Generation of CTL responses using Kunjin replicon RNA

The Kunjin replicon was used to express a polytope that consisted of seven hepatitis C virus cytotoxic T lymphocyte epitopes and one influenza cytotoxic T lymphocyte epitope for vaccination studies. The self-replicating nature of, and expression from, the ribonucleic acid was confirmed in vitro . Initial vaccinations with one dose of Kun-Poly ribonucleic acid showed that an influenza-specific cytotoxic T lymphocyte response was elicited more efficiently by intradermal inoculation compared with intramuscular delivery. Two micrograms of ribonucleic acid delivered in the ear pinnae of mice was sufficient to elicit a detectable cytotoxic T lymphocyte response 10 days post-vaccination. Further vaccination studies showed that four of the seven hepatitis C virus cytotoxic T lymphocyte epitopes were able to elicit weak cytotoxic T lymphocyte responses whereas the influenza epitope was able to elicit strong, specific cytotoxic T lymphocyte responses following three doses of Kun-Poly ribonucleic acid. These studies vindicate the use of the Kunjin replicon as a vector to deliver encoded proteins for the development of cell-mediated immune responses.

Fas ligand and tumour counter-attack in colorectal cancer stratified according to microsatellite instability status

Expression of membrane-bound Fas ligand (FasL) by colorectal cancer cells may allow the development of an immune-privileged site by eliminating incoming tumour-infiltrating lymphocytes (TILs) in a Fas-mediated counter-attack. Sporadic colorectal cancer can be subdivided into three groups based on the level of DNA microsatellite instability (NISI). High-level NISI (NISI-High) is characterized by the presence of TILs and a favourable prognosis, while microsatellite-stable (MSS) cancers are TIL-deficient and low-level MSI (MSI-Low) is associated with an intermediate TIL density. The purpose of this study was to establish the relationship between MSI status and FasL expression in primary colorectal adenocarcinoma. Using immunohistochemistry and a selected series of 101 cancers previously classified as 31 MSI-High, 30 NISI-Low, and 40 MISS, the present study sought to confirm the hypothesis that increased TIL density in MSI-High cancers is associated with low or absent membrane-bound FasL expression, while increased FasL in MSS cancers allows the killing of host TILs. TUNEL/CD3 double staining was also used to determine whether MSS cancers contain higher numbers of apoptotic TILs in vivo than MSI-High or MSI-Low cancers. Contrary to the initial hypothesis, it was found that MSI-High cancers were associated with higher FasL expression (p = 0.04) and a stronger intensity of FasL staining (p = 0.007). In addition, mucinous carcinomas were independently characterized by increased FasL expression (p = 0.03) and staining intensity (p = 0.0005). Higher FasL expression and staining intensity did not correlate with reduced TIL density or increased numbers of apoptotic TILs. However, consistent with the hypothesis that curtailment of the host anti-tumour immune response contributes to the poor prognosis in MSS cancers, it was found that apoptotic TILs were most abundant in MSS carcinomas and metastatic Dukes' stage C or D tumours (p = 0.004; p = 0.046 respectively). This study therefore suggests that MSS colorectal cancers are killing incoming TILs in an effective tumour counter-attack, but apparently not via membrane-bound FasL. Copyright (C) 2003 John Wiley Sons, Ltd.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2(d)), C57BL6 (H-2(b)), DBA/2J (H-2(d)) and CBA/CaH (H-2(k)) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4(+) and CD8(+) T-cell subsets, CD14(+) macrophages and CD19(+) B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8(+) T cells and CD19(+) B cells were found in any of the lesions. The percentages of CD4(+) cells, CD14(+) cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14(+) cells in sham-immunized mice. The percentage of CD14(+) cells was higher than that of CD4(+) cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4(+) and CD14(+) cells predominated in immunized CBA/CaH mice and CD4(+) cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14(+) cells and CD4(+) cells in sham-immunized mice. IgG1(+) plasma cells were more dominant than IgG2a(+) cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a(+) plasma cells were more obvious in sham-immunized mice. IgG2a(+) plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

Impulse-response function of splanchnic circulation with model-independent constraints: theory and experimental validation

Modeling physiological processes using tracer kinetic methods requires knowledge of the time course of the tracer concentration in blood supplying the organ. For liver studies, however, inaccessibility of the portal vein makes direct measurement of the hepatic dual-input function impossible in humans. We want to develop a method to predict the portal venous time-activity curve from measurements of an arterial time-activity curve. An impulse-response function based on a continuous distribution of washout constants is developed and validated for the gut. Experiments with simultaneous blood sampling in aorta and portal vein were made in 13 anesthetized pigs following inhalation of intravascular [O-15] CO or injections of diffusible 3-O[ C-11] methylglucose (MG). The parameters of the impulse-response function have a physiological interpretation in terms of the distribution of washout constants and are mathematically equivalent to the mean transit time ( T) and standard deviation of transit times. The results include estimates of mean transit times from the aorta to the portal vein in pigs: (T) over bar = 0.35 +/- 0.05 min for CO and 1.7 +/- 0.1 min for MG. The prediction of the portal venous time-activity curve benefits from constraining the regression fits by parameters estimated independently. This is strong evidence for the physiological relevance of the impulse-response function, which includes asymptotically, and thereby justifies kinetically, a useful and simple power law. Similarity between our parameter estimates in pigs and parameter estimates in normal humans suggests that the proposed model can be adapted for use in humans.

Haemodynamic response to TENS applied to upper thoracic nerve roots in normal subjects

This study investigated the haemodynamic response to the 90-minute application of 85 Hz transcutaneous electrical nerve stimulation (TENS) to the T1 and T5 nerve roots. Comparison was made between 20 healthy subjects who had TENS stimulation and a separate group of 20 healthy subjects who rested for 90 minutes. Pulse and blood pressure were measured just prior to the start of TENS stimulation, after 30 minutes of stimulation, and after 90 minutes of stimulation (immediately after stopping TENS) or at completion of the rest time depending on group allocation. The rate pressure product was calculated from the pulse and systolic blood pressure data. Multivariate repeated measures analysis showed a significant group effect for TENS (p = 0.048). Univariate repeated measures analyses showed a significant group by time effect due to TENS on systolic blood pressure over the 90-minute time period (p = 0.028). Separate group repeated measures ANOVA showed a significant decline in heart rate (p = 0.000), systolic blood pressure (p = 0.013) and rate pressure product (p = 0.000) for the TENS group, while the control resting group showed a significant decline in heart rate only (p = 0.04). The application of 85 Hz TENS to the upper thoracic nerve roots causes no adverse haemodynamic effects in healthy subjects.

Crack detection in hollow section structures through coupled response measurements

Detection of a circumferential crack in a hollow section beam is investigated using coupled response measurements. The crack section is represented by a local flexibility matrix connecting two undamaged beam segments. This matrix defines the relationship between the displacements and forces across the crack section and is derived by applying fundamental fracture mechanics theory. The suitability of the mode coupling methodology is first demonstrated analytically. Laboratory test results are then presented for circular hollow section beams with artificially generated cracks of varying severity. It is shown that this method has the potential as a damage detection tool for mechanical structures. (C) 2002 Elsevier Science Ltd. All rights reserved.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NES1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G2/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G2/M checkpoint.

Induction of metallothionein in human skin by routine exposure to sunlight: Evidence for a systemic response and enhanced induction at certain body sites

Expression of metallothionein, an antioxidant induced by a variety of stimuli including ultraviolet light, was quantitated by immunohistochemistry in the skin of males aged over 50 who had known short- and long-term exposures to sunlight. Skin punch biopsies were taken from two sites in each subject: the hand in all subjects and a range of other sites matched to patients with a previously excised primary melanoma. Metallothionein expression (strongest in the basal layers of the epidermis and primarily nuclear) was associated with both short- and long-term exposure to sunlight. A plateau of staining intensity was reached after 3 h sun exposure, within the previous 3 d before biopsy. Expression was also elevated in the nonexposed skin sites of subjects who had recent sun exposure, indicating a systemic response to exposure of remote sites. Using the skin of the hand to normalize responses to chronic exposure between individuals, the systemically modulated response to sunlight was significantly greater on the unexposed back than on other sites. The possibility of ultraviolet-induced cytokines selectively modifying the response of skin on a site-specific basis was investigated. The circulating leukocytes, but not lymphocytes, of two individuals exposed to 1 minimal erythema dose whole-body solar-simulated ultraviolet showed increased interleukin-6 mRNA 4 h after exposure. Interleukin-6 was not directly induced in these cell populations 4 h after ultraviolet A or ultraviolet B irradiation ex vivo . Leukocytes may therefore contribute to and amplify the systemic effects of ultraviolet-induced interleukin-6 and metallothionein expression.

Immunological changes after cancer treatment and participation in an exercise program

Purpose: The purpose of this investigation was to evaluate the impact of undertaking peripheral blood stem cell transplantation (PBST) on T-cell number and function, and to determine the role of a mixed type, moderate intensity exercise program in facilitating the recovery of T-cell number and function. Methods: Immunological measures of white blood cell, lymphocyte, CD3(+), CD4(+), and CD8(+) counts, and CD3(+) cell function were assessed pretransplant (PI), immediately posttransplant (PII), and 1 month (II), 2 months (12) and 3 months (PIII) posttransplant. After PII, 12 patients were divided equally into a control group (CG) or exercise intervention group (EG). Results: Lower total T-cell, helper T-cell, and suppressor T-cell counts (P < 0.01), as well as lower T-cell function (P < 0.01), when compared with normative data, were found at PI. More specifically, 88% of the group had CD3(+), CD4(+), and CD8(+) counts that were more than 40%, 20%, and 50% below normal at PI, respectively. Undertaking a PBST caused further adverse changes to the total leukocyte, lymphocyte, CD3(+), CD4(+) and CD8(+) count. and the helper/suppressor ratio. Although CD8(+) counts had returned to normal by PIII, CD3(+), CD4(+), and the CD4(+)/CD8(+) ratio remained significantly lower than normative data (P < 0.01), with 66%, 100%, and 100% of the subject group reporting counts and ratios, respectively, below the normal range. Conclusion: The PBST patients were immunocompromised before undertaking the transplant, and the transplant procedure imposed further adverse changes to the leukocyte and lymphocyte counts. The leukocyte and CD8(+) counts returned to normal within 3 months posttransplant; however, the other immunological parameters assessed demonstrated a delayed recovery. Although participation in the exercise program did not facilitate a faster immune cell recovery, neither did the exercise program hinder or delay recovery.

Early pregnancy factor treatment suppresses the inflammatory response and adhesion molecule expression in the spinal cord of SJL/J mice with experimental autoimmune encephalomyelitis and the delayed-type hypersensitivity reaction to trinitrochlorobenzene in normal BALB/c mice

Early pregnancy factor (EPF) is a secreted protein, present in serum during early pregnancy and essential for maintaining viability of the embryo. It is a homologue of chaperonin 10 (Cpn10) but, unlike Cpn10, it has an extracellular role. EPF has immunosuppressive and growth regulatory properties. Previously we have reported the preparation of recombinant EPF (rEPF) and shown that treatment with rEPF will suppress clinical signs of MBP-EAE in Lewis rats and PLP-EAE in SJL/J mice. In the present study, these findings have been extended to investigate possible mechanisms involved in the action of EPF. Following treatment of mice with rEPF from the day of inoculation, there were fewer infiltrating CD3+ and CD4+ cells in the parenchyma of the spinal cord during the onset of disease and after the initial episode, compared with mice treated with vehicle. Expression of the integrins LFA-1, VLA-4 and Mac-1 and of members of the immunoglobulin superfamily of adhesion molecules ICAM-1 and VCAM-1 was suppressed in the central nervous system (CNS) following rEPF treatment. The expression of PECAM-1 was not affected. To determine if rEPF suppressed T cell activation in the periphery, the delayed-type hypersensitivity (DTH) reaction of normal BALB/c mice to trinitrochlorobenzene (TNCB) following treatment with rEPF was studied. The results showed that treatment with rEPF suppressed the DTH reaction, demonstrating the ability of EPF to downregulate the cell-mediated immune response. These results indicate that suppression of immunological mechanisms by rEPF plays a major role in the reduction of clinical signs of disease in experimental autoimmune encephalomyelitis (EAE). (C) 2003 Elsevier Science B.V. All rights reserved.

Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10

Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. ReIB activation is required for myeloid DC differentiation. Here, we show that antigen-exposed DCs in which ReIB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. DCs generated from CD40-deficient mice similarly confer suppression. Regulatory CD4(+) T cells induced by the DCs transfer antigen-specific Infectious tolerance to primed recipients in an interleukin10-dependent fashion. Thus CD40, regulated by ReIB activity, determines the consequences of antigen presentation by myeloid DCs. These observations have significance for autoimmune immunotherapy and suggest a mechanism by which peripheral tolerance might be constitutively maintained by RelB(-) CD40(-) DCs.

IL-10 Mediates Suppression of the CD8 T Cell IFN-γ Response to a Novel Viral Epitope in a Primed Host

Priming to Ag can inhibit subsequent induction of an immune response to a new epitope incorporated into that Ag, a phenomenon referred to as original antigenic sin. In this study, we show that prior immunity to a virus capsid can inhibit subsequent induction of the IFN-gamma effector T cell response to a novel CD8-restricted antigenic epitope associated with the virus capsid. Inhibition does not involve Ab to the virus capsid, as it is observed in animals lacking B cells. CD8-restricted virus-specific T cell responses are not required, as printing to virus without CTL induction is associated with inhibition. However, IL-10(-/-) mice, in contrast to IL-10(+/+) mice, generate CD8 T cell and Ab responses to novel epitopes incorporated into a virus capsid, even when priming to the capsid has resulted in high titer Ab to the capsid. Furthermore, capsid-primed mice, unable to mount a response to a novel epitope in the capsid protein, are nevertheless able to respond to the same novel epitope delivered independently of the capsid. Thus, inhibition of responsiveness to a novel epitope in a virus-primed animal is a consequence of secretion of IL-10 in response to presented Ag, which inhibits local generation of new CD8 IFN-gamma-secreting effector T cells. Induction of virus- or tumor Ag-specific CD8 effector T cells in the partially Ag-primed host may thus be facilitated by local neutralization of IL-10.

Site-directed mutagenesis of the ATM promoter: Consequences for response to proliferation and ionizing radiation

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. (C) 2003 Wiley-Liss, Inc.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.