Sialyl-glycoconjugates in cholesterol-rich microdomains of P388 cells are the triggers for apoptosis induced by Rana catesbeiana oocyte ribonuclease.

SBL/RC-RNase was originally isolated from frog (Rana catesbeiana) oocytes and purified as a novel sialic acid-binding lectin (SBL) that displayed strong anti-cancer activity. SBL was later shown to be identical to a ribonuclease (RC-RNase) from oocytes of the same species. The administration of SBL/RC-RNase induced apoptosis (with nuclear condensation and DNA fragmentation) in mouse leukemia P388 cells but did not kill umbilical vein endothelial or fibroblast cells derived from normal tissues. The cytotoxic activity of SBL/RC-RNase was inhibited by desialylation of P388 cells and/or the co-presence of free bovine submaxillary mucin. FACS analysis showed that SBL/RC-RNase was incorporated into cells after attachment to cholesterol-rich microdomains. Addition of the cholesterol remover methyl-ß-cyclodextrin reduced SBL/RC-RNase-induced apoptosis. Apoptosis occurred through the caspase-3 pathway following activation of caspase-8 by SBL/RC-RNase. A heat shock cognate protein (Hsc70) and a heat shock protein (Hsp70) (each 70 kDa) on the cell membrane were shown to bind to SBL/RC-RNase by mass spectrometric and flow cytometric analyses. Quercetin, an inhibitor of Hsc70 and Hsp70, significantly reduced SBL/RC-RNase-induced apoptosis. Taken together, our findings suggest that sialyl-glycoconjugates present in cholesterol-rich microdomains form complexes with Hsc70 or Hsp70 that act as triggers for SBL/RC-RNase to induce apoptosis through a pathway involving the activation of caspase-3 and caspase-8.

Net effect of lymphaticovenous anastomosis on volume reduction of peripheral lymphoedema after complex decongestive physiotherapy.

OBJECTIVE: The results of reported lymphaticovenous anastomoses include some effects of complex decongestive physiotherapy (CDP). The present study aimed to determine the net effect of lymphaticovenous side-to-end anastomosis (LVSEA) in patients with lower limb lymphoedema treated by preoperative CDP. DESIGN: Retrospective observational study. MATERIALS: 37 LVSEAs in 31 patients. METHODS: Volumes of the thigh and leg with oedema were compared between the time of initial examination, and before (application of CDP) and after LVSEA. The patients were divided into two groups based on the number of anastomoses and lymphoscintigraphic findings. RESULTS: Preoperative CDP resulted in a reduction of 593 ml (both leg and thigh; p < 0.001). After CDP, LVSEA (1-8 anastomoses; average of 5) reduced the volume by 109 ml (52 ml for the thigh (p = 0.01) and 57 ml for the leg (p = 0.002)). There was no significant difference in volume reduction on lymphoscintigraphy. Volume was significantly reduced (by 55 ml in the thigh, p = 0.049; 96 ml in the leg, p = 0.006) in the group that underwent 6-8, but not 1-5 LVSEAs. CONCLUSIONS: The net effect of LVSEA on volume reduction was confirmed, but was not particularly large. The need for CDP decreased in some patients postoperatively, and these patients should be considered for evaluation.

Contributions of FASN and SCD gene polymorphisms on fatty acid composition in muscle from Japanese Black cattle.

The fatty acid synthase (FASN) and stearoyl-CoA desaturase (delta-9-desaturase) (SCD) genes affect fatty acid composition. This study evaluated the contributions of polymorphisms of these genes on fatty acid composition in muscle in two different populations: 1189 and 1058 Japanese Black cattle from the Miyagi and the Yamagata populations respectively. We sampled intramuscular fat from the longissimus thoracis muscle in the Miyagi population and from the trapezius muscle in the Yamagata population. The collective contributions of FASN and SCD polymorphisms to total additive genetic variance for oleic acid were 13.46% in the Miyagi population and 16.29% in the Yamagata population and to phenotypic variance were 5.45% and 6.54% respectively. Although the individual effects of FASN and SCD polymorphisms on fatty acid composition were small, overall gene substitution may effectively improve fatty acid composition. In addition, we found that gene polymorphism contributions of fatty acids varied by population even in the same breed.

Classification of lymphoscintigraphy and relevance to surgical indication for lymphaticovenous anastomosis in upper limb lymphedema.

Upper limb lymphedema that develops after breast cancer surgery causes physical discomfort and psychological distress, and it can require both conservative and surgical treatment. Lymphaticovenous anastomosis has been reported to be an effective treatment; however the disease severity criteria that define indications for this treatment remain unclear. Here, we examined lymphoscintigraphic findings in 78 patients with secondary upper limb lymphedema and classified them into 5 major types (Type I-V) and 3 subtypes (Subtype E, L, and 0). Results revealed that this classification is related to the clinical stage scale of the International Society of Lymphology. Based on intraoperative examination findings in 20 of the 78 patients, lymphatic pressure is likely to be further elevated in Type II-V cases which are characterized by the presence of dermal back flow. Therefore, lymphaticovenous anastomosis should be considered as a treatment option for lymphedema in Type II-V cases. Furthermore, there are only limited lymph vessel sites usable for lymphaticovenous anastomosis in more severe lymphedema types [Types IV and Type V (which is characterized by dermal backflow only in the hand)]. The findings in Type IV-V cases suggest that therapeutic strategies for severe upper limb lymphedema need further consideration.

[Intermittent opening of a mechanical mitral valve prosthesis due to pannus formation; report of a case].

A 76-year-old woman with a history of severe mitral valve stenosis had undergone mitral valve replacement with a 27 mm St. Jude Medical (SJM) valve in 1991. Follow-up transthoracic echocardiography revealed an increase in the pressure gradient across the mitral prosthesis 16 years after the surgery. Prosthetic valve dysfunction was suspected, but transesophageal echocardiography and cineradiography failed to show mechanical valve dysfunction. Two years later, she presented with dyspnea on exertion and leg edema. Cineradiography revealed intermittent restriction of the opening of the mechanical valve leaflet approximately every 10 beats. Thus, we diagnosed intermittent prosthetic valve dysfunction and performed a reoperation. On inspection of the prosthesis, we observed semicircular pannus formation around the posterior leaflet in the ventricular side. It was considered that the pannus tissue had interfered with 1 leaflet opening of the mitral valve prosthesis, resulting in intermittent valve dysfunction. We replaced the prosthesis with a new 25 mm SJM valve. The patient was discharged after confirmation of normal prosthetic function.

Distinct mechanisms of neonatal tolerance induced by dendritic cells and thymic B cells.

To assess the role of different types of antigen-presenting cells (APC) in the induction of tolerance, we isolated B cells, macrophages, and dendritic cells from thymus and spleen, and injected these into neonatal BALB/c mice across an Mls-1 antigenic barrier. One week after injection of APC from Mls-1-incompatible mice or from control syngeneic mice, we measured the number of thymic, Mls-1a-reactive, V beta 6+ T cells and the capacity of thymocytes to induce a graft-vs.-host (GVH) reaction in popliteal lymph nodes of Mls-1a mice. Injection of thymic but not spleen B cells deleted thymic, Mls-1a-reactive V beta 6+ T cells and induced tolerance in the GVH assay. The thymic B cells were primarily of the CD5+ type, and fluorescence-activated cell sorter-purified CD5+ thymic B cells were active. Injection of dendritic cells from spleen or thymus also induced tolerance, but the V beta 6 cells were anergized rather than deleted. Macrophages from thymus did not induce tolerance. Dendritic cells and thymic B cells were also effective in inducing tolerance even when injected into Mls-, major histocompatibility complex-incompatible, I-E- mice, but only thymic B cells depleted V beta 6-expressing T cells. Therefore, different types of bone marrow-derived APC have different capacities for inducing tolerance, and the active cell types (dendritic cells and CD5+ thymic B cells) can act by distinct mechanisms.

[Graft harvesting in coronary artery bypass grafting].

The technique of graft harvesting during coronary artery bypass grafting (CABG) consists of 2 main components: the vessel exposure at an adequate layer and the division of branches. Recently, the ultrasonic scalpel has been used for skeletonization of arterial grafts. A hook-type tip is used for the internal thoracic artery and the radial artery graft, while a shear type tip is used for the right gastroepiploic artery graft. The ultrasonic scalpel is useful both for graft vessel exposure and for the division of branches. The cavitation phenomenon is useful for the vessel exposure, while ultrasonic protein coagulation is useful for the division of branches. In endoscopic saphenous vein graft harvesting, electrocautery scissors are used for the division of branches. Avoiding thermal damage to the graft vessel is important in the use of either device. In any graft harvesting, direct contact with the main trunk should be avoided as much as possible to prevent damage. A thorough knowledge of the anatomy of the graft vessel and the surrounding organs is necessary for graft harvesting and to avoid complications. Furthermore, an understanding of characteristics of the harvesting devices is also important.

Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs.

A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80 degrees C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, beta-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-alpha- and methyl-beta-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k(ass)) and dissociation rate constant (k(diss)) were determined for the lectin to be 4.3 x 10(5) M(-1) x sec(-1) and 2.2 x 10(-3) sec(-1), respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.

ICRP Publication 140: Radiological Protection in Therapy with Radiopharmaceuticals.

Radiopharmaceuticals are increasingly used for the treatment of various cancers with novel radionuclides, compounds, tracer molecules, and administration techniques. The goal of radiation therapy, including therapy with radiopharmaceuticals, is to optimise the relationship between tumour control probability and potential complications in normal organs and tissues. Essential to this optimisation is the ability to quantify the radiation doses delivered to both tumours and normal tissues. This publication provides an overview of therapeutic procedures and a framework for calculating radiation doses for various treatment approaches. In radiopharmaceutical therapy, the absorbed dose to an organ or tissue is governed by radiopharmaceutical uptake, retention in and clearance from the various organs and tissues of the body, together with radionuclide physical half-life. Biokinetic parameters are determined by direct measurements made using techniques that vary in complexity. For treatment planning, absorbed dose calculations are usually performed prior to therapy using a trace-labelled diagnostic administration, or retrospective dosimetry may be performed on the basis of the activity already administered following each therapeutic administration. Uncertainty analyses provide additional information about sources of bias and random variation and their magnitudes; these analyses show the reliability and quality of absorbed dose calculations. Effective dose can provide an approximate measure of lifetime risk of detriment attributable to the stochastic effects of radiation exposure, principally cancer, but effective dose does not predict future cancer incidence for an individual and does not apply to short-term deterministic effects associated with radiopharmaceutical therapy. Accident prevention in radiation therapy should be an integral part of the design of facilities, equipment, and administration procedures. Minimisation of staff exposures includes consideration of equipment design, proper shielding and handling of sources, and personal protective equipment and tools, as well as education and training to promote awareness and engagement in radiological protection. The decision to hold or release a patient after radiopharmaceutical therapy should account for potential radiation dose to members of the public and carers that may result from residual radioactivity in the patient. In these situations, specific radiological protection guidance should be provided to patients and carers.

Inferring causal structures and comparing the causal effects among calving difficulty, gestation length and calf size in Japanese Black cattle.

The objectives of this study were to infer phenotypic causal networks involving gestation length (GL) and calving difficulty (CD) for the primiparity of 1850 Japanese Black heifers, and the birth weight (BWT), withers height (WH) and chest girth (CHG) of their full blood calves, and to compare the causal effects among them. The inductive causation (IC) algorithm was employed to search for causal links among these traits; it was applied to the posterior distribution of the residual (co)variance matrix of a multiple-trait sire-maternal grand sire (MGS) model. The IC algorithm implemented with 95% and 90% highest posterior density intervals detected only one structure with links between GL and BWT (WH or CHG) and between BWT (WH or CHG) and CD, although their directions were not resolved. Therefore, a possible causal structure based on the networks obtained from the IC algorithm [GL→BWT (WH or CHG)→CD] was fitted using a structural equation model to infer causal structure coefficients between the traits. The structural coefficients of GL on BWT and of BWT on GL on the observable scale showed that an extra day of GL led to a 270-g gain in BWT, and a 1-kg increase in BWT increased the risk for dystocia by 1.1%, in the causal structure. Similarly, an increase in GL by 1 day resulted in a 2.1 (2.0)-mm growth in WH (CHG), and a 1-cm increase in WH (CHG) increased the risk of dystocia by 1.2% (0.9%). The structural equation model was also fitted to alternative causal structures, which involved the addition of a directed link from GL to CD, or GL→CD to the structures described above. The inferred structural coefficients with the alternative structures were almost the same as the corresponding ones that had GL→BWT (WH or CHG)→CD. However, the direct causal effect of the extra link from GL on CD was similar to the indirect causal effect of GL through the mediating effect of BWT (WH or CHG) on CD and significant (P<0.05). This suggest that maternal genetic effects might not be removed completely from the residual variance components in the sire-MGS model, and the application of the IC algorithm to the variances from the model could detect an incorrect structure. Nonetheless, fitting the structural equation model to the causal structure provided useful information such as the magnitude of the causal effects between the traits.

Induction of renal pelvic carcinoma by phenacetin in hydronephrosis-bearing rats of the SD/cShi strain.

Carcinogenicity of phenacetin (PH) to the urinary tract was tested with the use of spontaneously hydronephrosis-bearing rats. In Experiment 1, 55 SD/cShi male rats were fed with 2% PH-containing diet for 85 weeks, and 32 SD/cShi male rats fed basal diet for 85 weeks served as controls. Forty-three of 53 rats fed with PH had renal pelvic carcinoma with lung metastases in three. The mean induction time was 78 weeks. Ureteral carcinoma and urinary bladder carcinoma were observed in 2 and 6 of 53 rats given PH, respectively. No urinary tract carcinoma was found in control animals. In Experiment 2, early lesions of the kidney affected by PH were also evaluated with the use of SD/cShi and Sprague-Dawley (SD) rats. Two groups of animals containing 6 SD/cShi or 6 SD male rats per group were fed 2% PH-containing diet for 8 weeks. Control animals containing 6 SD/cShi rats or 6 SD rats were fed basal diet for 8 weeks. Simple hyperplasia was found in 5 of 6 SD/cShi rats given PH and 2 of 6 SD/cShi control rats. Papillary necrosis was seen in 4 of 6 SD/cShi and 2 of 6 SD rats given PH. SD/cShi rats, especially those treated with PH, showed higher but not significant 5-bromo-2'-deoxyuridine labeling indices in the covering epithelium of the renal pelvis and papillae. In this short term experiment PH and its metabolites, N-hydroxyphenacetin and N-acetyl-p-aminophenol, were measured in urine and plasma by using high performance liquid chromatography. Significantly higher PH and slightly higher metabolites were detected in urine and plasma of SD/cShi rats compared to SD rats. These results indicated that the renal pelvis of SD/cShi rats had more sensitivity to PH carcinogenicity. This paper provides experimental proof of PH carcinogenicity toward the renal pelvis in an animal model.

Scintigraphic detection of overexpressed c-erbB-2 protooncogene products by a class-switched murine anti-c-erbB-2 protein monoclonal antibody.

Class-switched monoclonal antibody SV2-61r recognized the extracellular domain of c-erbB-2 protooncogene products separate from the epidermal growth factor receptor. We studied the potential of SV2-61r for evaluating the amplification of c-erbB-2 protooncogene on cancer cells, which has been reported to have prognostic value in adenocarcinoma patients. Radiolabeled SV2-61r specifically bound to various adenocarcinoma cells in addition to c-erbB-2-transfected NIH-3T3 cells (A4) with the affinity constant of 4.4 x 10(8) M-1. SV2-61r injected i.v. localized well to A4 cells xenografted in nude mice. Tumor uptake and localization index of radioiodinated SV2-61r were lower than those of 111In-labeled SV2-61r, probably due to the internalization and dehalogenation of formed antibody-antigen complexes. Biodistribution and specificity of targeting were assessed by comparison among three cells, A4, lung cancer SBC-3 (c-erbB-2 weakly positive) and B-lymphoblastoid Manca cells (c-erbB-2 negative). Tumor:blood ratios, obtained 48 h after injection, were 5.63, 1.45, and 0.68, respectively, indicating the potential of 111In-labeled SV2-61r for evaluating the amplification of c-erbB-2 protooncogene on cancer cells. Because of its close relationship with carcinogenesis and the uniform expression, c-erbB-2 protooncogene products seem to be the optimal target of imaging and therapy of adenocarcinoma patients.

Inhibition of cell proliferation by Rana catesbeiana and Rana japonica lectins belonging to the ribonuclease superfamily.

Two frog egg lectins [Rana catesbeiana lectin (SBL-C) and Rana japonica lectin] preferentially agglutinate a large variety of human and animal tumor cells but not blood cells, lymphocytes, or fibroblasts. These lectins belong to the superfamily of pyrimidine base-specific RNases. The two lectins bound to a heparin-Sepharose column and were eluted from the column by an increase of NaCl molarity. Both their tumor cell-agglutinating activity and RNase activity were inhibited by heparin, and also by polyamines, such as spermine. Both lectins inhibited P388 leukemia cell proliferation. The inhibitory activity of SBL-C was blocked by addition of heparin. SBL-C inhibited protein synthesis by P388 cells, but RNase A did not. No lectin-induced antiproliferative effect was observed after sialidase treatment of cells. The antiproliferative activity of SBL-C was also inhibited by ammonium chloride treatment. These results suggest that internalization of the lectins by lectin receptor (sialoglycoconjugate)-mediated endocytosis is followed by cell death due to inhibition of protein synthesis. Administration of SBL-C i.p. delayed time to death in mice receiving i.p. transplants of Sarcoma 180 and Mep II cells.

Characterization of a Rana catesbeiana lectin-resistant mutant of leukemia P388 cells.

Sialic acid-binding lectin (SBL-C) from Rana catesbeiana eggs inhibits the growth of tumor cells such as P388 and L1210 leukemia cells (K. Nitta et al., Cancer Res., 54: 920-927, 1994). Here we report the establishment of an SBL-resistant P388 variant cell line, RC-150. Both P388 and RC-150 cells were agglutinated by SBL-C; however, growth of RC-150 cells was unaffected by SBL-C. Cytoplasmic free Ca2+ concentration and transglutaminase activity of RC-150 cells were 0.5 (110 nM) and 3 times (0.62 nmol/mg/min) as high as those of P388 cells, respectively. Microvilli and microplicae were observed on the surface of P388 cells by scanning electron microscopy but were rarely seen on RC-150 cells. Dansylcadaverine-labeled SBL-C bound to both P388 and RC-150 cells. Binding of SBL-C to these tumor cells appears to be mediated by two species of wheat germ agglutinin-stained cell membrane sialoglycoproteins. Labeled SBL-C entered P388 but not RC-150 cells, suggesting that internalized SBL-C acts as an inhibitor of cell proliferation.

[New proximal anastomotic system in coronary artery bypass grafting].

The Heartstring proximal anastomotic system is a device designed to facilitate the creation of a clampless hand-sewn proximal anastomosis. Thirty-four patients who underwent coronary artery revascularization had 40 proximal anastomoses using the Heartstring device. There were 26 men and 8 women, with the mean age of 70 +/- 8.9 years. Thirty-one patients underwent coronary artery bypass grafting through off-pump procedures and 3 patients on-pump beating procedures. In all patients, saphenous vein grafts were anastomosed to the aorta using the Heartstring device, the median number of distal anastomoses being 2.4 +/- 0.7. Either emergent or urgent surgery was required in 14 patients (41%). Diseased aorta was found in 11 patients (32%). One patient (2.9%) died postoperatively due to ischemic necrosis of the small intestine and the colon. There was no occurrence of postoperative stroke. Of 40 saphenous vein grafts anastomosed with the Heartstring system, 39 (97.5%) were patent. The occluded saphenous vein was not considered to be device related. Our clinical experience demonstrated that the Heartstring system allow us to create clampless and reproductive hand-sewn proximal anastomosis and to decrease the incidence of neurological complication.

[Aortic root replacement in Marfan syndrome with hemophilia A].

A 34-year-old man with Marfan syndrome was admitted to our hospital for surgical treatment of aortic regurgitation due to annuloaortic ectasia. He had no history of bleeding complications. Preoperative investigation revealed a slight prolongation of an activated partial thromboplastin time, which went unnoticed. He underwent aortic root replacement with a composite valve graft. During the operation, he had excessive bleeding due to coagulopathy after the termination of cardiopulmonary bypass, and needed a large amount of blood transfusion to obtain hemostasis. Before his discharge from our hospital, he was diagnosed as mild hemophilia A because of the decline in his factor VII level. To our knowledge, there has been no published case of cardiac operations in Marfan syndrome with hemophilia A.

Tandem repeat structure of rhamnose-binding lectin from catfish (Silurus asotus) eggs.

The primary structure of catfish (Silurus asotus) egg lectin (SAL) was determined. SAL cDNA contained 1448-bp nucleotides and 308 amino acid residues, deduced from open reading frame. The SAL mature protein composed of 285-amino acid residues was followed by a predicted signal sequence having 23 residues. The mRNA of SAL was found to be expressed in eggs, but not in liver. SAL is composed of three tandem repeat domain structures divided into exactly 95 amino acid residues each, and all cysteine positions of each domain were completely conserved. Sequence homologies between the three domains, termed D1 (1-95), D2 (96-190) and D3 (191-285), were as follows; D1-D2, 28%; D2-D3, 33%; D1-D3, 43%. Two conserved peptide motifs, -(AN)YGR(TD)S(T)XCS(TGR)P- and -DPCX(G)T(Y)KY(L)-, appear to exist at the N- and C-terminal regions of each domain, respectively. The kinetic parameters of SAL obtained by measuring surface plasmon resonance were as follows: K(a) (M(-1)) for neohesperidosyl-BSA, 7. 1 x 10(6); for melibiosyl-BSA, 4.9 x 10(6); and for lactosyl-BSA, 5. 2 x 10(5). These results show that RBLs including SAL comprise a family of alpha-galactosyl binding lectins having characteristic tandem repeat domain structures.

Influence of different pneumoperitoneal pressures on tumor cell distribution in rats.

BACKGROUND: The effect of different pneumoperitoneal pressures on tumor cell distribution was investigated. METHODS: Donryu rats were allocated to receive carbon dioxide pneumoperitoneum at 5, 10, or 15 mmHg for 60 min or to serve as a control. During the procedure, each rat was inoculated with radiolabeled ascites hepatoma cells via the portal vein (experiment 1) or femoral vein (experiment 2). In both experiments, the rats were killed 30, 60, 90, or 120 min after tumor cell inoculation, and the liver and lungs were extirpated for radioactivity count (n = 5 or 6 for each time point in each group). RESULTS: In experiment 1, the percentage of injected dose (% ID) for the liver was greater than for the other three groups 120 min after tumor cell inoculation. There were no significant differences in the %IDs of the lungs at any time point among the groups. In experiment 2, there were no significant differences in the %IDs of the liver and lungs at any time point among the groups. CONCLUSIONS: These results suggest that an elevated insufflation pressure facilitates the location of intraportally injected tumor cells in the liver, and that pulmonary location of the tumor cells may not depend on insufflation pressures in this animal model.

Autostimulatory lymphoid cells in aging mice: induction of syngeneic host-versus-graft reaction by radioresistant adherent cells in normal recipients.

When young (6-week-old) BALB/c and also young C57BL/6 mice were inoculated into footpads with spleen cells from normal, syngeneic, aged donors, their response to the inoculum resulted in the enlargement of popliteal lymph nodes (PLN). The degree of PLN enlargement increased as the age of donor mice increased up to age one year. Spleen and lymph node cell populations were highly effective in eliciting the PLN enlargement. Thymus cells and bone marrow cells were moderately effective, but erythrocytes were ineffective. Resident peritoneal exudate cells and spleen adherent cells were much more effective than a whole spleen cell population. The syngeneic response seems to be attributable to a host-versus-graft reaction, since the PLN response was not affected by 2000 rad irradiation of inoculum cells and since the nylon wool-passed spleen T cell population was ineffective as the stimulator. The response was significantly reduced 3 weeks after thymectomy of recipients. PLN enlargement was also observed in older (7-month-old) mice which received spleen cells of younger mice. In this case, however, the response is ascribable to a graft-versus-host reaction, since the inoculation of radiosensitive spleen T cells into footpads resulted in the PLN enlargement. On the other hand, such a stimulatory activity was not found in either lymph node cells or thymus cells. These results suggest that the antigenicity of adherent cells changes with age and that the change is discernible by spleen-locating and short-lived T cells of young mice.

Termination by early deletion of V beta 8 + T cells of aged mice in response to staphylococcal enterotoxin B.

The changes in the T cell repertoire of aging BALB/c mice include an increase of V beta 8 + T cells, most of which have a relatively low density of T cell receptors (TCR). We investigated the response of V beta 8 + T cells to staphylococcal enterotoxin B (SEB), a superantigen from a common bacterium, the anamnestic response to which is thought usually to be part of the defense against infection. The injection of an amount of SEB optimum for V beta 8 + T cell proliferation in young mice induced little or no proliferative response in aged mice, and within one or two days they died in shock with apoptotic cells in the spleen, a sign of T cell-shock caused by SEB. Flowcytometry analysis (FCA) 15 h after SEB injection, when cell division had not yet started, revealed the loss of 90% of V beta 8 + T cells in the blood and of 50% in the spleen in mice of all ages tested. However, conspicuous in the remaining V beta 8 + T cells in the spleens of the young mice but not in those of the aged mice, was an increased cellular complexity, as shown by the fact that light was strongly side scattered in FCA, indicating intracellular re-organization. The remaining T cells in the young could include progenitors for the expanding population of V beta 8 + T cells. As seen in lethal shock, V beta 8 + T cells in the aged are not unresponsive to SEB in vitro. They responded to the antigen by increasing the amount of TCR up to the level of that in young mice, but without proliferation. The proliferation arrest of V beta 8 + T cells of aged mice was found to be an intrinsic defect in in vitro cell mixture experiments, in which they were cocultured with young spleen cells which provided a complete immune microenvironment. It was simultaneously found in vitro that most of the V beta 8 + T cells from aged mice disappeared after antigen stimulation and that their disappearance was prevented by the presence of spleen cells from young mice, although they still did not proliferate. Taken all together the findings suggest that V beta 8+ T cells in the aged are at the end state of maturation and terminate by apoptotic death, causing T-cell shock in response to SEB.