Genetic variants of immunoglobulin γ and κ chains influence humoral immunity to the cancer-testis antigen XAGE-1b (GAGED2a) in patients with non-small cell lung cancer.

GM (γ marker) allotypes, genetic variants of immunoglobulin γ chains, have been reported to be associated strongly with susceptibility to lung cancer, but the mechanism(s) underlying this association is not known. One mechanism could involve their contribution to humoral immunity to lung tumour-associated antigens. In this study, we aimed to determine whether particular GM and KM (κ marker) allotypes were associated with antibody responsiveness to XAGE-1b, a highly immunogenic lung tumour-associated cancer-testis antigen. Sera from 89 patients with non-small cell lung cancer (NSCLC) were allotyped for eight GM and two KM determinants and characterized for antibodies to a synthetic XAGE-1b protein. The distribution of various GM phenotypes was significantly different between XAGE-1b antibody-positive and -negative patients (P = 0·023), as well as in the subgroup of XAGE-1b antigen-positive advanced NSCLC (P = 0·007). None of the patients with the GM 1,17 21 phenotype was positive for the XAGE-1b antibody. In patients with antigen-positive advanced disease, the prevalence of GM 1,2,17 21 was significantly higher in the antibody-positive group than in those who lacked the XAGE-1b antibody (P = 0·026). This phenotype also interacted with a particular KM phenotype: subjects with GM 1,2,17 21 and KM 3,3 phenotypes were almost four times (odds ratio = 3·8) as likely to be positive for the XAGE-1b antibody as the subjects who lacked these phenotypes. This is the first report presenting evidence for the involvement of immunoglobulin allotypes in immunity to a cancer-testis antigen, which has important implications for XAGE-1b-based immunotherapeutic interventions in lung adenocarcinoma.

The effects of ADL on recovery of swallowing function in stroke patients after acute phase.

This study aimed to examine the association between the degree of recovery from dysphagia and changes in functional independence measure (FIM) items in stroke patients after acute phase by conducting a historical cohort study, because none explains the effects of activities of daily living (ADL) on recovery of swallowing function. Study patients included hospitalised stroke patients after acute phase in whom dysphagia was confirmed (n = 72). Change in nutritional intake method score was examined for association with age, days from stroke onset to admission, length of hospital stay and change in FIM score. Moreover, to examine characteristics of patients who were removed from tube feeding, all patients who required tube feeding at the time of admission were divided into two groups comprising those who required tube feeding at discharge and those who did not. A significant and positive association was observed between change in nutritional intake method and FIM for all items other than self-care of bathing, locomotion of stairs and problem solving. Patients who were removed from tube feeding were significantly younger than those who required tube feeding at the time of discharge (P < 0.041) and also showed significantly higher FIM scores for transfer and all cognitive FIM items at the time of admission (P < 0.05). This study demonstrated that nutritional intake methods improve in conjunction with FIM improvements in patients with dysphagia following the acute phase of stroke. Our results suggest that the age and cognitive function may influence the recovery of patient ability of oral intake.

NY-ESO-1 antibody as a novel tumour marker of gastric cancer.

BACKGROUND: NY-ESO-1 antibodies are specifically observed in patients with NY-ESO-1-expressing tumours. We analysed whether the NY-ESO-1 humoral immune response is a useful tumour marker of gastric cancer. METHODS: Sera from 363 gastric cancer patients were screened by enzyme-linked immunosorbent assay (ELISA) to detect NY-ESO-1 antibodies. Serial serum samples were obtained from 25 NY-ESO-1 antibody-positive patients, including 16 patients with curative resection and 9 patients who received chemotherapy alone. RESULTS: NY-ESO-1 antibodies were detected in 3.4% of stage I, 4.4% of stage II, 25.3% of stage III, and 20.0% of stage IV patients. The frequency of antibody positivity increased with disease progression. When the NY-ESO-1 antibody was used in combination with carcinoembryonic antigen and CA19-9 to detect gastric cancer, information gains of 11.2% in stages III and IV, and 5.8% in all patients were observed. The NY-ESO-1 immune response levels of the patients without recurrence fell below the cutoff level after surgery. Two of the patients with recurrence displayed incomplete decreases. The nine patients who received chemotherapy alone continued to display NY-ESO-1 immune responses. CONCLUSION: When combined with conventional tumour markers, the NY-ESO-1 humoral immune response could be a useful tumour marker for detecting advanced gastric cancer and inferring the post-treatment tumour load in seropositive patients.

Effects of reclining posture on velopharyngeal closing pressure during swallowing and phonation.

Velopharyngeal closure plays an important role in preventing air pressure leakage during swallowing and phonation from oropharynx to nasopharynx. Levator veli palatini muscle activity is influenced by oral and nasal air pressure, volume of the swallow bolus and postural changes. However, it is unclear how velopharyngeal closing pressure is affected by reclining posture. The purpose of this study was to investigate the effects of reclining posture on velopharyngeal closing pressure during swallowing and phonation. Nine healthy male volunteers (age range, 27-34 years) participated in this study. Velopharyngeal closing pressure during a dry swallow, a 5-mL liquid swallow, a 5-mL honey-thick liquid swallow and phonations of /P∧/ and /K∧/ were evaluated in an upright posture and at reclining postures of 60° and 30°. A manometer catheter was inserted transnasally onto the soft palate, and each trial was repeated three times. A solid-state manometer catheter with an intra-luminal transducer was used to evaluate the amplitude and duration of each trial, and data were statistically analysed. Average amplitudes during dry and liquid swallows were significantly lower in reclining postures compared with the upright posture, but the amplitude was not significantly different during the thick liquid swallow. Average durations were not affected by postural changes. The amplitudes during phonations were lower in reclining postures, but the differences were not significant. Velopharyngeal closure is significantly affected by reclining posture. This suggests that velopharyngeal closing pressure may be adjusted according to afferent inputs, such as reclining posture and bolus viscosity.

Effect of in vivo administration of Lyt antibodies. Lyt phenotype of T cells in lymphoid tissues and blocking of tumor rejection.

After transplantation of B6RV2 leukemia, initial tumor growth was followed by tumor regression in B6 (CB6F1) female, but not male, mice. This indicated that H-Y antigen is involved in B6RV2 rejection by syngeneic female recipient mice. In the case of another leukemia, BALB.RL male 1, and Ir gene, probably identical to the Rgv-1 gene, is responsible for RL male 1 rejection. Thus, F1 hybrids of BALB/c with certain other strains of mice can reject RL male 1. Using these two different systems of tumor rejection, we investigated the effects of in vivo administration of Lyt and Thy-1 monoclonal antibodies (mAb). Results showed that Lyt-2 and -3 mAb blocked both B6RV2 rejection by B6 female mice and BALB.RL male 1 rejection by CB6F1 mice. The specificity of blocking was confirmed by use of Lyt-2 and -3 mAb to reciprocal alleles and mice from B6 Lyt-congeneic stocks. No blocking was observed with Lyt-1 and Thy-1 mAb. The Lyt phenotype of T cells in lymphoid tissues from mice treated with mAb was then studied. Blocking of the Lyt-2+3+ population was observed in the lymph node and spleen, but not in the thymus. These results indicate the involvement of Lyt-2+3+ cells (or Lyt-2,3 antigen) in tumor rejection. The precise mechanism of blocking is unknown, but it was observed after even a single injection of Lyt-2,3 mAb on day 9 after tumor transplantation, suggesting that effector cells were functionally blocked, rather than that the generation of these cells was inhibited.

Effector cells in allelic H-2 class I-incompatible skin graft rejection.

The cellular mechanisms of skin graft rejection with allelic H-2 class I differences were studied by examining the effect on graft survival of in vivo administration of anti-Lyt-2.2 mAb, anti-L3T4 mAb, or both to recipient mice. The injections of anti-Lyt-2.2 mAb and anti-L3T4 mAb caused selective depletions of Lyt-2+ cells and L3T4+ cells, respectively. Injection of anti-Lyt-2.2 mAb significantly prolonged graft survival in 7 of 12 combinations of H-2D-end difference, but did not prolong graft survival in 5 other combinations of H-2D-end difference, or in 2 combinations of H-2K-end difference. Injection of anti-L3T4 mAb did not prolong graft survival in any combinations with class I difference tested. Injection of anti-L3T4 mAb plus anti-Lyt-2.2 mAb markedly prolonged graft survival in the combinations with class I difference in which anti-Lyt-2.2 mAb had no effect and overcame the effect of anti-Lyt-2.2 mAb in those in which anti-Lyt-2.2 mAb had an effect in prolonging graft survival. These results indicated that in combinations in which anti-Lyt-2.2 mAb did not prolong graft survival, class I antigen stimulated L3T4+ effector cells when Lyt-2+ cells were blocked and Lyt-2+ effector cells when L3T4+ cells were blocked. On the other hand, in the combinations in which anti-Lyt-2.2 mAb prolong graft survival, these antigens initially caused preferential stimulation of Lyt-2+ but not L3T4+ effector cells, although delayed activation of L3T4+ effector cells occurred when Lyt-2+ cells were blocked. Furthermore, a significant correlation was found between the effect of anti-Lyt-2.2 mAb in prolonging graft survival and the failure of recipient mice to produce H-2 antibody. These results can be taken as evidence that L3T4+ effector cells are not involved in the initial phase of graft rejection in these combinations.

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.

Identification of a unique antigen peptide pRL1 on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes and its relation to the Akt oncogene.

BALB/c radiation leukemia RL male 1 is an immunogenic tumor. We established bulk and cloned cytotoxic T lymphocyte (CTL) lines from regressor (BALB/c x C57BL/6)F1 (CB6F1) spleen cells that recognized RL male 1 specifically. We then obtained antigen peptide recognized by CTL from RL male 1 by acid extraction. Analysis of the acid extract by reversed-phase high performance liquid chromatography (HPLC) on a semipreparative C18 column revealed that fractions eluted in 23 min (peak a) and 26 min (peak b) showed sensitization activity on the P815 target for specific CTL. On further purification of these fractions by HPLC and direct sequencing by Edman degradation, we identified the CTL-recognizing RL male 1 peptide pRL1a (IPGLPLSL) in peak a and its possible precursor peptide pRL1b (SIIPGLPLSL) in peak b. Sequence homology indicated that these peptides were derived from the 5' untranslated region of c-akt oncogene.

TL antigen as a transplantation antigen recognized by TL-restricted cytotoxic T cells.

In contrast to broadly expressed classical class I antigens of the major histocompatibility complex, structurally closely related TL antigens are expressed in a highly restricted fashion. Unlike classical class I antigens, TL antigens are not known to be targets of cytotoxic T cells or to mediate graft rejection. Whereas classical class I antigens function as antigen-presenting molecules to T cell receptors (TCR), the role of TL is yet to be defined. To elucidate the function of TL, we have derived transgenic mice expressing TL in most tissues including skin by introducing a TL gene, T3b of C57BL/6 mouse origin, driven by the H-2Kb promoter. By grafting the skin of transgenic mice, we demonstrate that TL can serve as a transplantation antigen and mediate a TCR-alpha/beta+ CD8+ cytotoxic T cell response. This T cell recognition of TL does not require antigen presentation by H-2 molecules. Furthermore, we show that C57BL/6 F1 mice develop CD8+ T cells that are cytotoxic for C57BL/6 TL+ leukemia cells, providing further support for the concept that aberrantly expressed nonmutated proteins such as TL can be recognized as tumor antigens.

Differential involvement of CD4+ cells in mediating skin graft rejection against different amounts of transgenic H-2K(b) antigen.

Differential involvement of CD4+ cells in mediating class I-disparate skin graft rejection was investigated using quantitatively different Kb transgenic mice as donors under conditions in which CD8+ cells were blocked in vivo by administration of anti-CD8 monoclonal antibody (mAb). Tg.H-2Kb-1 and -2 are C3H transgenic mice with 14 and 4 copies, respectively, of the H-2Kb gene. Cell surface expression of Kb antigen and the Kb antigenicity of skin for eliciting graft rejection with homozygous and heterozygous transgenic mice were correlated with the copy number. In vivo administration of anti-Lyt-2.1 (CD8) mAb markedly prolonged survival of heterozygous and homozygous C3H Tg.H-2Kb-2 skin grafted onto C3H mice, but prolonged survival of heterozygous Tg.H-2Kb-1 skin grafts much less and did not prolong survival of homozygous Tg.H-2Kb-1 grafts. Administration of anti-L3T4 (CD4) mAb alone did not have any effect on skin graft rejection. Administration of anti-L3T4 (CD4) mAb with anti-Lyt-2.1 (CD8) mAb blocked rejection in all combinations. These findings indicate that a quantitative difference of class I antigen caused differential activation of CD4+ cells under conditions in which CD8+ cells were blocked.

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.

Hepatitis C virus NS4 protein impairs the Th1 polarization of immature dendritic cells.

Dendritic cells (DCs) in chronic hepatitis C patients display impaired function, although the details remain unclear. To investigate the hepatitis C virus (HCV) protein that has the most impact on DC function, we compared five recombinant proteins and seven HCV protein genes in modulating DC phenotype and function. Immature DCs (iDCs) were established from healthy donor peripheral blood monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4. Lipopolysaccharide was used to establish mature DCs (mDCs). Cells were then pulsed with HCV recombinant proteins or transfected with HCV plasmids and subsequently assayed for cell surface marker expression by flow cytometry. For cytokine and proliferative T-cell response analysis, DCs were cultured with autologous CD4 T cells and tuberculin purified protein derivative (PPD). Mean fluorescent intensity of CD86 was reduced in HCV protein-pulsed iDCs. Proliferative T-cell responses and Th1 cytokine concentrations were reduced with HCV nonstructural proteins (NS), particularly with HCV NS4. HCV nonstructural proteins, particularly NS4, change the iDC phenotype and reduce antigen-specific T-cell stimulatory function with Th1 cytokine reductions.

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin.

F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.

Diagnostic accuracy of a rapid E1-antigen test for chikungunya virus infection in a reference setting.

OBJECTIVES: Rapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses. METHODS: An immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test. RESULTS: Sensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5-98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5). CONCLUSIONS: The E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes.

Predictive Factors Associated with Oral Intake Ability in Gastrostomy Patients Under Long-Term Care.

OBJECTIVE: To determine the physical indicators associated with oral intake status and swallowing function in gastrostomy patients under long-term care. DESIGN: Cross-sectional study. SETTING: Thirty-one hospitals that perform gastrostomy insertion, replacement and management. PARTICIPANTS: A total of 117 respondents from 31 hospitals in Japan underwent gastrostomy tube replacement and management between September 2012 and January 2014. Each participant underwent a gastrostomy at least 6 months prior to the study, and received long-term care either at home, a care facility, or a hospital. MEASUREMENTS: We conducted a questionnaire survey at Japanese hospitals and used the data obtained from 117 respondents for analysis. The survey was conducted using a questionnaire form that collected information about the following items: oral intake status, sex, age, disease history, number of days elapsed since gastrostomy, residence status, modified Rankin Scale score, consciousness, oral hygiene status, articulation and phonation, voluntary saliva swallow, Modified Water Swallow Test, and Food Test. RESULTS: Results revealed significant differences in modified Rankin Scale scores, sputum production, articulation and phonation, and voluntary saliva swallowing between patients who were orally fed and those who were not. Moreover, sputum production and voluntary saliva swallowing were strongly associated with oral intake status. Finally, sputum production, articulation and phonation, and voluntary saliva swallowing were strongly associated with swallowing function test results. CONCLUSION: Results from this study suggested that sputum production, articulation and phonation, and voluntary saliva swallowing could be used as indicators for estimating oral intake status and swallowing function in gastrostomy patients under long-term care.

Detection of a unique antigen on radiation leukemia virus-induced leukemia B6RV2.

Radiation leukemia virus-induced leukemia of a male C57BL/6 mouse, B6RV2, is immunogenic to female BALB/c X C57BL/6 F1 mice. In these mice, B6RV2 tumors regressed after initial growth, and after tumor regression the mice were resistant to repeated inocula of up to 10(8) B6RV2 cells. Serum from these mice reacted with B6RV2 in mixed hemadsorption or protein A assays, and absorption analysis indicated that the antigen was restricted to B6RV2; it could not be detected in normal thymocytes or spleen concanavalin A blasts from different inbred strains, nor in 16 C57BL/6 or BALB/c leukemias. Spleen cells from mice in which the tumor had regressed were cytotoxic to B6RV2 after in vitro stimulation with B6RV2, as shown by 51-chromium release assay. This cytotoxicity was eliminated by pretreatment of the cells with anti-Thy-1.2, anti-Lyt-2.2, anti-Lyt-3.2, and complement, indicating that the effector cells were T-cells. The specificity of T-cell killing of B6RV2 was examined by competitive inhibition assays with unlabeled cells; only B6RV2 inhibited killing, while eight other C57BL/6 leukemias did not inhibit. Thus, the antigen on B6RV2 defined serologically and by cytotoxic T-cells is a unique antigen. However, it was not revealed by antibody-blocking test whether the unique determinant defined serologically was related to that recognized by T-cells; B6RV2 antiserum did not block lytic activity in the absence of added complement, irrespective of whether the target cells were untreated or anti-H-2b-treated B6RV2. H-2Kb antisera, but not H-2Db antisera, blocked lysis. This indicated that the H-2Kb molecule was exclusively involved in recognition of B6RV2 by cytotoxic T-cell.

Rejection antigen peptides on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes: derivation from the normally untranslated 5' region of the c-akt proto-oncogene activated by long terminal repeat.

Tumor antigen peptides on BALB/c leukemia RL male 1 that were recognized by cytotoxic T lymphocytes were shown to be derived from a normally untranslated region of the akt proto-oncogene (Uenaka, A. et al., J. Exp. Med., 180: 1599, 1994). We show here that the murine leukemia virus (MuLV) long terminal repeat (LTR) was inserted directly into the exon of c-akt in RL male 1 leukemia and that transcription started from the cap site of the LTR. Translation appeared to start from the ATG codon created in the six nucleotides of unknown origin, which were inserted into the LTR/akt junction. The deduced molecular size is approximately M(r) 59,000 due to the addition of 33 amino acid residues to the normally expressed c-AKT protein. Western blot analysis demonstrated the presence of M(r) 59,000 molecules in an RL male 1 lysate, and their expression at about ten times the level of normal AKT molecules of M(r) 56,000, which is consistent with the increased expression of akt mRNA demonstrated by Northern blot analysis. The findings show that the molecular alteration of AKT protein by insertion of MuLV LTR is the mechanism for creating rejection antigen peptides derived from the untranslated region of akt.

Vaccination with multiple antigen peptide as rejection antigen peptide in murine leukemia.

pRL1a (IPGLPLSL) is the Ld-binding tumor rejection antigen peptide recognized by CTLs on BALB/c radiation leukemia RL(male)1. We demonstrated that in vivo and in vitro sensitization with pRL1a multiple antigen peptide (MAP), but not with the pRL1a peptide itself, generated pRL1a-specific CTLs in the spleen cells of BALB/c mice. No enhancement of cytotoxicity was observed by emulsifying pRLla MAP in incomplete Freund's adjuvant or in complete Freund's adjuvant for in vivo sensitization. Selective depletion of CD4+ T cells in mice by treatment with anti-L3T4 (CD4) monoclonal antibody and that of macrophages by treatment with carrageenan on in vivo sensitization with pRL1a MAP abrogated CTL generation. The findings suggest that CD4+ T cells and antigen-presenting cells were necessary for the in vivo priming of CD8+ T cells with pRL1a MAP. Furthermore, we demonstrated that in vivo sensitization of BALB/c mice with pRL1a MAP, but not with pRL1a peptide, showed an inhibitory effect on RL(male)1 tumor growth. No growth-inhibitory effect was observed on control RVA, RVD, or Meth A tumors.

Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody.

Immune regulation has been shown to be involved in the progressive growth of some murine tumors. In this study, we demonstrated that a single in vivo administration of an amount less than 0.125 mg of anti-CD25 interleukin 2 receptor alpha monoclonal antibody (mAb; PC61) caused the regression of tumors that grew progressively in syngeneic mice. The tumors used were five leukemias, a myeloma, and two sarcomas derived from four different inbred mouse strains. Anti-CD25 mAb (PC61) showed an effect in six of the eight tumors. Administration of anti-CD25 mAb (PC61) caused a reduction in the number of CD4+ CD25+ cells in the peripheral lymphoid tissues. The findings suggested that CD4+ CD25+ immunoregulatory cells were involved in the growth of those tumors. Kinetic analysis showed that the administration of anti-CD25 mAb (PC61) later than day 2 after tumor inoculation caused no tumor regression, irrespective of depletion of CD4+ CD25+ immunoregulatory cells. Two leukemias, on which the PC61-treatment had no effect, seemed to be incapable of eliciting effective rejection responses in the recipient mice because of low or no antigenicity.

Two isotypes of murine nm23/nucleoside diphosphate kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a murine melanoma line.

A series of sublines of a murine melanoma B16 of C57BL/6 origin were established and examined regarding their metastatic capacity and expression of nm23. The number of pulmonary metastases developed by these sublines was inversely correlated with the expression of two isotypes of nm23, nm23-M1 and nm23-M2. The cDNAs of nm23-M1, nm23-M2, and a combination of both were transfected into the highly metastatic melanoma subline FE7, with low nm23 expression. FE7 transfectants of any of these cDNAs expressed transfected genes, and their metastatic capacity was suppressed when compared with parental FE7 or FE7 transfected with a control neo gene. These cell lines, however, did not change in terms of in vitro growth in the presence of 3 or 10% fetal bovine serum and in vivo growth when injected s.c. into C57BL/6-nu/nu mice. Similar experiments were also performed using FE7 transfectants of human nm23 genes. Transfectants of nm23-H1, nm23-H2, and their combination did not present altered metastatic potential. These findings indicated that two murine isotypes of nm23 but not those of humans are intimately related with the suppression of metastasis in the murine body.