Phenotyping of proliferating lymphocytes in angioimmunoblastic lymphadenopathy and related lesions by the double immunoenzymatic staining technique.

Biopsy specimens of lymph nodes with the histologic characteristics of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) were obtained from 9 cases (4 cases of AILD and 5 cases of AILD-like T lymphoma [AILD-T]) and histologically analyzed by the use of a double immunoenzymatic staining technique with the combination of a monoclonal antibody against lymphocyte membrane antigen and that against human DNA polymerase alpha (pol alpha), which is detectable in the nucleus of the cells in G1, S, and G2 phases. In all 9 cases, the pol alpha + proliferating cells had a peripheral T-cell phenotype with T11 and Leu-4 antigens, whereas proliferating B cells with B1 antigen were rarely observed. As for T-cell subset antigens, the proliferating T cells had T4+ helper/inducer phenotype in 7 cases, while T8+ suppressor/killer T cells proliferated in 2 cases, although a significant number of T4+ proliferating cells were also recognized. The study on malignant lymphomas that evolved in the 2 cases showed that the T-subset antigens on major proliferating tumor cells were the same as those found in the preceding AILD lesions, suggesting that lymphoma T cells originate from the AILD lesion. The results suggested that AILD without histologic manifestations of malignancy and AILD-T may be a neoplastic disease derived from either subset of peripheral T cells.     

Breast cancer with reactive multinucleated giant cells: report of three cases

We report here three cases of breast cancer with reactive multinucleated giant cells. The patients were among the 605 patients with breast cancer seen in the past 17 years at Tenri Hospital; the incidence of this variety of breast cancer was 0.5%. Enzyme histochemical and electron microscopic examination suggested that the giant cells were of histiocytic origin. However, results of immunohistochemical technique, S-100 protein, lysozyme, nonspecific cross-reacting antigen with carcinoembryonic antigen, alpha-1-antitrypsin, and alpha-1-antichymotrypsin, all currently used as markers of histiocytes, were negative. Because of the rarity of this variety of breast cancer, the biological significance of these unusual findings remains unknown.     

Rat costochondral cell characteristics on poly (L-lactide-co-epsilon-caprolactone) scaffolds

This study was designed to examine the adhesion, proliferation, and morphology of chondrocytes on new scaffolds; and to examine these cells histologically for the ability of the chondrocytes to maintain chondrogenic properties after subcutaneous implantation into nude mice. Both 75:25 poly (L-lactide-co-epsilon-caprolactone) (75PLC) and 50:50 poly (L-lactide-co-epsilon-capro-lactone) scaffold (50PLC) were tested as a scaffold for rat costochondral resting zone chondrocytes in comparison with a type I collagen sponge scaffold (collagen scaffold). Both of the poly (L-lactide-co-epsilon-caprolactone) scaffolds (75PLC and 50PLC) were coated with type I collagen solution and the effects of the collagen coat (hybrid-PLC) were also examined. The hybrid-75PLC bound the same number of cells as the collagen scaffold, whereas the 75PLC and the 50PLC bound 60% and 50% fewer cells than the collagen scaffold, respectively. The cell growth on the scaffolds progressed with culture time in all scaffolds. Cell morphology was assessed by scanning electron microscopy for differences in the structure of cellular interaction. Chondrocytes on every scaffold maintained a spherical shape. The hybrid-PLCs were superior to the PLCs with respect to the number of cells attached. The PLCs had an advantageous degradation characteristic in that they retained their original shape better than the collagen scaffold. Additionally, in the PLCs seeded, the cells retained their integrity 4 weeks after implantation, although the volume of collagen scaffold decreased by 50%.     

Tumor necrosis factor-alpha in the placenta is not elevated in pre-eclamptic patients despite its elevation in peripheral blood

PROBLEM: Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells at the early and late stages of gestation and promotes the regulation of trophoblast growth and invasion. We evaluated whether TNF-alpha levels in the placenta and blood of pre-eclamptic women differed from those with normal pregnancies. METHOD OF STUDY: The subjects were 39 pregnant women carrying single fetuses (21 normal-pregnant and 18 pre-eclamptic patients). Their average gestational age at entry was 38-39 weeks. Peripheral blood was collected before the onset of labor and separated serum was stored at -20 degrees C. A tissue segment of the placenta was cut and frozen in liquid nitrogen immediately after delivery at -80 degrees C. The frozen placental tissue was added to phosphate-buffered saline. The tissue was fully homogenized and centrifuged. Separated supernatant was stored at -80 degrees C. TNF-alpha levels in separated serum and TNF-alpha and total protein (TP) levels in separated supernatant were measured. The presence of TNF-alpha in the placenta was evaluated by immunohistochemistry in five pre-eclamptic and five normal-pregnant patients. RESULTS: Serum TNF-alpha levels were higher in pre-eclampsia than in normal pregnancies. However, TNF-alpha/TP levels in the placenta did not differ significantly between the two groups. As for TNF-alpha immunostaining of trophoblastic cells in the placenta, it was weak in three and moderate in two of the normal pregnancies, while it was absent in two, weak in one, and moderate in two in the pre-eclampsia group. CONCLUSIONS: We demonstrated no significant increase in TNF-alpha/TP levels in the placenta in pre-eclampsia despite a significant increase in serum TNF-alpha levels. There was no strong immunostaining for TNF-alpha detected by immunohistochemistry in the pre-eclampsia group. These findings suggest that TNF-alpha in the placenta is not a key cytokine to interfere with normal trophoblast invasion into the myometrium in pre-eclampsia, and that sources other than the placenta may contribute to the elevated levels of TNF-alpha found in the circulation of pre-eclamptic patients.     

B-cell repertoire specific for an unfolded self-determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.     

Characteristics of murine non-specific killer cells induced in vivo by recombinant human interleukin-2.

We have examined the induction of murine non-specific killer cells in vivo and in vitro by purified recombinant human interleukin-2 (rIL-2), and compared their characteristics with respect to killing ability, cell surface phenotypes, and antibody-dependent cell-mediated cytotoxicity (ADCC). C57BL/6 spleen cells cultured with rIL-2 were remarkably cytotoxic against a variety of tumour cells in a 4-hr 51Cr-release assay. Treatment with various antibodies (anti-Thy 1, anti-Lyt 1, anti-Lyt 2, and anti-asialo GM1) plus complement (C) showed that anti-Thy 1 or anti-asialo GM1 antibody plus C removed a majority of killer activity (80% and 66%, respectively). In addition, an increase in ADCC was detected in the spleen cells cultured with rIL-2. These ADCC effector cells were indistinguishable from non-specific killer cells by the cell surface phenotypes. A single administration of rIL-2 in vivo induced only transient and marginal enhancement of non-specific killer activity of spleen cells in C57BL/6 mice. On the other hand, when 10 micrograms of rIL-2 were administered daily by bolus to C57BL/6 mice, the activity increased gradually for about 10 days and reached a plateau. This enhanced non-specific killer activity rapidly decreased and returned to normal by 72 hr after the administration was stopped. The non-specific killer cells induced in vivo in this manner were not only greatly cytotoxic against natural killer (NK)-sensitive tumour cells but were also significantly cytotoxic against NK-resistant tumour cells. Most of the killer activity (more than 90%) was specifically removed by treatment with anti-Thy 1 or anti-asialo GM1 antibody plus C. An increase in ADCC was detected concurrently with an increase in non-specific killer activity in vivo, and both effector cells were indistinguishable by their cell surface phenotypes. These results indicate that a majority of non-specific killer cells induced both in vivo and in vitro by rIL-2 have some common features. Our results also suggest that these cells belong to the same lineage as NK cells, although they are thought to be at different stages from resident NK cells.     

The effect of immunoglobulin G1 structure on macrophage binding to supported planar lipid monolayers.

We have studied the antibody-dependent binding of macrophages to supported planar lipid monolayers containing haptenated phospholipids (Tnp-Cap-DPPE). Eight monoclonal anti-TNP IgG1s, which had similar affinities to the TNP residues in solution and in the membranes, were used in the experiment. The results showed that mouse macrophages (P388D1 and J774.1) bound with different affinities to these IgG1-coated lipid monolayers. The monoclonal antibody shown to be deficient in macrophage binding was also relatively ineffective in activating complement. These results indicated that individual monoclonal antibodies of a given subclass may prove deficient in terms of the biological activities associated with the group as a whole.     

Relative frequency of VP4 gene alleles among human rotaviruses recovered over a 10-year period (1982-1991) from Japanese children with diarrhea.

The relative frequencies of the Wa (corresponding to serotype P1A), DS-1(P1B), M37(P2), and AU-1(P3) alleles of the VP4 gene from rotaviruses collected from the stools of individuals in Japan between 1982 and 1991 were determined to be 83.1, 15.6, 0, and 1.3%, respectively, by a polymerase chain reaction-based typing assay.