Hyperthermia by near infrared radiation induced immune cells activation and infiltration in breast tumor.

Breast cancer is the most common cancer that causes death in women. Conventional therapies, including surgery and chemotherapy, have different therapeutic effects and are commonly associated with risks and side effects. Near infrared radiation is a technique with few side effects that is used for local hyperthermia, typically as an adjuvant to other cancer therapies. The understanding of the use of near NIR as a monotherapy, and its effects on the immune cells activation and infiltration, are limited. In this study, we investigate the effects of HT treatment using NIR on tumor regression and on the immune cells and molecules in breast tumors. Results from this study demonstrated that local HT by NIR at 43 °C reduced tumor progression and significantly increased the median survival of tumor-bearing mice. Immunohistochemical analysis revealed a significant reduction in cells proliferation in treated tumor, which was accompanied by an abundance of heat shock protein 70 (Hsp70). Increased numbers of activated dendritic cells were observed in the draining lymph nodes of the mice, along with infiltration of T cells, NK cells and B cells into the tumor. In contrast, tumor-infiltrated regulatory T cells were largely diminished from the tumor. In addition, higher IFN-γ and IL-2 secretion was observed in tumor of treated mice. Overall, results from this present study extends the understanding of using local HT by NIR to stimulate a favourable immune response against breast cancer.

Microparticles produced by human papillomavirus type 16 E7-expressing cells impair antigen presenting cell function and the cytotoxic T cell response.

High-risk, cancer-causing human papillomaviruses (HPV) cause infections of the epidermis that may progress to cancer, including cervical cancer. Viral persistence, contributed to by viral evasion of the host immune response, is associated with the likelihood of cancer developing. Langerhans cells (LCs) are the only professional antigen presenting cells located in the epidermis, therefore may influence the antiviral immune response. Microparticles, or microvesicles, are small membrane particles shed by cells that can exert effects on other cells at both a local and systemic level. We found increased numbers of microparticles were shed from human or mouse keratinocytes expressing the HPV16 E7 oncoprotein, compared with control keratinocytes. Co-culture of LCs with microparticles from E7-expressing cells suppressed the cytotoxic T cell response. We attributed this, at least in part, to the reduction in surface of CD40 and intracellular pro-inflammatory cytokine IL-12 p40 subunit that we measured in the LCs. The evidence provided here shows that co-culture of E7-microparticles with LCs inhibits antigen-specific cytotoxicity. This is an important finding, suggesting that microparticles from HPV-infected cells could suppress the T cell response by regulating LCs, potentially contributing to persistence of HPV infection and cancer.

Suppression of the CD8 T cell response by human papillomavirus type 16 E7 occurs in Langerhans cell-depleted mice.

Human papillomavirus (HPV) is an epitheliotropic virus that is the primary causal agent for cervical cancer. Langerhans cells (LC) are skin antigen presenting cells that are reduced in number in HPV-infected skin. The aim of this study was to understand the immune-modulatory effects of HPV16 E7 on LC and on the CD8 T cell response to a skin-expressed antigen. To test this, HPV16 E7 was expressed in mouse skin keratinocytes with the model antigen ovalbumin (Ova). Similar to what is observed in HPV-infected human skin, LC numbers were significantly reduced in E7-expressing mouse skin. This shows that expression of the E7 protein alone is sufficient to mediate LC depletion. Expression of E7 with Ova in keratinocytes strongly suppressed the Ova-specific CD8+ T cell response in the skin draining lymph node. When tested in LC-ablated mice, the CD8 T cell response to skin-expressed Ova in control mice was not affected, nor was the T cell response to Ova restored in E7-expressing skin. These data indicate a role for E7 in regulation of LC homeostasis in the skin and in suppression of antigen specific CD8 T cell expansion, but suggest that these two effects occur independent of each other.

Delivery of Echinococcus granulosus antigen EG95 to mice and sheep using recombinant vaccinia virus.

The tapeworm Echinococcus granulosus is the causative agent of hydatid disease and affects sheep, cattle, dogs and humans worldwide. It has a two-stage life cycle existing as worms in the gut of infected dogs (definitive host) and as cysts in herbivores and humans (intermediate host). The disease is debilitating and can be life threatening where the cysts interfere with organ function. Interruption of the hydatid life cycle in the intermediate host by vaccination may be a way to control the disease, and a protective oncosphere antigen EG95 has been shown to protect animals against challenge with E. granulosus eggs. We explored the use of recombinant vaccinia virus as a delivery vehicle for EG95. Mice and sheep were immunized with the recombinant vector, and the result monitored at the circulating antibody level. In addition, sera from immunized mice were assayed for the ability to kill E. granulosus oncospheres in vitro. Mice immunized once intranasally developed effective oncosphere-killing antibody by day 42 post-infection. Antibody responses and oncosphere killing were correlated and were significantly enhanced by boosting mice with either EG95 protein or recombinant vector. Sheep antibody responses to the recombinant vector or to EG95 protein mirrored those in mice.

Deregulation of E-cadherin by human papillomavirus is not confined to high-risk, cancer-causing types.

BACKGROUND: E-cadherin is a tumour suppressor protein, which is normally expressed on keratinocytes and antigen-presenting Langerhans cells (LCs) in the epidermis. We have previously shown that E-cadherin is lost from tissues infected with the high-risk cancer-causing human papillomavirus (HPV) type 16. OBJECTIVES: To test if E-cadherin dysregulation is associated with the cancer risk of the infecting HPV and to establish if it is conserved among HPVs in the α, ß, γ and µ genera. METHODS: Forty-seven lesions infected with low- or high-risk HPV types spanning four HPV genera were stained for E-cadherin, P-cadherin and CD1a to detect LCs. RESULTS: Surface E-cadherin was reduced in tissues infected with members of the α4, α7 and α9 species and the γ and µ genera but was equivalent to normal epidermis in the ß only-infected lesions tested and patchy in α10-infected tissues. There was a direct relationship between atypical E-cadherin expression and a significant reduction in LCs. Expression of P-cadherin, a protein that is increased in the E-cadherin constitutive knockout mouse, was increased in lesions with reduced E-cadherin. CONCLUSIONS: These data show that E-cadherin dysregulation by HPV is widely conserved across the majority of HPV genera. E-cadherin expression was reduced or lost in epidermis irrespective of the cancer risk of the infecting HPV type or the ability of the virus to degrade retinoblastoma protein or p53. A correlation between dysregulated E-cadherin and reduced numbers of LCs supports viral regulation of surface E-cadherin contributing to viral evasion of the host immune system.

p53 promotes adenoviral replication and increases late viral gene expression.

The tumor suppressor protein, p53, plays a critical role in viro-oncology. However, the role of p53 in adenoviral replication is still poorly understood. In this paper, we have explored further the effect of p53 on adenoviral replicative lysis. Using well-characterized cells expressing a functional p53 (A549, K1neo, RKO) and isogenic derivatives that do not (K1scx, RKOp53.13), we show that virus replication, late virus protein expression and both wtAd5 and ONYX-015 virus-induced cell death are impaired in cells deficient in functional p53. Conversely, by transfecting p53 into these and other cells (IIICF/c, HeLa), we increase late virus protein expression and virus yield. We also show, using reporter assays in IIICF/c, HeLa and K1scx cells, that p53 can cooperate with E1a to enhance transcription from the major late promoter of the virus. Late viral protein production is enhanced by exogenous p53. Taken together, our data suggest that functional p53 can promote the adenovirus (Ad) lytic cycle. These results have implications for the use of Ad mutants that are defective in p53 degradation, such as ONYX-015, as agents for the treatment of cancers.

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein.

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully chracterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.

The human papillomavirus type 16 E7 protein is associated with the nucleolus in mammalian and yeast cells.

In this study we show, by immunofluorescence and electron microscopy immuno-gold labelling, that the major transforming protein of Human Papillomavirus type 16 E7 is associated with the nucleolus of cells derived from the HPV16-positive cervical carcinoma line CaSki. The E7 nucleolar staining appeared to be cell cycle dependent, being considerably reduced in the G2 phase. The total level of the protein in the cell, however, remained constant during all phases. We also show that the cellular protein Rb1, which is targeted by E7, is localised in the nucleus and nucleolus in CaSki cells. Thus, it is possible that the presence of E7 in the nucleolus correlates with a hypothetical function(s) of Rb1 in this particular intranuclear compartment. The nucleolar localisation of HPV16 E7 protein was also observed in the fission yeast Schizosaccharomyces pombe, suggesting that a targeting mechanism of HPV16 E7 protein into the nucleolus is common to both mammalian and yeast systems. Nucleolar localisation of HPV16 E7 protein may be independent from Rb1 since no Rb1 related proteins have been identified in fission yeast.

Diagnostic phase antibody response to the human papillomavirus type 16 E2 protein is associated with successful treatment of genital HPV lesions with systemic interferon alpha-2b.

BACKGROUND AND OBJECTIVE: Systemic interferon alpha-2b treatment reduces relapses of genital human papillomavirus (HPV) lesions in some but not all females. The aim of the present study was to investigate possible predictive pretreatment factors for the outcome of therapy. MATERIAL AND METHODS: HPV DNA status and HPV antibody response were evaluated in 100 randomized patients treated with laser ablation and systemic interferon alpha-2b or placebo, and followed up to 6 months. RESULTS: Overall, adjuvant therapy with systemic interferon-alpha did not differ from placebo. However, detectable diagnostic phase levels of serum antibodies to e.g. HPV16 open reading frame (ORF) E2 derived peptide 141EEASVTVVEGQVDYY155 predicted 10-fold difference in the risk of recurrence of HPV infection following adjuvant interferon alpha-2b therapy as compared with placebo (odds ratio, OR, 0.5, 95% confidence interval (CI), 0.1-2.3; OR, 4.6, 95% CI 0.5-41, respectively). This trend was statistically significant in the whole study population (2P < 0.05), and in patients with high viral load (2P < 0.01). CONCLUSIONS: Evaluation of the E2 antibody responses may help to identify women with genital HPV lesions who respond to systemic interferon alpha-2b treatment.

Human T helper cell epitopes overlap B cell and putative cytotoxic T cell epitopes in the E2 protein of human papillomavirus type 16.

Activation of T helper cells is a prerequisite for the function of cytotoxic T lymphocytes (CTL) and the maturation of the B cell response. Because epitopes recognized by each of these cells may overlap, we scanned the E2 protein of human papillomavirus (HPV) type 16 to identify the T helper cell epitopes. Four major T helper cell epitopes (mapping between aa:s 11-25, 141-155, 191-205 & 231-245) and adjacent human B cell epitopes were found. The first peptide-defined epitope (RLNV) 11CQDKILTHYENDSTD25 overlapped five putative HLA-I (A1, A2, A0205, A3 & A11) binding motifs (CQDKILTHY, RLNVCQDKI, NVCQDKIL, RLNVCQDK & RLNVCQDK). This epitope is also part of an N-terminal alpha-helix which may form four HLA-II (DR2, DR4, DR7 & DR8) specific agretopes for structures recognizable by the T cell receptor (e.g. KILT). The second epitope 141EEASVTVVEGOVDYY155 (GLYY) overlapped the putative HLA-A1 & HLA-Bw37 binding motifs (VVEGQVDYY/QVDYYGLYY and EEASVTVV), and two HLA-II (DR1 & DR3) specific agretopes. The third and fourth epitopes were not associated with more than one putative CTL epitope each. Only the first epitope shared considerable aa-homology with corresponding regions of other genital HPV types.

The interaction between human papillomavirus type 16 E1 and E2 proteins is blocked by an antibody to the N-terminal region of E2.

Replication of papillomavirus DNA requires two virally encoded proteins, E1 and E2. We expressed human papillomavirus (HPV) type 16 E1 and E2 in bacteria and showed that purified full-length E2 protein interacted directly with E1, in the absence of HPV16 DNA. It was established that the first 142 amino acids of E1 were not required for binding as E2 protein was able to interact with E1 devoid of this region. The interaction of E2 with E1 could be blocked by a monoclonal antibody that bound E2 in the region of amino acids 18-41 of E2 whereas a monoclonal antibody reactive with a nearby part of the molecule (amino acids 2-17) only partially blocked this interaction. These results suggest that a region in the N-terminus of E2 around amino acids 18-41 is a site of interaction with the E1 protein.

Antibodies against linear and conformational epitopes of human papillomavirus type 16 that independently associate with incident cervical cancer.

In a seroepidemiological study of incident cervical cancer, 94 cases and 188 population-based controls were used to evaluate the disease-association of IgG and IgA antibody responses against 6 human papillomavirus (HPV) type-16 antigens. Nine of the tested antibody responses were positively associated with cervical cancer, with odds ratios (ORs) ranging from 2.5 to 15.0. The antibody responses most strongly associated with cervical cancer were IgA against E6:10, an epitope derived from the carboxyterminal part of the HPV16 E6 [OR = 15.0, confidence intervals (CI) = 5.9-48.6], IgG against HPV16 virus-like particles (OR = 9.5, CI = 3.9-28.0) and IgG against the E1:19 epitope in the middle part of the E1 protein of HPV16 (OR = 7.7, C1 = 3.9-16.5). When the 3 serological assays that showed the strongest association with cervical cancer were combined, positivity for 2 assays was found among 52% of cases at an OR of 29.9. We conclude that antibody responses to several linear and conformational HPV epitopes are independently associated with cervical cancer and that combined analysis of several HPV antibody responses can result in better predictive values for HPV-associated cancer.

The influence of maternal separation on humoral and cellular immunity in farmed deer.

The effect of maternal separation on in vivo and in vitro immune parameters was studied in young hybrid deer. Groups of fawns separated from their mothers either 2 days prior to or 7 days post immunization with keyhole limpet hemocyanin were compared with a control group of immunized, unseparated fawns. Animals separated prior to antigenic challenge had significantly higher concentrations of antigen-specific IgG in their serum than control animals. There was no influence on the humoral immune response in animals separated following immunization. In contrast, Con A transformations were transiently depressed in separated animals compared to the control group. The time separation was imposed relative to challenge and therefore influenced the subsequent immune response.

Generation and characterisation of murine monoclonal antibodies specific for cervine immunoglobulin light chain, IgM and IgG.

Monoclonal antibodies (mAb) which react with cervine immunoglobulin (Ig) light chain, IgM and IgG were produced using conventional cell fusion technology. Hybridoma supernatants were initially screened for specificity against cervine Ig using an enzyme-linked immunosorbent assay (ELISA). The specificity of supernatants against size-fractionated cervine Ig was further determined. Supernatants were characterised using western blotting and autoradiographic techniques. The mAb OU1G, OU2G and OU3G were specific for cervine gamma-chain of IgG, whereas OU1L was specific for light chain of Ig. A further mAb (OU1M) bound IgM and not IgG. These mAb were found to have varying cross-reactivity against Ig from other species.

The effect of adjuvants on active and passive immunity in pregnant deer and their offspring.

Experiments were carried out to determine the effects of a range of adjuvants on immunoglobulin M (IgM) and immunoglobulin G (IgG) serum concentrations to a protein antigen administered subcutaneously to farmed female deer following mating. The antibody responses of animals immunised with keyhole limpet hemocyanin (KLH) in Freund's Incomplete Adjuvant (FIA), diethylaminoethyl dextran (DEAE-dextran) and aluminium hydroxide (alum) were compared with the response to antigen administered in the absence of adjuvants. Animals were subsequently challenged with a subcutaneous immunisation of the antigen in saline. Following parturition, the concentration of passively transferred antigen-specific antibody was measured in the serum of the offspring. The polyionic adjuvant, DEAE-dextran, produced the greatest enhancement of both primary and secondary IgG responses to KLH. Offspring suckling from mothers immunised with antigen in DEAE-dextran consequently had higher concentrations of specific antibodies in their serum than other fawns in the experiment. The adjuvants FIA and alum were approximately 20-fold less effective in enhancing antigen-specific IgG than DEAE-dextran but induced greater amounts of antigen-specific IgM. From the results presented in this paper, there is evidence that immunisation of deer during pregnancy may be an effective way of reducing morbidity in both mothers and offspring.

Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer.

The purification and characterisation of cervine IgM and IgG.

A procedure is described for the isolation of immunoglobulin G (IgG) and immunoglobulin M (IgM) from hyperimmune cervine serum. Hybrids of red deer (Cervus elaphus) and wapiti (Cervus canadensis) were immunised with keyhole limpet hemocyanin (KLH). An immunoglobulin-containing fraction was precipitated from the hyperimmune serum using ammonium sulphate. The antigen-specific immunoglobulins were purified by KLH-conjugated sepharose affinity chromatography and further separated into IgM and IgG by gel-filtration chromatography. Purified immunoglobulin was analysed by polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights and isoelectric points of the composite chains of cervine IgG and IgM are presented.

Altered humoral immunity during pregnancy in the guinea pig.

A guinea pig model was developed to determine whether humoral immune responsiveness is altered during pregnancy. Pregnant animals were immunised at mid-gestation with haptenated protein. The humoral response to antigen was measured as numbers of antibody-producing cells in the spleen, the affinity of the antibody produced by spleen cells and the levels of IgG in the serum. The values obtained were compared with those from an age matched non-pregnant control group. Early in the primary response, there was a significant decrease in the number of IgM antibody-producing cells with an associated decrease in serum IgM levels in pregnant animals. Late in the primary response, pregnant and control animals had similar levels of IgM antibody-producing cells. During the later stages of the response, many of the pregnant animals did not respond with IgG antibody-producing cells or IgG in the serum. When IgG-producing cells were detected, the antibody was of lower affinity than that observed with the control group. A selective lack of responsiveness was detected in the primary response of pregnant guinea pigs. The reduced number of IgG antibody-producing cells in gravid animals suggests that an immune switch from IgM to IgG is impaired in pregnancy. The low affinity of antibody produced indicates the immunoglobulin produced in pregnancy may also be functionally limited.