Cytological markers for predicting ALK-positive pulmonary adenocarcinoma.

BACKGROUND: ALK gene rearrangement is an important class of gene mutations in pulmonary adenocarcinoma. ALK-positive pulmonary adenocarcinoma exhibits characteristic histological features, such as signet ring cell carcinoma (SRCC) and a mucinous cribriform structure. However, when insufficient histological specimens are obtained, ALK-positivity must be predicted based on cytological features. The purpose of this study was to clarify the cytological characteristics of ALK-positive pulmonary adenocarcinoma. METHODS: We compared the cytological findings of 16 ALK-positive cases with 40 ALK-negative cases. We examined various cytoplasmic features of SRCC, including the presence of pink, yellow, or orange mucin; green, vacuolar, or vesicular cytoplasm; and green globular cytoplasmic secretions. We also examined whether the SRCC cells exhibited a pattern of individually scattered cells, the formation of cell clusters, and formation of a mucinous cribriform pattern. RESULTS: A univariate analysis showed that significantly frequent cytological findings included pink mucin, green cytoplasm, vacuolar cytoplasm, vesicular cytoplasm, green globular cytoplasmic secretions, an individually scattered pattern, cluster formation, and a mucinous cribriform structure (all, P < .05). A stepwise multivariate logistic regression analysis identified three significant contributing factors: pink mucin (P = .03), vesicular cytoplasm (P = .06), and an individually scattered pattern (P = .01) of SRCC. If the specimens showed two or three of these features, the sensitivity and specificity were both 88% for the prediction of ALK-positive cancers. CONCLUSION: Three cytological features of SRCC (pink mucin, vesicular cytoplasm, and an individually scattered pattern) could be useful cytological markers for the prediction of ALK-positive pulmonary adenocarcinoma.

Comparison of preoperative chemotherapy using docetaxel, cisplatin and fluorouracil with cisplatin and fluorouracil in patients with advanced carcinoma of the thoracic esophagus.

We retrospectively compared preoperative docetaxel, cisplatin, and fluorouracil (DCF) with cisplatin and fluorouracil (CF) in patients with esophageal cancer. The study included patients with advanced thoracic esophageal carcinoma (excluding T4 tumors) receiving preoperative chemotherapy. In the DCF group, five patients received two courses of treatment every 4 weeks, and 33 patients received three courses every 3 weeks. In the CF group, 38 patients received two courses of treatment every 4 weeks. Patients underwent curative surgery 4-5 weeks after completing chemotherapy. Patient demographic characteristics did not differ between the two study groups. The incidence of a grade 3 or 4 hematologic toxicity was significantly higher in the DCF group (33 patients) than in the CF group (five patients; P < 0.001). Curative resection was accomplished in 79% of patients in the DCF group and 66% in the CF group (P = 0.305). There were no in-hospital deaths. The incidence of perioperative complications did not differ between the groups. A grade 2 or 3 histological response was attained in a significantly higher proportion of patients in the DCF group (63%) than in the CF group (5%; P < 0.001). Progression-free survival and overall survival were significantly higher in the DCF group (P = 0.013, hazard ratio 0.473; P = 0.001, hazard ratio 0.344). In conclusion, a grade 3 or 4 hematologic toxicity was common in the DCF group but was managed by supportive therapy. Histological response rate, progression-free survival, and overall survival were significantly higher in the DCF group compared with the CF group.

Pyrethroid insecticide exposure and reproductive hormone levels in healthy Japanese male subjects.

The associations between serum levels of reproductive hormones (follicle-stimulating hormone, luteinizing hormone, testosterone, sex hormone-binding globulin, inhibin B and calculated free testosterone) and urinary metabolite concentration of pyrethroid insecticides [3-phenoxybenzoic acid (3-PBA)] were explored in 322 male university students in suburban Tokyo. The subjects constituted part of a large cross-sectional survey on the reference value of semen quality of Japanese men. Urinary 3-PBA was detectable in 91% of the subjects demonstrating ubiquitous exposure among the general population. However, there were no associations between urinary 3-PBA and serum hormone levels. This result was inconsistent with those reported in China and the USA for subjects who had similar levels of urinary 3-PBA to the present subjects. One of the possible reasons of the inconsistency might be different composition of pyrethroid insecticides to which the subjects were exposed; 3-PBA is a common metabolite of a number of pyrethroids and thus lacks specificity to compounds that may have different potentials of reproductive toxicity. Another reason might be related to the fact that our subjects were university students who were not aware of their own fertility, whereas the previous study subjects were infertility patients. However, the multiple regression models could explain only a limited fraction of total variance in serum levels of hormones. Identification of other contributors is warranted.

Obstacles of non-heart-beating donor kidney transplantation in Japan to date and future perspectives.

In Japan, multiple organ retrieval from brain-dead heart-beating donors has been gradually increasing since the law was adopted in 1997 and amended in 2009. However, almost more than 90% of total deceased donor kidney transplantation (DDKT) in Japan are still obtained from non-heart-beating donors (NHBD). The majority of NHBD are Maastricht categories IV and III. In category IV, we usually place a double balloon arterial and a venous drainage catheter via the femoral vessels after the diagnosis of clinical brain death and acquisition of informed consent from the family. After controlled cardiac arrest, the double balloons are inflated and in situ cold perfusion started as soon as possible to minimize warm ischemic time (WIT), seeking to achieve a zero to within a few minutes WIT in most cases. In category III, it is impossible to place the device prior to cardiac arrest. In these cases, after declaration of cardiac death, cardiopulmonary compression is accompanied by systemic heparinization, immediate laparotomy, and insertion of a cold perfusion catheter at the aortic and caval bifurcations to minimize WIT. NHBD kidney retrieval is critical; extirpation must be performed as rapidly as possible. The results of NHBD kidney transplantation in Japan are excellent, according to the advancement and utilization of in situ cannulation, organ perfusion, and sophisticated retrieval techniques. The patient and graft survival rates of DDKT at 1, 3, and 5 years in most recent 2001 to 2007 era were 95.4%, 92.2%, 89.1% (n = 945) and 89.2%, 83.7%, 77.8% (n = 919), respectively.

Terahertz optical clock generation with tunable repetition rate and central wavelength using variable-bandwidth spectrum shaper.

Recently, a number of high-speed optical clock generation technologies have been developed due to their potential useful applications in different fields. Here, we propose a new terahertz optical clock generation technique with tunable repetition rate and central wavelength. The proposed optical clock generator consists of an frequency comb light source and a variable-bandwidth spectrum shaper (VBS). The VBS can generate arbitrary repetition rate pulse trains and waveform by controlling each spectral mode. We experimentally demonstrated optical clock generation with repetition rates of 1.28, 2.56, 3.0, and 4.0 THz.

Cellular interactions during the development of autoimmunity in a fetal lamb model of self-antigen deprivation.

Anti-thyroid autoimmune responses have been examined in fetal lambs, the immune systems of which had matured in the absence of exposure to thyroid-specific antigens. The lymphocytic infiltrate in self-thyroid tissue reintroduced into autoimmune lambs showed well-differentiated B and T cell domains. However, T cells from these fetuses were not sensitized against ovine thyroglobulin nor did serum antibodies appear against ovine thyroglobulin or thyroid peroxidase. In the light of these observations, it is inferred that the primary abnormality in the immune systems of fetuses deprived of exposure to thyroid autoantigens is likely to be a failure of the development of a normal T cell subpopulation responsible for down-regulation of autoreactivity. It is also concluded that overt autoimmunity develops only when these fetuses are challenged with thyroid tissue and that B cells may undertake an antigen-presentation role in its induction.

CD4-CD8- T cell receptor alpha beta T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumor rejection.

The generation of an in vitro major histocompatibility complex class I specific response of CD4-CD8- T cell receptor (TCR) alpha beta cytotoxic T lymphocytes (CTL) and their allogeneic tumor rejection were investigated. Inocula of BALBRL male 1 were rejected in C57BL/6 (B6) mice treated with minimum essential medium (MEM) (control), anti-L3T4 (CD4) monoclonal antibody (mAb) or anti-Lyt-2.2 (CD8) mAb and CTL against the tumor were generated in vitro. No rejection and no induction of CTL were observed in B6 mice treated with anti-L3T4 (CD4) plus anti-Lyt-2.2 (CD8) mAb. CTL with the classical Thy-1+ CD3+CD4-CD8+ TCR alpha beta phenotype were generated in mixed lymphocyte tumor cell culture (MLTC) spleen cells from B6 mice treated with MEM (control) or anti-L3T4 (CD4) mAb, whereas CTL with an unusual Thy-1+CD3+CD4-CD8- TCR alpha beta phenotype were generated in MLTC spleen cells from anti-Lyt-2.2 (CD8) mAb-treated B6 mice. Both types of CTL were reactive with both H-2Kd and Dd (Ld) class I antigen. These findings suggest that when CD4+ cells were blocked by anti-L3T4 (CD4) mAb, CD8+ CTL mediated rejection, and when CD8+ cells were blocked by anti-Lyt-2.2 (CD8) mAb, CD4+ cells were capable of mediating rejection, although less efficiently than CD8+ cells, by inducing CD4-CD8- TCR alpha beta CTL. The finding that adoptive transfer of CD4 and CD8-depleted MLTC spleen cells, obtained from anti-Lyt-2.2 (CD8) mAb-treated B6 mice that had rejected BALBRL male 1, resulted in rejection of BALBRL male 1 inoculated into B6 nu/nu mice confirmed the above notion. CTL clones with the CD4-CD8- TCR alpha beta phenotype specific for Ld were established.

Participation of a dominant cytotoxic T cell population defined by a monoclonal antibody in syngeneic anti-tumor responses.

Cytotoxic T lymphocyte (CTL) clones against a syngeneic Friend virus-induced erythroleukemia (FBL-3) were generated in C57BL/6 (B6) mice. A monoclonal antibody (mAb, N9-127) was then raised from spleen cells of a B6 mouse immunized syngenically against one of these CTL clones. This mAb detected the epitope (127Ep) of the T cell antigen receptor (TcR) on the immunizing CTL clone in tests of immunoprecipitation, specific blocking and proliferation, and induction of TcR-mediated nonspecific lysis of the clone. In addition, more than 10% of the FBL-3-specific CTL clones isolated independently from B6 mice were 127Ep+. Further investigations revealed that up to 30% of B6 anti-FBL-3 T cell blasts from mixed lymphocyte tumor cell cultures were positive for this epitope, and that its expression was confined to CD8+ T cells. This epitope was not detected in naive lymphoid cells from the spleen, lymph nodes or thymus or in T cell clones specific for tumors other than FBL-3. The FBL-3-specific CTL clones were next grouped into 127Ep+ and 127Ep- clones. Sequence analyses of the CTL clone used for immunization showed the rearrangements of V alpha 1J alpha 112-2 and V beta 10D beta 2.1J beta 2.7. Southern blot analysis of all the 127Ep+ CTL clones examined showed the same DNA rearrangement bands of both the TcR alpha and beta genes. These findings suggested that mAb N9-127 recognized the shared determinant of the TcR molecule which was expressed by the dominant CTL population in the response to FBL-3.

[Functional alterations of human platelets following 111In labeling with different ligands and incubation media].

We studied the effects of various 111In-water soluble chelates and incubation media on labeling efficiency of platelets and in vitro platelet aggregability. High labeling efficiency of platelets in ACD-saline was achieved with 111In-oxine sulfate, 111In-tropolone and 111In-MPO (2-mercaptopyridine-N-oxide). In the condition with 4.8 x 10(6)/mm3 platelets in ACD-plasma, 111In-oxine-sulfate had low labeling efficiency and inconsistent labeling, while 111In-tropolone and 111In-MPO had high labeling efficiency. In vitro platelet aggregability (ADP 2 microM) was reduced when platelets were labeled in the absence of plasma. However, there was no significant difference in platelet aggregability among 111In-platelets labeled by three different chelates. In conclusion, to maintain aggregation activity of the platelets with relatively high labeling efficiency, the best result was obtained by using MPO or tropolone chelate in plasma at 4.8 x 10(6)/microliters platelet concentration.

Roles of CD4+ and CD8+ cells, and the effect of administration of recombinant murine interferon gamma in listerial infection.

Studies were made on the effects of in vivo administration of anti-CD4 mAb, anti-CD8 mAb, or a combination of both mAbs on multiplication of bacteria, the levels of serum transaminases, and mortality in mice infected with Listeria monocytogenes. Results showed that in sublethal infection, CD8+ cells enhanced the peak of bacterial multiplication and liver cell necrosis, and CD4+ cells suppressed CD8+ cell-mediated enhancement. Results also showed that either CD4+ or CD8+ cells were necessary for, and capable of, mediating clearance of the bacteria. CD8+ cells were more efficient than CD4+ cells, but for optimal clearance both were necessary. In lethal listeriosis, treatment of mice with anti-CD8 mAb or a combination of both anti-CD4 and anti-CD8 mAbs, but not anti-CD4 mAb only, protected mice from death by decreasing multiplication of bacteria in the liver and spleen after a peak of approximately 10(8) CFU, and lowering the elevated serum levels of transaminases. These findings indicated that CD8+ cells were responsible for causing irreversible systemic Listeria infection and severe liver necrosis. In lethal listeriosis, administration of rMuIFN-gamma markedly prolonged survival by decreasing multiplication of bacteria and promoting recovery from liver necrosis.

Platelet aggregability and in vivo platelet deposition in patients with ischemic cerebrovascular disease--evaluation by indium-111-platelet scintigraphy.

In ischemic cerebrovascular disease, it is not clear whether platelet function in vitro actually reflects the situation in vivo. Using indium-111 platelet scintigraphy as a method for detecting platelet activation in vivo, we tried to elucidate this problem. Twenty eight patients with chronic stage of ischemic cerebrovascular disease (CVD) and 17 control subjects were examined. Platelet scintigrams were positive in 9 of 28 patients in CVD, while all were negative in control. A comparison of the results obtained from qualitative platelet imaging and platelet aggregability was performed to evaluate whether threshold aggregation concentration (TAC) grade differed across the three groups (control, CVD patients without platelet deposition and CVD patients with platelet deposition). CVD patients with platelet deposition showed a higher TAC than those patients who did not show platelet deposition (P less than 0.05) or control subjects without platelet deposition (P less than 0.05). These results suggest that some patients in chronic stages of CVD may have active platelet deposition on carotid atheromatous lesions, and presence of platelet deposition in vivo could contribute to reduce platelet reactivity in peripheral blood.

Blocking of cytotoxic T-cell function of MOLF/Ei mice by anti-Ly 35.1 monoclonal antibody.

Ly 35.1 antigen is an alloantigen expressed only on T cells of Mus musculus molossinus-derived inbred strains. Previous findings indicated that the genetic locus coding for Ly 35 antigen was closely linked to Ly 2/3 on chromosome 6 and that epitopes detected by Ly 2.1 and Ly 35.1 monoclonal antibodies (mAb) were closely associated, as shown by binding inhibition assay. In this study, we examined the blocking effects of anti-Ly 2.1 and anti-Ly 35.1 mAb on cytotoxic T-cell function of MOLF/Ei mice generated against BALB/c. MOLF/Ei cytotoxicity was blocked by Ly 35.1 mAb, but not by anti-Ly 2.1 mAb. Additional tests showed that cytotoxicity was blocked by Ly 3.1 mAb, but not Ly 3.2 or Ly 2.2 mAb. These results suggested that MOLF/Ei mice express Ly 3.1, but not Ly 2.1 antigen at a functional level, and that Ly 35.1 may be a functional epitope of Ly 2 antigen in the MOLF/Ei strain.

The roles of CD8+ and CD4+ cells in tumor rejection.

In vivo administrations of anti-Lyt-2.2 (CD8) mAb and anti-L3T4 (CD4) mAb selectively eliminated CD8+ cells and CD4+ cells, respectively. The relative potencies of CD8+ cells and CD4+ cells and their roles in primary tumor rejections were studied by investigating the effects of these mAbs on tumor growth. CD8+ cells were themselves fully capable of mediating rejection in 5 different tumor rejection systems: two radiation leukemia virus (RadLV)-induced leukemias, B6RV2 and BALBRVD, a radiation-induced leukemia BALBRL male 1, and a plasmacytoma BALBMOPC-70A in CB6F1 mice, and a Friend virus-induced leukemia B6FBL-3 in B6 mice. On the other hand, CD4+ cells were capable of resisting tumor growth of B6FBL-3, but not of the other four tumors. Furthermore, for efficient rejection of CB6F1UV female 1 sarcoma by CB6F1 mice, synergy of CD8+ and CD4+ cells was necessary. Blocking of UV female 1 rejection was abrogated by delayed administration of anti-L3T4 (CD4) mAb but not anti-Lyt-2.2 (CD8) mAb, indicating the involvement of CD4+ cells in only the initial phase of rejection.

Proliferative response of cloned CTL line 10B-5 upon stimulation with soluble clonotypic monoclonal antibody and its blocking by anti-Lyt-2 antibody.

Proliferation of the cloned cytotoxic T lymphocyte (CTL) line 10B-5 induced by anticlonotypic antibody and its blocking by anti-Lyt-2 mAb were studied. Clone 10B-5 was derived from spleen cells of a (BALB/c x C57BL/6)F1, (CB6F1)-nu/+ mouse immunized with UV female 1 sarcoma. 10B-5 cells lysed UV female 1 specifically and proliferated upon stimulation with UV female 1 and feeder cells in the presence of IL 2. Anti-clonotypic monoclonal antibody (mAb) N1-56 was produced by a hybridoma established by fusion of spleen cells from a CB6F1-nu/+ mouse that had been immunized by five injections of 10B-5 cells prefixed with 0.1% formaldehyde. N1-56 mAb immunoprecipitated 90 kd molecules cleavable to 45 kd molecules under reducing conditions, indicating its reaction with T cell antigen receptor (TCR). N1-56 mAb in its soluble form induced a proliferative response of 10B-5 cells. Thus, the antigen binding site of N1-56 mAb appeared to substitute for the specific antigen determinant on UV female 1 sarcoma. The F(ab')2, but not the Fab fragment of purified N1-56 mAb, stimulated proliferation, indicating that cross-linking of TCR molecules was necessary for stimulation. The proliferative response of 10B-5 cells induced by soluble N1-56 mAb was blocked by addition of anti-Lyt-2.2 mAb to the cultures. The specificity of Lyt-2 blocking was confirmed by an absorption test. The proliferative response of 10B-5 cells induced by Con A, but not that induced by IL 2, was blocked by anti-Lyt-2.2 mAb. These results indicated that blocking by anti-Lyt-2.2 mAb was at an early stage and that it could be bypassed by stimulation with IL2.

Immunoreactive glucagon in the smooth muscle cells of the rat cerebral artery: an immunohistochemical analysis.

The presence of immunoreactive glucagon (IRG) in a smooth muscle of the cerebral arteries of rats was demonstrated immunohistochemically using two antisera against pancreatic glucagon, OAL -123 and Unger 's 30K . Based on the results and on our previous radioimmunoassay and gel filtration study, the smooth muscle cells of the blood vessels may be one of the extrapancreatic sources of IRG in the plasma.

Suppression of the luteinizing hormone releasing effect of luteinizing hormone releasing hormone by arginine-vasotocin in immature male dogs.

Arginine-vasotocin (0.1 or 10 ng/kg body wt) was administered into the carotid artery of anaesthetized immature male dogs 3 h before the administration of a standard dose of luteinizing hormone releasing hormone (LH-RH, 5 microgram/kg body wt) into the same vessel. The rate of secretion of 17-oxosteroids by the testes in vivo served as an index of luteinizing hormone (LH) secretion. The administration of LH-RH into the carotid artery of control dogs which had been injected with isotonic saline caused a slight but definite increase in the secretion of testicular 17-oxosteroids. This effect of LH-RH on the testicular secretion of steroids was markedly reduced by pretreatment with arginine-vasotocin. However, the testicular response to the i.v. administration of human chorionic gonadotrophin (5 i.u./kg body wt) was unaffected by pretreatment with arginine-vasotocin (10 ng/kg body wt). These results indicate that in immature male dogs, arginine-vasotocin is able to inhibit the action of LH-RH by acting directly on the anterior pituitary gland.

Suppression of the luteinizing hormone releasing effect of luteinizing hormone releasing hormone by arginine-vasotocin.

The concentrations of 17-oxosteroids in the spermatic venous blood of anaesthetized dogs were used as an index of LH release to assess the effects of arginine-vasotocin on the response of the canine pituitary gland to exogenous luteinizing hormone releasing hormone (LH-RH). When injected into the carotid artery, arginine-vasotocin (1.0 microgram/kg body wt) caused no significant alterations in the testicular output of 17-oxosteroids. The administration of LH-RH (5 microgram/kg body wt, a standard dose) into the carotid artery produced typical stimulation of testicular 17-oxosteroid secretion. Administration of arginine-vasotocin (0.01, 0.1 or 1.0 microgram/kg body wt) into the carotid artery 3 h before the administration of a standard dose of LH-RH inhibited the testicular secretion of 17-oxosteroids normally induced by LH-RH. However, pretreatment with arginine-vasotocin (1.0 microgram/kg body wt) did not affect the testicular response to i.v. administration of human chorionic gonadotrophin (5 i.u./kg body wt). These results indicate that in the dog, arginine-vasotocin inhibits the LH-RH-induced release of LH by acting acting directly on the anterior pituitary gland.