Using methylcholanthrene-induced fibrosarcomas to study tumor immunology.

Mouse models of cancer are essential in furthering our understanding both of the mechanisms that drive tumor development and the immune response that develops in parallel, and also in providing a platform for testing novel anti-cancer therapies. The majority of solid tumor models available rely on the injection of existing cancer cell lines into naïve hosts which, while providing quick and reproducible model systems, typically lack the development of a tumor microenvironment that recapitulates those seen in human cancers. Administration of the carcinogen 3-methylcholanthrene (MCA), allows tumors to develop in situ, forming a tumor microenvironment with an established stroma and vasculature. This article provides a detailed set of protocols for the administration of MCA into mice and the subsequent monitoring of tumors. Protocols are also provided for some of the routinely used downstream applications that can be used for MCA tumors.

Deletion of GARP on mouse regulatory T cells is not sufficient to inhibit the growth of transplanted tumors.

GARP is a transmembrane protein that presents latent TGF-ß1 on the surface of regulatory T cells (Tregs). Neutralizing anti-GARP monoclonal antibodies that prevent the release of active TGF-ß1, inhibit the immunosuppressive activity of human Tregs in vivo. In this study, we investigated the contribution of GARP on mouse Tregs to immunosuppression in experimental tumors. Unexpectedly, Foxp3 conditional garp knockout (KO) mice challenged orthotopically with GL261 tumor cells or subcutaneously with MC38 colon carcinoma cells did not show prolonged survival or delayed tumor growth. Also, the suppressive function of KO Tregs was similar to that of wild type Tregs in the T cell transfer model in allogeneic, immunodeficient mice. In conclusion, garp deletion in mouse Tregs is not sufficient to impair their immunosuppressive activity in vivo.

Feasibility and acceptability of introducing routine antenatal contraceptive counselling and provision of contraception after delivery: the APPLES pilot evaluation.

OBJECTIVE: To determine the feasibility and acceptability of routine antenatal contraceptive counselling and contraception provision including long-acting reversible contraception (LARC) postpartum. DESIGN: Health service research evaluation. SETTING: Community antenatal clinics and hospital maternity settings in National Health Service, Scotland UK. POPULATION: Women booked for antenatal care. METHODS: Contraceptive counselling with a community midwife (22 weeks' gestation) and provision of contraception (with facilitated access to LARC methods) prior to discharge from maternity hospital. Evaluation consisted of (i) self administered questionnaire (32-34 weeks) of women's views of antenatal contraceptive counselling, (ii) database review of contraceptive methods provided at discharge, and (iii) focus groups with midwives and obstetricians. MAIN OUTCOME MEASURES: Women's views on antenatal contraceptive counselling. Secondary outcomes included (i) uptake of LARC methods and (ii) barriers and facilitators to providing antenatal counselling and contraception. RESULTS: There were 1369 women in the cohort. Questionnaires were distributed to 1064 women (78%) and completed by 794 (75%). In all, 78% of respondents (n = 621) discussed contraception antenatally with a community midwife and 74% (n = 461) found this helpful. Although 43% of respondents (n = 341) were planning to use LARC, only 9% of the cohort (118 of 1369) received LARC prior to discharge. Community midwives indicated that antenatal contraceptive counselling was now embedded in their role, but hospital staff indicated that workloads impacted upon ability to provide contraception for women. CONCLUSIONS: Antenatal contraceptive counselling, delivered by community midwives, is feasible and highly acceptable to women. However, providing contraception and LARC for women before they are discharged home remains a challenge. TWEETABLE ABSTRACT: Giving contraceptive advice antenatally is feasible and acceptable.

Note: Improved heater design for high-temperature hollow cathodes.

We present an improved heater design for thermionic cathodes using a rhenium filament encased in a boron nitride ceramic sleeve. This heater is relatively simple to fabricate, yet has been successfully used to reliably and repeatably light a lanthanum hexaboride (LaB6) hollow cathode based on a previously published design without noticeable filament degradation over hundreds of hours of operation. The high decomposition temperature of boron nitride (2800 C for inert environments) and melting point for rhenium (3180 C) make this heater especially attractive for use with LaB6, which may require operating temperatures upwards of 1700 C. While boron nitride decomposes in air above 1000 C, the heater was used only at vacuum with an inert gas discharge, and no degradation was observed. Limitations of current state of the art cathode heaters are also discussed and compared with the rhenium-boron nitride combination.

A cavity ring-down spectroscopy sensor for real-time Hall thruster erosion measurements.

A continuous-wave cavity ring-down spectroscopy sensor for real-time measurements of sputtered boron from Hall thrusters has been developed. The sensor uses a continuous-wave frequency-quadrupled diode laser at 250 nm to probe ground state atomic boron sputtered from the boron nitride insulating channel. Validation results from a controlled setup using an ion beam and target showed good agreement with a simple finite-element model. Application of the sensor for measurements of two Hall thrusters, the H6 and SPT-70, is described. The H6 was tested at power levels ranging from 1.5 to 10 kW. Peak boron densities of 10 ± 2 × 10(14) m(-3) were measured in the thruster plume, and the estimated eroded channel volume agreed within a factor of 2 of profilometry. The SPT-70 was tested at 600 and 660 W, yielding peak boron densities of 7.2 ± 1.1 × 10(14) m(-3), and the estimated erosion rate agreed within ~20% of profilometry. Technical challenges associated with operating a high-finesse cavity in the presence of energetic plasma are also discussed.

Validation and evaluation of a novel time-resolved laser-induced fluorescence technique.

We present a novel technique to measure time-resolved laser-induced fluorescence signals in plasma sources that have a relatively constant Fourier spectrum of oscillations in steady-state operation, but are not periodically pulsed, e.g., Hall thrusters. The technique uses laser modulation of the order of MHz and recovers signal via a combination of band-pass filtering, phase-sensitive detection, and averaging over estimated transfer functions calculated for many different cycles of the oscillation. Periodic discharge current oscillations were imposed on a hollow cathode. Measurements were validated by comparison with independent measurements from a lock-in amplifier and by comparing the results of the transfer function average to an independent analysis technique triggering averaging over many oscillation cycles in the time domain. The performance of the new technique is analyzed and compared to prior techniques, and it is shown that this new technique has a niche in measurements where the analog photomultiplier signal has a nonwhite noise spectral density and cycles of oscillation are not sufficiently repeatable to allow for reliable triggering or a meaningful average waveform in the time domain.

Highly prevalent colorectal cancer-infiltrating LAP⁺ Foxp3⁻ T cells exhibit more potent immunosuppressive activity than Foxp3⁺ regulatory T cells.

Although elevated CD4⁺Foxp3⁺ regulatory T cell (Treg) frequencies within tumors are well documented, the functional and phenotypic characteristics of CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ T cell subsets from matched blood, healthy colon, and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intratumoral CD4⁺Foxp3⁺ T cells (Tregs) were Helios⁺ and expressed higher levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD39 than Tregs from colon and blood. Moreover, ∼30% of intratumoral CD4⁺Foxp3⁻ T cells expressed markers associated with regulatory functions, including latency-associated peptide (LAP), lymphocyte activation gene-3 (LAG-3), and CD25. This unique population of cells produced interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß), and was ∼50-fold more suppressive than Foxp3⁺ Tregs. Thus, intratumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies.

External auditory canal paraganglioma: an atypical presentation.

OBJECTIVE: We present the first published description of a painful paraganglioma of the external auditory canal. Atypical histopathology made the diagnosis difficult. We discuss the potential pitfalls of clinical diagnosis and treatment of such a case. CLINICAL PRESENTATION: A 49-year-old woman presented with left-sided otalgia, hearing loss and tinnitus. Physical examination revealed a firm swelling arising from the superior portion of the left external auditory canal. A clinical diagnosis of otitis externa was made. INTERVENTION: There was minimal response to medical treatment. The swelling was aspirated, leading to brisk bleeding. A tumour was suspected from the computed tomography scan, and confirmed by a biopsy. The patient underwent excision of the paraganglioma. The histopathology was atypical, making diagnosis difficult. CONCLUSION: Such unusual masses of the external ear should always be borne in mind, especially when dealing with atypical presentations of commonly encountered diseases. Clinicians should have a low threshold for early intervention with imaging and biopsy.

Positive and negative influences of regulatory T cells on tumour immunity.

Clinicians and scientists have long questioned whether the immune system has a role in destroying cancerous tissue. Studies performed in animal models have, however, recently revealed that the immune system can, at least in principle, effectively control tumours. In parallel with these findings, a large body of evidence indicates that although the immune system has the capacity to control tumours, there are also regulatory mechanisms that subdue these responses. A major challenge of tumour immunotherapy, therefore, is to find ways of disabling these regulatory functions while restoring or priming any immune responses that are protective.

The impact of regulatory T cells on carcinogen-induced sarcogenesis.

Burnet proposed in the 1950's that the immune system is engaged in identifying and destroying abnormal cancerous cells. This process, termed immune surveillance, has been at the centre of intense debate for decades. Results using immunodeficient mice lend support to the immune surveillance hypothesis. We surmised that immune surveillance would be hampered by the inhibitory effect of naturally occurring FoxP3(+) regulatory T cells, a population of T cells shown to be present at an increased frequency in a variety of human tumours. The carcinogen, methylcholanthrene was injected subcutaneously into mice and the steady development of fibrosarcomas was observed over approximately 200 days. These fibrosarcomas were strikingly infiltrated with FoxP3(+) regulatory T cells implying that these cells impinge upon immune-mediated rejection of the tumour. This was confirmed by partial ablation of FoxP3(+) regulatory T-cell activity, which resulted in a marked reduction in tumour incidence. The reduction of tumour incidence was ablated in mice that lacked interferon gamma. These data offer strong support for the concept of immune surveillance and indicate that this process is limited by the inhibitory effect of FoxP3(+) regulatory T cells.

Eosinophilic angiocentric fibrosis as a cause of nasal obstruction.

We present the tenth case of Eosinophilic Angiocentric Fibrosis (EAF) in the english literature, which presented as nasal obstruction in a patient of Indian descent. The histopathological and clinical features of this underreported condition is discussed as well as other lesions that may show similar features to EAF.

Deletion of the CD4 silencer element supports a stochastic mechanism of thymocyte lineage commitment.

The mechanism of T cell lineage commitment remains controversial; to examine it we deleted the CD4-silencer element in the germ line of a mouse using a combination of gene targeting and Cre/LoxP-mediated recombination. We found that these mice were unable to extinguish CD4 expression either in immature thymocytes or mature CD8+ cytotoxic T cells (CTLs), which resulted in the development of major histocompatibility complex class II-restricted double-positive CTLs in the periphery. This finding strongly supports a stochastic over an instructive mechanism of coreceptor down-regulation.

Intra-parenchymal thyroid epidermal cyst presenting with a left recurrent laryngeal nerve palsy.

We present a rare case of an intra-parenchymal thyroid epidermal cyst presenting with a left recurrent laryngeal nerve palsy. There was a complete recovery of the nerve function following surgical excision of the lesion. Theories of aetio-pathogenesis of the cyst and underlying mechanisms responsible for the nerve paralysis are explored.

Normal pathogen-specific immune responses mounted by CTLA-4-deficient T cells: a paradigm reconsidered.

CTLA-4 is a critical negative regulator of T cell responses and CTLA-4-deficient (CTLA-4(-/-)) mice die of a lymphproliferative disease. Nevertheless, RAG-2-deficient mice reconstituted with a mixture of CTLA-4(-/-) and normal (CTLA-4(+/+)) bone marrow survive in the absence of any signs of disease, although 50% of their T cells do not express CTLA-4. Using such mixed chimeras, we analyzed the role of CTLA-4 in specific T cell responses to lymphocytic choriomeningitis virus, Leishmania major and mouse mammary tumor virus, which cause acute, chronic and persistent infections, respectively. The populations of antigen-specific CTLA-4(-/-)CD4(+) and CTLA-4(-/-)CD8(+) T cells became activated, expanded and contracted indistinguishably from CTLA-4(+/+)CD4(+) and CTLA-4(+/+)CD8(+) T cells after infection with all three pathogens. Thus, CTLA-4 is not involved in the down-regulation of specific T cell responses and peripheral deletion in a T cell-autonomous fashion.

Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine.

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-naïve human volunteers using the needle-free PowderJect system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 microg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg(335-343) tetramers, secreted IFN-gamma, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans.

Functionally distinct CD8+ memory T cell subsets in persistent EBV infection are differentiated by migratory receptor expression.

Human memory T lymphocytes have recently been re-defined as central or effector memory cells (Sallusto, F., Lenig, D., Forster, R., Lipp, M. and Lanzavecchia, A., Nature 1999. 401: 708-712). Effector memory cells (T(em)) are targeted to the peripheral tissues and show rapid effector function in response to antigenic stimulation. Central memory (T(cm)) cells are targeted to the lymph nodes and cannot be immediately activated. In this report HLA-A2-Epstein-Barr virus (EBV) peptide tetramers have been used to characterize the EBV-specific CD8+ T cell subsets in persistent EBV infection. In short-term activation studies two populations of tetramer-positive T cells were identified. One group resembled T(em) cells in that they rapidly produced IFN-gamma and lacked the lymph node homing receptor, CD62L, the second was similar to T(cm) cells since they were CD62L+ but could not be immediately induced to express IFN-gamma.

Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection.

It has been shown that certain pathogens can trigger efficient T cell responses in the absence of CD28, a key costimulatory receptor expressed on resting T cells. Inducible costimulator protein (ICOS) is an inducible costimulator structurally and functionally related to CD28. Here, we show that in the absence of CD28 both T helper cell type 1 (Th1) and Th2 responses were impaired but not abrogated after infection with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and the nematode Nippostrongylus brasiliensis. Inhibition of ICOS in CD28-deficient mice further reduced Th1/Th2 polarization. Blocking of ICOS alone had a limited but significant capacity to downregulate Th subset development. In contrast, cytotoxic T lymphocyte (CTL) responses, which are regulated to a minor and major extent by CD28 after LCMV and VSV infection, respectively, remained unaffected by blocking ICOS. Together, our results demonstrate that ICOS regulates both CD28-dependent and CD28-independent CD4(+) subset (Th1 and Th2) responses but not CTL responses in vivo.

MHC class I-restricted killing of neurons by virus-specific CD8+ T lymphocytes is effected through the Fas/FasL, but not the perforin pathway,.

Induction of MHC class I genes in neurons of the central nervous system requires signals by pro-inflammatory cytokines, in particular IFN-gamma, and the blockade of electric activity, which is known to suppress induction of MHC related genes in a highly ordered, but unusual fashion [1], [2]. The present experiments explore the immunological function of neuronal MHC class I antigens expressed under permissive conditions. MHC class I proteins were induced in electrically silenced murine hippocampal neurons by treatment with the sodium channel blocker tetrodotoxin and recombinant IFN-gamma, conditions which also resulted in the induction of Fas molecules. The MHC class I positive neurons were challenged with CD8+ cytotoxic T lymphocytes (CTL) specific for the H2-Db binding peptide GP33, a dominant epitope of the lymphocytic choriomeningitis virus envelope glycoprotein, or with alloreactive CTL. Single primed neurons, attacked by GP33-specific CTL, were continuously monitored for changes in intracellular calcium ([Ca2+]i), an indicator of cytotoxic damage. MHC class I-induced neurons pulsed with the GP33 peptide, but not a control peptide, showed a gradual and sustained increase in [Ca2+]i within 3 h following attack by GP33-specific CTL, while in astrocytes [Ca2+]i elevation was rapid. The slow course of the neuronal response was consistent with a delayed apoptotic killing mechanism rather than rapid granule-mediated plasma membrane lysis. Indeed, the attacked neurons bound annexin V, indicating membrane alterations preceding apoptotic cell death. In further support of apoptotic cell death, this sustained increase of [Ca2+]i levels was also observed following attack by perforin-deficient CTL, but was not detected in neurons derived from mutant lpr mice, which lack functional Fas molecules.

Effect of epitope flanking residues on the presentation of N-terminal cytotoxic T lymphocyte epitopes.

We here demonstrate that placing two distinct influenza virus nucleoprotein epitopes at the N terminus of a cytosolic protein selectively blocks their presentation to specific cytotoxic T lymphocytes. The block is a cytosolic phenomenon, which can be overcome by distancing the epitope from the protein N terminus by two or more amino acids. Shortening the protein's C terminus fails to relieve the antigen presentation block. These results demonstrate that events at the N terminus of the target protein, rather than at its C terminus, are responsible for the lack of presentation of N-terminal epitopes. We also show that lack of presentation of N terminal epitopes is associated with a modification of the target protein which affects its electrophoretic mobility and isoelectric focusing point. This modification can be prevented by mutating the epitope's N-terminal flanking sequence, which results in an efficient presentation of the N-terminal epitope. Lack of presentation of the N-terminal epitopes results in a reduced ability of influenza-primed mice to clear acute infection with vaccinia virus encoding an N-terminal nucleoprotein epitope. Our results demonstrate that presentation of epitopes localized at the N terminus of cytosolic proteins can be modulated by events occurring at early stages of antigen processing.