Lynne Hampson

This chapter outlines the dual killing mechanism used by the immune system and suggests a way in which effector cells may act with impunity while remaining susceptible to regulation by apoptosis. It deals with cell death caused by granular serine proteases, granzymes, which gain access to the interior of target cells and activate the apoptotic process. The chapter then examines evidence showing that a member of the intracellular serpin family called granzyme B inhibitor is expressed in immune cells and may protect them from endogenous granzymes. The principal effectors of the mammalian immune system are cytotoxic T cells and natural killer cells. Granzyme B shares with the caspase family of cysteine proteases a specificity for cleavage at aspartate residues. Caspases are activated during apoptosis and cleave key cellular proteins which render cell death irreversible. Apoptosis is a critical process in cellular function and is also tightly controlled.

There are >200 different types of human papilloma virus (HPV) of which >51 infect genital epithelium, with ~14 of these classed as high-risk being more commonly associated with cervical cancer. During development of the disease, high-risk types have an increased tendency to develop a truncated non-replicative life cycle, whereas low-risk, non-cancer-associated HPV types are either asymptomatic or cause benign lesions completing their full replicative life cycle. HPVs can also be present as non-replicative so-called “latent” infections and they can also show superinfection exclusion, where cells with pre-existing infections with one type cannot be infected with a different HPV type. Thus, the HPV repertoire and replication status present in an individual can form a complex dynamic meta-community which changes with respect to both time and exposure to different HPV types. In light of these considerations, it is not clear how current prophylactic HPV vaccines will affect this system and the potential for iatrogenic outcomes is discussed in light of recent outcome data.

Summary: We investigate the accumulated microbial and autoantigen antibody repertoire in adult-onset dermatomyositis patients sero-positive for TIF1γ (TRIM33) autoantibodies. We use an untargeted high-throughput approach which combines immunoglobulin disease-specific epitope-enrichment and identification of microbial and human antigens. Increased microbial diversity was observed in dermatomyositis. Viruses were over-represented and species of the Poxviridae family were significantly enriched. The autoantibodies identified recognised a large portion of the human proteome, including interferon regulated proteins; these proteins were clustered in specific biological processes. Apart from TRIM33, autoantibodies against eleven further TRIM proteins, including TRIM21, were identified. Some of these TRIM proteins shared epitope homology with specific viral species including poxviruses. Our data suggest antibody accumulation in dermatomyositis against an expanded diversity of microbial and human proteins and evidence of non-random targeting of specific signalling pathways. Our findings indicate that molecular mimicry and epitope spreading events may play a significant role in the pathogenesis of dermatomyositis.

Head and neck squamous cell carcinoma (HNSCC) is a widely prevalent cancer globally with high mortality and morbidity. We report here changes in the genomic landscape in the development of HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Frequent likely pathological mutations are restricted to a relatively small set of genes including TP53, CDKN2A , FBXW7 , FAT1 , NOTCH1 and KMT2D ; these arise early in tumour progression and are present in PPOLs with NOTCH1 mutations restricted to cell lines from lesions that subsequently progressed to HNSCC. The most frequent genetic changes are of consistent somatic copy number alterations (SCNA). The earliest SCNAs involved deletions of CSMD1 (8p23.2), FHIT (3p14.2) and CDKN2A (9p21.3) together with gains of chromosome 20. CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines (promoter hypermethylation ~63%, hemizygous deletions ~75%, homozygous deletions ~18%). Forced expression of CSMD1 in the HNSCC cell line H103 showed significant suppression of proliferation (p=0.0053) and invasion in vitro (p=5.98X10 ⁻⁵ ) supporting a role for CSMD1 inactivation in early head and neck carcinogenesis. In addition, knockdown of CSMD1 in the CSMD1 -expressing BICR16 cell line showed significant stimulation of invasion in vitro (p=1.82 x 10 ⁻⁵ ) but not cell proliferation (p=0.239). HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1 , hsa-miR-548k and TP63 in the metastases group. GISTIC peak SCNA regions showed significant enrichment (adj PCSMD1 gene in early premalignant lesions; we further show that this is likely to result in increased ability of the cells to spread and possibly, multiply faster as well.

Background: The idiopathic inflammatory myopathies are heterogeneous autoimmune disorders characterised by inflammation of muscle tissue and are thought to result from environmental exposures in genetically susceptible individuals. We identified recently genetic association to the chemokine receptor 5 gene in inclusion body myositis (IBM); this suggests a potential mechanism relating to disease pathogenesis whereby reduced frequency of the CCR5Δ32 deletion in IBM increases viral entry into host cells. Similarly, antibodies against tripartite motif-containing proteins reduce ability to restrict infectivity and replication of retroviruses, suggesting a mechanism relating to disease pathogenesis in dermatomyositis patients with anti-TIF1 antibodies. We propose to identify disease-subtype specific signatures of past...

The oncogenic retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in some countries although its prevalence and relationship with other sexually transmitted infections in Sub-Saharan Africa is largely unknown. A novel endpoint PCR method was used to analyse the prevalence of HTLV-1 proviral DNA in genomic DNA extracted from liquid based cytology (LBC) cervical smears and invasive cervical carcinomas (ICCs) obtained from human immunodeficiency virus-positive (HIV+ve) and HIV-negative (HIV−ve) Kenyan women. Patient sociodemographic details were recorded by structured questionnaire and these data analysed with respect to HIV status, human papillomavirus (HPV) type (Papilocheck^®) and cytology. This showed 22/113 (19.5%) of LBC’s from HIV+ve patients were positive for HTLV-1 compared to 4/111 (3.6%) of those from HIV−ve women (p = 0.0002; odds ratio (OR) = 6.42 (2.07–26.56)). Only 1/37 (2.7%) of HIV+ve and none of the 44 HIV−ve ICC samples were positive for HTLV-1. There was also a significant correlation between HTLV-1 infection, numbers of sexual partners (p < 0.05) and smoking (p < 0.01). Using this unique method, these data suggest an unexpectedly high prevalence of HTLV-1 DNA in HIV+ve women in this geographical location. However, the low level of HTLV-1 detected in HIV+ve ICC samples was unexpected and the reasons for this are unclear.

Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp's being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types.

Cervical cancer is the most common female malignancy in the developing nations and the third most common cancer in women globally. An effective, inexpensive and self-applied topical treatment would be an ideal solution for treatment of screen-detected, pre-invasive cervical disease in low resource settings. Between 01/03/2013 and 01/08/2013, women attending Kenyatta National Hospital's Family Planning and Gynaecology Outpatients clinics were tested for HIV, HPV (Cervista®) and liquid based cervical cytology (LBC -ThinPrep®). HIV negative women diagnosed as high-risk HPV positive with high grade squamous intraepithelial lesions (HSIL) were examined by colposcopy and given a 2 week course of 1 capsule of Lopimune (CIPLA) twice daily, to be self-applied as a vaginal pessary. Colposcopy, HPV testing and LBC were repeated at 4 and 12 weeks post-start of treatment with a final punch biopsy at 3 months for histology. Primary outcome measures were acceptability of treatment with efficacy as a secondary consideration. A total of 23 women with HSIL were treated with Lopimune during which time no adverse reactions were reported. A maximum concentration of 10 ng/ml of lopinavir was detected in patient plasma 1 week after starting treatment. HPV was no longer detected in 12/23 (52.2%, 95%CI: 30.6–73.2%). Post-treatment cytology at 12 weeks on women with HSIL, showed 14/22 (63.6%, 95%CI: 40.6–82.8%) had no dysplasia and 4/22 (18.2%, 95%CI: 9.9–65.1%) were now low grade demonstrating a combined positive response in 81.8% of women of which 77.8% was confirmed by histology. These data are supported by colposcopic images, which show regression of cervical lesions. These results demonstrate the potential of Lopimune as a self-applied therapy for HPV infection and related cervical lesions. Since there were no serious adverse events or detectable post-treatment morbidity, this study indicates that further trials are clearly justified to define optimal regimes and the overall benefit of this therapy. ISRCTN Registry 48776874

An overview of early investigational drugs for the treatment of human papilloma virus infection and associated dysplasia. . ???aop.label???. doi: 10.1517/13543784.2015.1099628

Introduction Human Papilloma Viruses (HPV) infection may occur in 40% of rectal adenocarcinomas (RCa). HPV positivity (mainly HPV16) at other tumour sites is a predictor for treatment response to chemo-radiotherapy (CRT). Here, we performed a ‘proof of concept’ study in patients with RCa testing the hypothesis that HPV16 tumour positivity is a treatment predictive biomarker for complete response (CR) to CRT. Method The study was a case-control design in 118 patients undergoing CRT for RCa (CR, 30: non-CR, 88) from two UK cancer centres (2008 to 2013). DNA was extracted from pre-treatment paraffin-embedded blocks with histologically verified carcinoma. High-sensitivity multiplex PCR for HPVs was performed using two sets of primers for each HPV to identify genotypes: HPV6, 16, 18, 33. The probability of a CR was expressed as odd ratios (ORs) using logistic regression models. Results Any HPV positivity was noted in 25% of tumours. Individual genotype frequencies were: HPV16, 16%; HPV18, 6%; HPV33, 3%; HPV6, 3%. In multivariate models that included age, gender, pre-treatment T-size and N status, there was no association between either any HPV positivity (OR = 1.095, 95% CIs: 0.394, 3.044) or HPV16 positivity (OR = 1.072, 95% CIs: 0.332, 3.465), and complete response to CRT. Conclusion In this stage 2 biomarker discovery study using high-sensitivity HPV multi-valent assays, we found tumour HPV positivity rates lower than those reported elsewhere in the literature. We found no clear ‘signal’ to take forward HPV genotyping for validation as a predictive biomarker for chemo-radiotherapy response in patients with rectal cancer. This study was generously supported by the BDRF. Disclosure of interest None Declared. References Baricevic-Jones, et al. High-sensitivity HPV genotyping reveals near universal positivity in anal squamous cell carcinoma: different implications for vaccine prevention and prognosis. Eur J Cancer [in press]