them underwent surgery after MRI. The area (including tumor) treated by MCT showed low to high intensity on T1WI, and low to isointensity on T2WI. No enhancement was obtained on dynamic MRI. Histologically, this area was supposed to be coagulation necrosis. On T1WI, only tumor showed high intensity within the MCT area in 8 patients, and nearly uniform intensity was observed in 5 patients. Histologically, residual cell nuclei were observed in the former, and nearly uniform coagulation necrosis in the latter. The marginal part of the MCT area exhibited low intensity on T1WI, and high intensity on T2WI. Strong enhancement was obtained on dynamic MRI, and histologically, granulation tissue was noted. In the hepatic parenchyma around the MCT area, a ring-or wedge-shaped high intensity part was observed in 7 patients on T2WI, and that part was enhanced on dynamic MRI. This finding was considered to reflect changes such as hepatic hyperperfusion. In terms of the capability of visualizing residual tumor after MCT, MRI was superior to CT. Furthermore, a clear distinction was seen between the MCT area and non-MCT area on T2WI and dynamic MRI. Thus, MRI was useful in the determination of additional therapy.

Differentiation of Salmonella typhi strains by ribosomal RNA gene restriction patterns.

Owing to the limited value of phage typing to determine the epidemiological association of Salmonella typhi (S. typhi) strains isolated from the source of typhoid fever, we analyzed ribosomal RNA (rRNA) gene restriction patterns to differentiate the independently isolated strains of identical phage type. The data showed that the restriction patterns of PstI was most polymorphic among four enzymes (BamHI, EcoRI, PstI, and SmaI) used, which revealed 13 types among 25 strains belonged to 4 phage types, 1 untypable and 2 not-determined strains. Total 25 strains of S. typhi were divided into 15 combination types by the rRNA restriction patterns with three enzymes (BamHI, PstI, and SmaI).

Remodeling mechanisms of transseptal fibers during and after tooth movement.

The remodeling mechanisms of transseptal fibers during and after tooth movement were investigated histologically. An autoradiographic study was conducted to assess the synthetic response. One hundred fifty male Wistar strain growing rats were divided into three groups--one control and two experimental groups. Animals in the experimental groups were subjected to tooth movement with 25 g and 150 g of force. Maxillary first and second molars were separated and retained mechanically. Transseptal fibers were stretched in proportion to the amount of force applied for up to 2 days. Dynamic remodeling with proliferation of fibroblasts during tooth movement and slow rearrangement during retention periods occurred in both experimental groups. Collagen phagocytosis within the fibroblasts was observed ultrastructurally during the experimental periods. The number of silver grains in fibroblasts in the collagen fibers increased 160% the first day and 206% during the first 3 days. Proliferating fibroblasts remodeled the transseptal fibers through the synthesization and degradation of collagen fibers.

[A study to establish the model of transseptal fibers in vitro by human gingival fibroblast].

The present study was attempted to establish the model of transseptal fibers in vitro to clarify the remodeling mechanisms of the fibers during orthodontic treatment. Human premolars were transversally cut. The thickness of films was 150 microns and a pair of dental rings was placed at intermediate of 500 microns in tissue culture dishes. Human gingival fibroblasts were seeded in these dishes and cultured. Culturing of these cells were changed at 1, 5, 10 and 20 days and used to histological observation. The following results were obtained: 1. It was observed that human gingival fibroblasts and fibrillar materials oriented approximately the surfaces of sliced teeth and extended the process of cells along the direction from sliced teeth to another one. The bridges of cells and fibers between two sliced teeth were completed after 10 days culturing. 2. The fibrillar materials were examined by TEM and bundles constructed from banded fibrils which each band was 60 nm in diameter. The red fibers were stained with Van Gieson's stain method and so many red fibers filled in the space between the two sliced teeth. This suggests that the most of fibrillar materials were collagen fibers formed from human gingival fibroblasts. The structural materials were assumed to be resembled to transseptal fibers in vivo. This culture system is usefull to study the remodeling mechanisms of transseptal fibers in tooth movement.

[An attempt of experimental study to clarify the rearrangement mechanisms of transseptal fibers during mechanical retention periods].

The present investigation has been attempted to clarify the remodeling mechanisms of transseptal fibers during mechanical retention periods. Orthodontic forces of 25 gm and 150 gm were respectively applied to upper molars of rats for 10 days and retained mechanically. Retention periods were 10, 30 and 60 days. The trend of transseptal fibers after these retention periods was observed by the methods of histology and histochemistry. Histological study was observed rearrangement of fibrous tissue by light and electron microscopy and histochemical one was observed acid phosphatase activity. The following results were obtained: 1. Very little was observed about the rearrangement of transseptal fibers by the orthodontic force applied for 10 days. 2. The mechanical retention was kept longer, stretched transseptal fibers were slowly restored to original shape. This means transseptal fibers were gradually rearranged. 3. The collagen containing fibroblast and fibroblast characterized by abundant rough endoplasmic reticulum and Golgi complex were ultrastructurally observed. 4. The reaction of ACPase activity in some fibroblasts can be observed. These results suggest that transseptal fibers were rearranged during mechanical retention periods and that fibroblasts were engaged in this remodeling by synthesizing and degrading collagen fibers.