IMMUNO-CYTOCHEMICAL STUDY ON THE DEVELOPMENT AND DIFFERENTIATION OF THE IMMUNE SYSTEM OF THE HUMAN FETUS

The lymphatic tissues of human fetus and infant were studied systematically from an immuno-cytochemical view-point. Among immunoglobulins, γA and γ M first appeared in the primitive mesenchymal cells around the mesenteric and hepatic hilar vessels at the 11th fetal week. These cells were considered to be the primordium of the gut wall lymphoid tissues; the thymus and the bursa type tissue. γM in the reticular cells and γA in the medium-sized lymphoid cells were identified in the thymus after the middle of fetal life. γG, maternal in origin, was observed in the eosinophilic cells, mostly in the thymus. After passing middle fetal life, these three types of immunoglobulins were found in the HassaU's corpuscles, increasing quantitatively toward the end of fetal life. The role of thymus in the development of the immune system during the human fetal life was discussed in relation to other lymphatic tissues.     

Age-related change of distribution of immunoglobulin containing cells in human bone marrow

Summary The number and mode of distribution of immunoglobulin containing cells in human bone marrow were investigated immunohistochemically using paraffin sections of bone marrow aspirates. In individuals without specific diseases, the number of immunoglobulin containing cells per unit field in bone marrow increased with advancing age until the 3rd decade and leveled off thereafter. The magnitude of the increase was great for Ig-G and Ig-A, but very slight for Ig-M. Such age-related change in the number of Ig-G and Ig-M containing cells in bone marrow was almost comparable to the age-related change of serum level of Ig-G and Ig-M. However, the magnitude of age-related increase of Ig-A containing cells in bone marrow was apparently higher than that of the serum level of Ig-A. Cluster formation of immunoglobulin containing cells increased with age in terms of both incidence and size. Three points were suggested for differentiation of benign monoclonal gammopathy (BMG) from multiple myeloma (MM). First, the ratio of serum level of M-component divided by the average number of immunoglobulin containing cells per unit field was higher in BMG than in MM; second, the number of cells per cluster of immunoglobulin containing cells was more than 50 in MM, but that in BMG less than 20; third, the small immunoglobulin containing cells with narrow cytoplasm were more prominent in MM than in BMG.     

The role of thymus in the aging of Th cell subpopulations and age-associated alteration of cytokine production by these cells

Mouse CD4⁺ T cells were subdivided into two subpopulations,naive (CD44^low CD45RB^high) and memory (CD44^highCD45RB^low) Tcells, by flow cytometric analysis. Examination of spleen andperipheral blood of C57BL/6 mice of various ages revealed thatthere was a reciprocal ageassociated change in these two subpopulations,i.e. naive T cells predominant in young mice decreased withage, while memory T cells increased. In order to investigatethe role of the thymus in the age change of naive and memoryT cells, we employed two experimental systems: radiation bonemarrow chimeras constructed between young and old mice, andgrafting of young or old thymus into nude mice. Data from thesetwo experiments suggested that the young thymus has a greaterability to provide naive T cells than the old thymus, whilethe old thymus favors the maintenance of memory T cells ratherthan naive T cells. In reference to cytokine production by enrichednaive and memory T cells, young naive T cells produced mainlyIL-2 and young memory T cells mainly IL-4. On the other hand,in old mice, memory T cells produced twice as much IL-2 thannaive T cells, although the level was significantly lower thanthat of young mice. In addition, old naive T cells producedtwice as much IL-4 than old memory T cells. These results suggesteda distinct age change in the profile of cytokine productionand functional heterogeneity of two T_h cell subpopulations.     

Expression of the cyclin dependent kinase inhibitor p21WAF1/CIP1 in oesophageal squamous cell carcinomas

 To elucidate the role of CDK inhibitor p21^WAF1/CIP1 in human oesophageal squamous cell carcinomas, we examined its expression immunohistochemically using surgically resected tissues from 25 patients, and have analyzed the relationship with alteration of p53 gene (F-SSCP analysis), proliferative activity (Ki-67 labelling index), frequency of apoptosis (in situ DNA nick end labelling), and degree of differentiation. P21 expression was observed in 11 cases (44%) with a percentage of positive cells ranging between 1% and 10%. Of the 25 cases, 4 cases showed >5% of positive cells. As for the relationship with p53 gene, all 7 p53-mutation positive cases were negative for p21 expression, whereas 11 out of 18 mutation negative cases showed positive for p21 expression. As for the relationship with degree of tumour differentiation, 6 out of 8 well differentiated type cases showed positive for p21 expression. By contrast, all 8 cases of poorly differentiated type were negative for p21 expression. Frequency of apoptotic cells was significantly higher in p21 positive cases than negative cases although Ki-67 labelling index was almost the same regardless of the expression of p21. P21 expressing cells were distributed mainly in the middle layers of the invading nests, especially around the keratinization, which was almost similar to the distribution of apoptotic cells. Our results suggest that expression of p21 in human oesophageal squamous cell carcinomas is induced by a p53-dependent pathway and affects apoptosis and differentiation of carcinoma cells. Received: 9 October 1996 / Accepted: 23 December 1996     

Differential expression of various cytokine receptors in the brain after stimulation with LPS in young and old mice.

The effect of lipopolysaccharides (LPS) on the expression of cytokine receptors was examined in the spleen, brain and pituitary gland, and compared in young and old mice. The level of mRNA for various cytokine receptors (IL-1RI, IL-2Ralpha, IL-3Ralpha, IL-6R, TNFalphaR and IFNgammaR) was found to be increased in the spleen of young but not in old mice within 2-6h of stimulation with LPS. Similar enhancement of cytokine receptor mRNA was also observed in the brain after LPS stimulation, but the magnitude varied according to the type of cytokine receptor, the site of brain and the age of the mice.In the hypothalamus, the level of mRNA for IL-1R, IL-3R, IL-6R and IFNgammaR increased in young but not in old mice. Reciprocally, in the cerebral cortex, mRNA for TNFalphaR and IFNgammaR increased in old but not in young mice. In the hippocampus, TNFalphaR mRNA expression, increased in young but not in old mice, and expression of the other cytokine receptors did not change greatly in either. In the pituitary gland, mRNA for IL-6R, TNFalphaR and IFNgammaR increased in both young and old mice, but IL-2Ralpha increased only in young mice.Thus, various cytokines produced by immune cells might directly or indirectly influence brain functions through the various cytokine receptors expressed in the brain. Moreover, interactions between the immune system and the brain at the time of infection would be expected to be different in young and old mice, because cytokine production changes with age, as does the expression of their receptors in the brain.     

Incidence of apoptosis increases with age in colorectal cancer

The incidence of cancer increases with advancing age, but the biological behavior of cancer is known to be less aggressive in elderly people. Thus, the proliferative activity and extent of apoptosis of cancer cells were assessed in samples from 163 cases of colorectal cancer focusing on the age of patients, using Ki-67 labeling index (LI) and apoptotic index (AI) by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end labeling method and staining for activated caspase-3.

The Ki-67 LI of colorectal cancer ranged from 2.33 to 80.4% (mean 32.2%), while the AI ranged from 0.00 to 14.8% (mean 3.57%). Concerning the aging effect, linear and positive correlations were found for the Ki-67 LI of cancer with age (p<0.05) and the AI of cancer with age (p<0.05). However, in normal colorectal mucosa, aging of patients revealed a significant correlation only with the AI but not with the Ki-67 LI. The AI in earlier stages of cancers (stages 0 and 1) revealed a significant difference between younger cases (age<65) and more elderly cases (age≥65) (p<0.05), however, the Ki-67 LI did not exhibit a significant difference. Therefore, an increased frequency of apoptosis in colorectal cancer tissues, especially in the earlier stages, may possibly explain the slower growth of colorectal cancers in the elderly. Next, the expressions of several regulatory molecules for the proliferation/apoptosis of tumor cells were determined. The results demonstrated a tendency for stronger and more frequent expressions of c-myc, Bak and Bax despite a rather weaker expression of Bcl-2 in cancer tissues from the elderly compared with those from the younger patients. The potential roles of these regulatory molecules on age-change in the proliferation/apoptosis of colorectal cancers are discussed.     

Expression of IAP-family proteins in adult acute mixed lineage leukemia (AMLL)

Inhibitor of apoptosis protein (IAP)-family proteins suppress apoptotic signaling in normal/neoplastic cells in various settings. To determine the apoptosis-resistant mechanism in adult acute mixed lineage leukemia (AMLL) with biphenotypic blasts responsible for resistance against chemotherapy, the expression levels of IAP-family proteins in AMLL bone marrow cells were analyzed by quantitative RT-PCR. The overall expression levels of IAPs were higher than those in control, AML, and ALL cells. A significant difference for the expression of survivin was observed between AMLL and AML (P <0.05), and differences between AMLL and ALL were significant for the expression of survivin (P <0.05), NAIP (P <0.05), and XIAP (P <0.05). These findings suggest that higher expression of various IAPs is associated with the chemotherapy-resistant nature of this specific type of leukemia.     

Experimental allergic orchitis in mice

Summary Experimental allergic orchids was induced in (C57BL/6J × A/J)F₁ mice by two injections of syngeneic testicular homogenate emulsified with adjuvants immediately followed by intravenous injection of pertussis vaccine, at a 2 week interval.Histologically, in the initial stage there was occasional focal degeneration and desquamation of both spermatogonia and Sertoli cells within limited parts of the seminiferous tubules, in the peripheral region of the testis. No inflammatory change was present. In some cases, however, inflammatory reaction in the rete testis and focal lymphocytic infiltration in the interstitium were also observed. Subsequently, marked infiltration of lymphocytes, monocytes, and polymorphs were found not only in the testes, but also in rete testis and epididymis. In later stages the inflammatory reaction gradually subsided, but the testes became atrophic due to progression of spermatogenic arrest. Many tubules were lined only with monolayers of Sertoli cells, surrounded by hyperplastic Leydig cells in the interstitium. At 5 months after the 2nd immunization, there was still variable depression of spermatogenesis and hyperplasia of Leydig cells with scattered fibrous scars in the seminiferous tubules, although good regeneration of germ cells appeared in some tubules.Immunological studies revealed that lymphocytes obtained from mice bearing developed orchitis showed a significantly enhanced response in the mixed culture with syngeneic testicular cells, and suggest that cellular immunity plays an important role in the induction of experimental allergic orchitis in mice.