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.

HIV-specific cytotoxic T-cells in HIV-exposed but uninfected Gambian women.

A crucial requirement in the rational design of a prophylactic vaccine against the human immunodeficiency virus (HIV) is to establish whether or not protective immunity can occur following natural infection. The immune response to HIV infection is characterized by very vigorous HIV-specific cytotoxic T-lymphocyte (CTL) activity. We have identified four HIV-1 and HIV-2 cross-reactive peptide epitopes, presented to CTL from HIV-infected Gambians by HLA-B35 (the most common Gambian class I HLA molecule). These peptides were used to elicit HIV-specific CTLs from three out of six repeatedly exposed but HIV-seronegative female prostitutes with HLA-B35. These women remain seronegative with no evidence of HIV infection by polymerase chain reaction or viral culture. Their CTL activity may represent protective immunity against HIV infection.

PIP: A crucial requirement in the rational design of a prophylactic vaccine against HIV is to establish whether or not protective immunity can occur following natural infection. The immune response to HIV infection is characterized by very vigorous HIV-specific cytotoxic T-lymphocyte (CTL) activity. Four HIV-1 and HIV-2 cross-reactive peptide epitopes were identified, presented to CTL from HIV-infected Gambian women by HLA-B35 (the most common Gambian class 1 HLA molecule). The study population consisted of 20 women: 14 had been prostitutes for more than 5 years and reported little condom usage and 6 were long-term sexual partners of HIV-infected men. Peptide-stimulated cultures were also set up from 8 known seropositive donors with HLA-B35 or B53, and from a control group of volunteers at low-risk of HIV infection with HLA-B35 (12 Gambian and 7 European) and 2 Gambians with HLA-B53. Specific CTL activity against one or more peptides was repeatedly detected after 10-14 days in the peptide-stimulated cultures from 3 of the 6 high-risk seronegative women with HLA-B35, but not in their three counterparts with HLA-B53 nor in any of the low-risk volunteers. The strongest responses were generated toward the HIV-1 pol peptide, which lies close to the active site of reverse transcriptase, and to the nef peptide, which is conserved between HIV-1 and -2. HIV-specific CTL in seronegative subjects could potentially be a response to acute HIV infection, before the development of antibodies, but the women were still seronegative and virus-culture negative 3 months after the CTL were first detected, making recent infection extremely unlikely. These women remain seronegative with no evidence of HIV infection by polymerase chain reaction or viral culture. Their CTL activity may represent protective immunity against HIV infection.

Early suppression of SIV replication by CD8+ nef-specific cytotoxic T cells in vaccinated macaques.

In order to develop a successful subunit vaccine against infection with the human immunodeficiency virus (HIV), protective immune effector functions must be identified. Until now, there has been only indirect evidence that HIV-specific cytotoxic T lymphocytes (CTLs) fulfill this role. Using the macaque simian immunodeficiency virus (SIV) model, the protective potential of nef-specific CTLs, stimulated by vaccination, was examined in animals challenged with a high intravenous dose of the pathogenic simian immunodeficiency virus, SIVmac251(32H)(pJ5). An inverse correlation was found between the vaccine-induced nef-specific CTL precursor frequency and virus load measured after challenge. In addition, the early decline in viraemia, observed in both vaccinated and unvaccinated control animals was associated with the development of virus-specific CTL activity and not with the presence of virus-specific neutralizing antibodies. The results imply that vaccines that stimulate strong CTL responses could protect against HIV infection.

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.

Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells.

The question of whether enhanced memory T cell responses are simply due to an increased frequency of specific cells or also to an improved response at the single cell level is widely debated. In this study, we analyzed T cell receptor (TCR) transgenic memory T cells and bona fide memory T cells isolated from virally infected normal mice using the tetramer technology. We found that memory T cells are qualitatively different from naive T cells due to a developmentally regulated rearrangement of the topology of the signaling machinery. In naive cytotoxic T cells, only a few CD8 molecules are associated with Lck and the kinase is homogeneously distributed inside the cell. However, in vivo priming of naive T cells induces the targeting of Lck to the CD8 coreceptor in the cell membrane and the consequent organization of a more efficient TCR signaling machinery in effector and memory cells.

Protective immunity does not correlate with the hierarchy of virus-specific cytotoxic T cell responses to naturally processed peptides.

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) stimulates major histocompatibility complex class I-restricted cytotoxic T cells (CTLs), which normally resolve the infection. Three peptide epitopes derived from LCMV have been shown to bind the mouse class I molecule H-2 Db and to stimulate CTL responses in LCMV-infected mice. This report describes the identity and abundance of each CTL epitope after their elution from LCMV-infected cells. Based on this information, peptide abundance was found to correlate with the magnitude of each CTL response generated after infection with LCMV. Subsequent experiments, performed to determine the antiviral capacity of each CTL specificity, indicate that the quantitative hierarchy of CTL activity does not correlate with the ability to protect against LCMV infection. This report, therefore, indicates that immunodominant epitopes should be defined, not only by the strength of the CTL response that they stimulate, but also by the ability of the CTLs to protect against infection.

Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.

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.

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.

The proteasome inhibitor lactacystin prevents the generation of an endoplasmic reticulum leader-derived T cell epitope.

The presentation of viral antigens on MHC class I molecules requires their intracellular fragmentation into peptides of appropriate length and anchor residue positions. Evidence has accumulated that the proteasome is the endoprotease in charge of the generation of MHC class I ligands in the cytoplasm. The generation of T cell epitopes derived from the leader peptides of endoplasmic reticulum (ER) targeted proteins, however. has been reported to be independent of the proteasome. Here we show that the H-2Db restricted antigen presentation of the immunodominant T cell epitope derived from the ER leader of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) is completely abolished by administration of the proteasome inhibitor lactacystin. Thus our data support the role of the proteasome in class I restricted antigen processing and extend it to an ER leader derived epitope from a viral glycoprotein.

Cytotoxic T cells--protection from disease progression--protection from infection.

We briefly review current evidence which indicates that major histocompatibility complex (MHC)-restricted, virus-specific cytotoxic T cells may be of immunological importance in protection from infection with immunodeficiency virus or with protection from disease progression which would finally result in acquired immunodeficiency syndrome (AIDS) and death. We suggest that prophylactic vaccines should elicit cytotoxic T lymphocyte (CTL) activity in naive individuals. Further, immunotherapy in infected individuals could be aimed at ensuring that levels of virus-specific CTL are kept high, broadening and redirecting their specificity towards conserved parts of the viral genome.

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.

Recurrent laryngeal papillomatosis: a case of florid papillomatosis following a remission of 30 years.

Laryngeal papillomatosis is the commonest benign tumour affecting the larynx. Two forms are found i.e. juvenile onset and adult onset. Typically the juvenile onset form has a greater rate of recurrence and often remits with the onset of puberty (Corbitt et al., 1988). The human papilloma virus (HPV) is the causative agent (Abramson et al., 1987; Corbitt et al., 1988), specifically types HPV6 and 11. Attempts have been made to correlate the clinical behaviour of these two modes with the viral serotype and other aetiological factors such as smoking and hormonal factors (Abramson et al., 1987; Rimmel et al., 1992). Studies, however have shown that there is considerable variation in behaviour (Steinberg et al., 1983; Corbitt et al., 1988; Crissman et al., 1988). It is widely accepted that the disease 'burns' itself out, particularly with respect to the juvenile form. It is interesting and unusual therefore when the disease reappears after many years of remission. The following case report illustrates this point.

Leiomyoma of the paranasal sinuses.

The paranasal sinuses are a rare site for tumours of myogenic origin. There has been only one previously reported case in the English literature. We present a case of a leiomyoma filling the anterior ethmoid sinus and middle meatus which was excised via a Patterson's external ethmoidectomy.

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.

Middle ear lipoma.

We report a case of primary middle ear lipoma diagnosed in the right ear of a five-year-old child with concurrent bilateral middle ear effusions. The lipoma occupied a site favoured by congenital cholesteatoma and was occlusive to the eustachian tube contributing to its dysfunction. This is the first case of de novo middle ear lipoma diagnosed in the UK, and the third in world literature. Our CT scans are suggestive of a similar but smaller lesion in the left ear of the same child.

Aspergillosis of the larynx.

Primary infection of the larynx with Aspergillus spp. is rare. It is more commonly seen as part of a wider infection involving the respiratory system in an immunocompromised host. In noncompromised patients laryngeal aspergillosis may represent colonization rather than invasion requiring no systemic anti-fungal treatment. The diagnosis is important as the presenting symptoms are suggestive of malignant laryngeal disease. We present a 62-year-old man with a short history of hoarseness. Direct laryngoscopy and biopsy confirmed the diagnosis of aspergillosis. Clinical presentation, diagnosis and the important pathological characteristics of this infection are discussed.

Laryngeal imaging by computerized tomography and magnetic resonance following radiation therapy: a need for caution.

For patients with laryngeal tumours, the use of computerized tomography (CT) or magnetic resonance imaging (MR) may facilitate accurate staging by the demonstration of cartilage invasion or tumour extension to areas such as the pre-epiglottic space. The role of imaging in the follow-up of patients after radiotherapy, however, has not been examined. A prospective study of 18 patients undergoing laryngectomy was performed. The results of pre-operative CT and MR imaging were correlated with the pathological findings from whole organ axial sections of the laryngeal specimens. In five patients (28 per cent) both CT and MR images were significantly impaired by movement artefact. In the eight patients without previous radiotherapy, seven had adequate quality imaging and both CT and MR accurately demonstrated the site, size and extent of laryngeal tumour. In eight of the ten patients following radiation therapy the presence of tumour was correctly identified, however there was a poor correlation between the imaging and pathological findings. Two patients had radionecrosis alone. Neither CT nor MR imaging could differentiate between radionecrosis and recurrent tumour.

Polycystic disease of salivary glands.

Polycystic disease of salivary glands is a rare condition which hitherto has been reported only in the parotid glands. We report a case in which an accessory salivary gland had evidence of polycystic disease.