Culture of chondrocytes in fibroin-hydrogel sponge

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.     

Abnormal distribution of cathepsins in the brain of patients with Alzheimer's disease

Formalin-fixed paraffin-embedded hippocampal sections of brains with early-onset and late-onset Alzheimer's disease were studied immunohistochemically with antisera against cathepsin D and cathepsin B. In addition to the staining of neuronal perikarya, some of the senile plaques visualized by Bielshowsky silver staining and some of reactive astrocytes were positively stained with the antisera against cathepsin D and cathepsin B in brains with Alzheimer's disease. Abnormal localization of cathepsin D and cathepsin B immunoreactivity in neuronal perikarya was observed in brains with early-onset Alzheimer's disease. These findings demonstrate that the distribution of lysosomal proteases was altered in brains with Alzheimer's disease, suggesting the primary and/or secondary involvement of the lysosomal proteases in the pathological process of Alzheimer's disease.     

AN AUTOPSY CASE OF METACHROMATIC LEUCODYSTROPHY

A case of metachromatic leucodystrophy was studied histologically and histochemically.
Metachromatic lipids were found in the liver, gallbladder, kidney, pancreatic islets and adrenal medulla, as well as in cerebral white matter. The main component of the stored lipids was histochemically proved to be sulfatide. However, besides sulfatide, cholesterol esters, fatty acids and a small amount of phosphatides were demonstrated in the enlarged cells in cerebral white matter. Histochemical characteristics of accumulated lipids in the metachromatic leucodystrophy were compared with those of various lipids in Tay-Sachs' disease, gargoylism, Gaucher's and Niemann-Pick's diseases. The acidic neutral red method was found effective for detecting sulfatide.     

General Pathology Of Kawasaki Disease

Systemic pathological alterations were studied in thirty-seven autopsied patients with Kawasaki disease. Systemic vasculitis was the most characteristic pathological finding and was present in all the patients. In addition to the vasculitis, there was a high incidence of inflammatory lesions in various organs and tissues: in the heart, endocarditis, myocarditis, and pericarditis; in the digestive system, stomatitis, sialoduct-adenitis, catarrhal enteritis, hepatitis, cholangitis, pancreatitis, and pancreas ductitis; in the respiratory system, bronchitis and segmental interstitial pneumonia; in the urinary system, focal interstitial nephritis, cystitis, and prostatitis; in the nervous system, aseptic leptomeningitis, choriomeningitis, ganglionitis, and neuritis; in the hematopoietic system, lymphadenitis, splenitis, and thymitis. Dermatitis, panniculitis or myositis were also observed in some patients. Therefore, Kawasaki disease is a systemic inflammatory disease which mainly affects the cardiovascular system. These systemic inflammatory lesions are considered to correspond to the variegated clinical manifestaitions. The relationship between Kawasaki disease and infantile polyarteritis nodosa (IPN) were discussed, based on the clinicopathological characteristics.     

The number of proliferating cell nuclear antigen positive cells in endometriotic lesions differs from that in the endometrium

Immunohistochemical expression of proliferating cell nuclear antigen (PCNA) was studied in the endometrium and in endometriotic lesions during the menstrual cycle and in post-menopausal patients. During the menstrual cycle, in the basal layer of the endometrium, an increase in the number of positive indices (PI) of PCNA was observed in epithelial cells from the menstrual phase. It reached a maximum in the proliferative phase and decreased in the secretory phase. However, no change was observed in the stromal cells of the basal layer. In the functional layer of the endometrium, the PI of the epithelial cells showed a high peak in the late proliferative phase, decreased sharply in the secretory phase and remained unchanged thereafter. The PI of the stromal cells in the functional layer showed two peaks, one in the late proliferative and the other in the mid and late secretory phase. In the endometriotic lesions, except for the proliferative phase, the number of PI was significantly higher than that of the corresponding endometrium and no significant changes were observed during the menstrual cycle. In post-menopausal endometriotic lesions, the number of PI was also higher than that of the corresponding endometrium. Thus the numbers of PI differed between the endometrium and endometriotic lesions in the same patients. These results imply that the endometriotic lesions are constantly more proliferative than the endometrium irrespective of the hormonal milieu during both the menstrual cycle and in a post-menopausal environment.     

Immune responses of interferon gamma (IFN-gamma) knock out mice to repeated Haemaphysalis longicornis (Acari: Ixodidae) nymph infestations

To investigate the immunological mechanisms of acquired resistance to tick infestation, interferon gamma (IFN-gamma) deficient mice (IFN-gamma mice) were used to assess interleukin-4 (IL-4) and antibody production levels against tick salivary gland antigen on three successive infestations with Haemoaphysalis longicornis Neumann nymphs. The engorged body weight of the ticks decreased during the second and third infestations. Similar observations were noted in IFN-gamma+/+ mice. However, the engorged body weight of the ticks from IFN-gamma +/+ mice were considerably lower than those from IFN-gamma-/- mice. A marked increase in antibody production during the second and third infestations was observed indicating that IFN-gamma-/- mice could acquire immunological resistance against H. longicornis nymphs. Moreover, IL-4 levels were higher during the first and third infestations but decreased during the second infestation. IL-4 levels were significantly higher in IFN-gamma-/- mice than in IFN-gamma+/+ mice. We have shown here that the statistically significant high IL-4 levels observed in IFN-gamma-/- mice may be a result of type 2 helper cell (Th2) polarization. However, the apparently higher IL-4 levels during the first and third infestations and the notable decline during the second infestation suggest that other cytokines or factors in the host immune system may play a part in regulating IL-4 levels.     

Effects of sedative odorant inhalation on patients with atopic dermatitis]

Atopic dermatitis (AD) has been clinically well-known to be frequently exacerbated by psychological and physiological stress. In this study, we examined effects of sedative odorant (modified valerian oil) inhalation on patients with AD. We investigated clinical scores, skin physiological parameters and psychological questionnaire (POMS) every 2 weeks. For first 2 weeks, we arranged non-inhalation period. Results for non-inhalation period were compared with these of 2- or 4-week inhalation. As results, sum of skin clinical scores significantly improved after odorant inhalation. Some patients improved for non-inhalation period, too. However, patients that had not improved for non-inhalation period significantly improved after odorant inhalation. Skin conductance and skin dryness/scaling score also improved after odorant inhalation without improving for non-inhalation period. Psychological parameter (POMS) also tended to improve after odorant inhalation. These results suggest that sedative odorants may be useful as a complementary therapy for AD through psychosomatic stress care.     

Development of T cells in SCID mice grafted with fetal thymus from AKR mice or F344 rats

To examine the development of T cells within an allogeneic or xenogeneic environment, we engrafted the fetal thymus from AKR mice or F344 rats under the kidney capsule of SCID mice (mTG and rTG mice). T lymphopoiesis developed in SCID mice 2 months after transplantation, although the ratio of CD4/CD8 in both experimental groups was different from that of normal control. T cells in mTG mice did not show in vitro proliferation or cytotoxicity against either host-type C.B-17 (H-2^d) or donor-type AKR (H-2^k) cells, while they exerted potent activities against third-party BIO (H-2^b) cells. In contrast, T cells in rTG mice exhibited proliferation against both host-type C.B-17 and donor-type F344 rat cells. Consistently, graft-vs.-host disease symptoms developed in these mice and histological examination showed impressive infiltration of lymphocytes into the skin or into the mucosal layers of the stomach. Activated state of T cells in rTG mice was also evidenced by the positive expression of interleukin-2 receptor. Taken together, fetal thymus appears to contain progenitor cells which are sufficient for in vivo reconstitution of T lymphopoiesis, but species-specific environment is important for the induction of tolerance. In mTG mice, Vβ6⁺ T cells reactive to donor Mls^a determinants and Vβ3⁺ T cells reactive to host Mls^c determinants were deleted, suggesting that tolerance was regulated mainly by clonal deletion. By contrast, Vβ11⁺ T cells reactive to Mls^f determinants were not deleted possibly due to the lack of their ligands.     

Comparison of various bone marrow fractions in the ability to participate in vascular remodeling after mechanical injury

In contrast to conventional assumption, recent reports propose the possibility that hematopoietic stem cells (HSCs) may have broader potential to differentiate into various cell types. Here, we tested the pluripotency of HSCs by comparing vascular lesions induced by mechanical injury after bone marrow reconstitution with total bone marrow (TBM) cells, c-Kit+ Sca-1+ Lin- (KSL) cells, or a single HSC cell (Tip-SP CD34-KSL cell, CD34- c-Kit+ Sca-1+ Lin- cell with the strongest dye-efflux activity) harboring green fluorescent protein (GFP). The lesions contained a significant number of GFP-positive cells in the TBM and KSL groups, whereas GFP-positive cells were rarely detected in the HSC group. These results suggest that transdifferentiation of a highly purified HSC seems to be a rare event, if it occurs at all, whereas bone marrow cells including the KSL fraction can give rise to vascular cells that substantially contribute to repair or lesion formation after mechanical injury.