The CD8+ T cell repertoire in beta 2-microglobulin-deficient mice is biased towards reactivity against self-major histocompatibility class I.

Beta 2-Microglobulin-deficient (beta 2m -/-) mice are reported to lack cell surface expression of major histocompatibility complex (MHC) class I molecules, CD8+ T cells, and the ability to mount MHC class I-specific T cell responses. We have observed that beta 2m -/- mice possess CD8+ T cells that can be induced to perform strong allospecific cytotoxic responses against nonself-MHC class I by in vivo priming. We report that these beta 2m -/- cytotoxic T lymphocyte (CTL) differ from those induced in beta 2m-positive littermates in that they cross-react and kill cells expressing self-MHC class I at normal ligand density with beta 2m. beta 2m -/- CTL could even be induced in primary mixed lymphocyte culture by self-MHC class I expressing stimulator cells, whereas allogeneic stimulator cells failed to elicit a response under similar conditions. Cells with a reduced cell surface MHC class I expression were less sensitive, while syngeneic beta 2m -/- cells were resistant to the beta 2m -/- CTL. This antiself-MHC reactivity could not be induced when beta 2m -/- T cells matured in an environment with normal MHC class I expression in bone marrow chimeric mice. Antiself-MHC reactivity was also observed against human peptide loading-deficient cells expressing the appropriate murine class I molecules, suggesting that affinity to self-MHC class I may occur irrespective of peptide content. The results fit with a model where positive and negative selection of CD8+ T cells in beta 2m -/- mice is mediated by low levels of MHC class I free heavy chains. In this model, low ligand density on selecting cells leads to positive selection of rare T cells that bind to low levels of MHC class I free heavy chains, resulting in a very small peripheral CD8+ compartment. Due to low density of the selecting ligand, negative selection does not remove T cells recognizing beta 2m-positive cells expressing self-MHC class I at normal ligand density, which generates a T cell repertoire that would be autoreactive in a beta 2m-positive littermate. The first "MHC deficient" animals thus paradoxically provide a tool for direct demonstration and analysis of self MHC bias in the T cell repertoire.

Recognition of the major histocompatibility complex restriction element modulates CD8(+) T cell specificity and compensates for loss of T cell receptor contacts with the specific peptide.

Triggering of a T cell requires interaction between its specific receptor (TCR) and a peptide antigen presented by a self-major histocompatibility complex (MHC) molecule. TCR recognition of self-MHC by itself falls below the threshold of detection in most systems due to low affinity. To study this interaction, we have used a read-out system in which antigen-specific effector T cells are confronted with targets expressing high levels of MHC compared with the selecting and priming environment. More specifically, the system is based on CD8(+) T cells selected in an environment with subnormal levels of MHC class I in the absence of beta2-microglobulin. We observe that the MHC restriction element can trigger viral peptide-specific T cells independently of the peptide ligand, provided there is an increase in self-MHC density. Peptide-independent triggering required at least four times the natural in vivo level of MHC expression. Furthermore, recognition of the restriction element at expression levels below this threshold was still enough to compensate for lack of affinity to peptides carrying alanine substitutions in major TCR contact residues. Thus, the specificity in TCR recognition and T cell activation is fine tuned by the avidity for self-MHC, and TCR avidities for peptide and MHC may substitute for each other. These results demonstrate a functional role for TCR avidity for self-MHC in tuning of T cell specificity, and support a role for cross-reactivity on "self" during T cell selection and activation.

Alteration of the natural killer repertoire in H-2 transgenic mice: specificity of rapid lymphoma cell clearance determined by the H-2 phenotype of the target.

The mechanism behind natural tumor resistance conveyed by a H-2Dd transgene to C57Bl/6 (B6) mice was investigated. Transgenic D8 mice were more efficient than control mice in natural killer (NK) cell mediated rapid elimination of intravenously inoculated radiolabeled lymphoma cells of B6 origin, such as RBL-5. There was no difference between D8 and B6 mice when elimination of YAC-1 targets was monitored. The effect of the transgene on the NK repertoire was related to the H-2 phenotype of the target: the differential elimination of RBL-5 lymphoma cells in D8 and B6 mice was not seen when a H-2 deficient variant of this line was used (efficiently eliminated in both genotypes), nor was it seen with a H-2Dd transfectant (surviving in both genotypes). The data show that a MHC class I transgene can directly control natural killing in vivo by altering the repertoire rather than the general levels of NK activity. Since the NK mediated elimination seen after introduction of a novel gene in the host was neutralized by introducing the same gene (H-2Dd), but not an unrelated class I gene (H-2Dp), in the tumor, the data support the concept of NK surveillance against missing self. This combined transgenic/transfectant system may serve as a tool for a molecular dissection of the interactions between NK cells and their targets in vivo.

Recruitment and activation of natural killer (NK) cells in vivo determined by the target cell phenotype. An adaptive component of NK cell-mediated responses.

Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon gamma in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I-deficient tumor cells were approximately 10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I-deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.

Restoration of a tumorigenic phenotype by beta 2-microglobulin transfection to EL-4 mutant cells.

It has frequently been suggested that loss of beta 2-microglobulin (beta 2m) in tumor cells may lead to malignant progression due to escape from immunological recognition. Here, we directly tested the role of beta 2m expression in tumorigenicity. A beta 2 m loss mutant (C4.4-25-), selected from the murine lymphoma EL-4, showed a marked reduction in tumorigenicity as compared with EL-4 in normal C57B1/6 (B6) mice. The reduced tumorigenicity was directly related to beta 2 m expression. Transfection of an intact murine beta 2m gene markedly increased the tumorigenic potential. The reduced tumorigenicity of C4.4-25- compared with beta 2m transfected cells was observed also in athymic B6 nu/nu mice, but was abolished in B6 mice depleted of natural killer (NK) 1.1-positive cells. These results show that restoration of beta 2m expression can promote tumorigenicity and demonstrate for the first time that induction of major histocompatibility complex class I expression by transfection can lead to escape from NK cells in vivo.

Purinergic P2X7 receptors regulate secretion of interleukin-1 receptor antagonist and beta cell function and survival.

AIMS/HYPOTHESIS: In obesity, beta cells activate compensatory mechanisms to adapt to the higher insulin demand. Interleukin-1 receptor antagonist (IL-1Ra) prevents obesity-induced hyperglycaemia and is a potent target for the treatment of diabetes, but the mechanisms of its secretion and regulation in obesity are unknown. In the present study, we hypothesise the regulation of IL-1Ra secretion by purinergic P2X(7) receptors in islets. METHODS: Production and regulation of P2X(7) were studied in pancreatic sections from lean and obese diabetic patients, non-diabetic controls and in isolated islets. IL-1Ra, IL-1beta and insulin secretion, glucose tolerance and beta cell mass were studied in P2x7 (also known as P2Rx7)-knockout mice. RESULTS: P2X(7) levels were elevated in beta cells of obese patients, but downregulated in patients with type 2 diabetes mellitus. Elevated glucose and non-esterified fatty acids rapidly activated P2X(7) and IL-1Ra secretion in human islets, and this was inhibited by P2X(7) blockade. In line with our results in vitro, P2x7-knockout mice had a lower capacity to secrete IL-1Ra. They exhibited severe and rapid hyperglycaemia, glucose intolerance and impaired beta cell function in response to a high-fat/high-sucrose diet, were unable to compensate by increasing their beta cell mass in response to the diet and showed increased beta cell apoptosis. CONCLUSIONS/INTERPRETATION: Our study shows a tight correlation of P2X(7) activation, IL-1Ra secretion and regulation of beta cell mass and function. The increase in P2X(7) production is one mechanism that may explain how beta cells compensate by adapting to the higher insulin demand. Disturbances within that system may result in the progression of diabetes.

Activity of the multikinase inhibitor dasatinib against ovarian cancer cells.

BACKGROUND: Here, we explore the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, including members of the Src kinase family, EphA2, and focal adhesion kinase for the treatment of ovarian cancer. METHODS: We examined the effects of dasatinib on proliferation, invasion, apoptosis, cell-cycle arrest, and kinase activity using a panel of 34 established human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions with chemotherapeutic drugs. RESULTS: Concentration-dependent anti-proliferative effects of dasatinib were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC(50) values (IC(50) range: 0.001-11.3 micromol l(-1)). Dasatinib significantly inhibited invasion, and induced cell apoptosis, but less cell-cycle arrest. At a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for dasatinib plus carboplatin (mean CI values, range: 0.73-1.11) or paclitaxel (mean CI values, range: 0.76-1.05). In this study, 24 out of 34 (71%) representative ovarian cancer cell lines were highly sensitive to dasatinib, compared with only 8 out of 39 (21%) representative breast cancer cell lines previously reported. Cell lines with high expression of Yes, Lyn, Eph2A, caveolin-1 and 2, moesin, annexin-1, and uPA were particularly sensitive to dasatinib. CONCLUSIONS: These data provide a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in patients with ovarian cancer.

Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.

Rejection of tumors in mice with severe combined immunodeficiency syndrome determined by the major histocompatibility complex. Class I expression on the graft.

This study addresses the role of MHC class I molecules in the rejection of tumor grafts by SCID mice. Tumor cell lines, their corresponding MHC class I transfectants, and MHC class I-deficient mutants were inoculated to SCID mice. This allowed a study of tumor rejection responses in an environment with normal numbers of natural killer cells but largely devoid of functional T and B cells. C.B-17 (H-2d) SCID mice were found to reject low (10(2)) but not high (10(4)) doses of allogeneic (H-2b) tumor cells. The introduction of H-2Dd into such allogeneic tumor cells abrogated the rejection response with progressive tumor growth as a consequence. Introduction of H-2Kd or Ld had no or only marginal effects. The protective ability of H-2Dd was mapped to the alpha 1/alpha 2 domains of the molecule. H-2Dd protected allogeneic tumors from rejection also in C3H SCID mice of the H-2k haplotype, demonstrating that this ability was not dependent on H-2Dd expression in the host. Expression of endogenous H-2Kb and/or Db molecules partially protected wild-type allogeneic tumor cells from rejection since mutant allogeneic cells, devoid of class I expression, were rejected even after high-dose inoculation. Introduction of either allogeneic or xenogeneic class I molecules did not lead to rejection of otherwise MHC class I syngeneic (H-2d) tumor cells. The observed tumor cell rejection in SCID mice was dependent on natural killer cells. After depletion of asialo-GM1+ cells, all inoculated tumor cell lines grew progressively, independently of MHC class I expression. These results are compatible with a model where expression of certain, but not all, class I molecules protect from natural killer cell-mediated rejection. There was no evidence for rejection occurring as a consequence of the expression of allogeneic or xenogeneic class I molecules on the grafted cells. MHC class I expression may thus influence tumor cell recognition in mice lacking T-cell receptor expression.

Impaired oxygenation during sleep in excessive polycythemia of high altitude: improvement with respiratory stimulation.

Although polycythemia of high altitude is usually due to excessive hypoxemia, in some patients the hematocrit is elevated out of proportion to the degree of hypoxemia measured awake. One possible explanation is that severe hypoxemia occurs during sleep in these subjects. We therefore monitored oxygen saturation (SaO2), breathing pattern, and electroencephalogram (EEG) during sleep in five normal high-altitude residents and in five patients with excessive polycythemia. The polycythemic patients were studied as part of a placebo--drug double-blind crossover trial of the respiratory stimulant drug medroxyprogesterone acetate (MPA). The polycythemic patients while taking placebo were much more hypoxemic during sleep than the normals (all-night mean SaO2: 79.4 +/- 1.7% versus 87.8 +/- 1.7%, p less than 0.01). Abnormalities in breathing patterns were observed in all the subjects, especially during REM stage sleep. In polycythemic subjects, this resulted in precipitous hypoxemia with SaO2 as low as 50%--70%. Severe hypoxemia was not observed in control subjects despite similar abnormalities in breathing. Significant improvement in nocturnal SaO2 occurred when the polycythemic patients were taking MPA, mean SaO2 rising from 79.4 +/- 1.7% to 83.7 +/- 0.7%, p less than 0.05. Of probably greater importance, MPA largely prevented the precipitous drops in SaO2, mean lowest SaO2 rising from 64.6 +/- 4.7% to 76.0 +/- 2.1% p less than 0.05. The severe decreases in SaO2 during sleep may explain elevations in hematocrit that are out of proportion to the awake SaO2 in man at high altitude. The therapeutic effect of MPA in this condition may be due to amelioration of sleep hypoxemia.

Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen.

Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE.

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.

Psychosocial interventions and cancer patients: psychological and immune responses may depend on cancer type.

Psychological and immunological responses of cancer patients to a psychosocial intervention program will be assessed over time. Previously it has been proposed that there are two large divisions in cancer histology (type I and II) and that the psychobiology of patients will vary accordingly.

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen.

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reactivation. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.