Fungal mannosylation enhances human papillomavirus 16 E7 therapeutic immunity against TC-1 tumors.

Cervical cancer, resulting from infection with human papillomavirus (HPV)16, remains the fourth most common cancer in women worldwide. Recently, three prophylactic HPV vaccines targeting high-risk HPVs (particularly HPV16 and HPV18) have been implemented to protect younger women. However, individuals with pre-existing infections have no benefit from prophylactic vaccines. Thus, there is an urgent need to develop therapeutic vaccines. HPV16 E7 has been widely utilized as a target for immune therapy of HPV16-associated lesions or cancers, reflecting the sustained existence of this virus in cancerous cells. We developed mannosylated HPV16 E7 (mE7) expressed from Pichia pastoris as a therapeutic vaccine against HPV16-associated cancer. Unmannosylated E7 (E7) was also generated from Pichia pastoris as a control. Mannosylation enhanced the uptake of mE7 by mannose receptors of bone marrow-derived dendritic cells (BMDCs), while the uptake of E7 was unaffected. mE7-uptake BMDCs in vitro induced more IFN-γ secretion by splenocytes of immunized mice than E7. Vaccination of C57BL/6 mice with mE7 combined with adjuvant monophosphoryl lipid A (MPL) elicited stronger Th1 (type 1 T helper cell) responses and E7-specific T cell responses than E7. The mE7 vaccine induced the increased production of IFN-γ, IL-2 and TNF-α, elicited more E7-specific IFN-γ-secreting CD8+ T cells in spleen and peripheral blood mononuclear cells (PMBCs) and promoted stronger E7-specific cytotoxic CD8+ T cell responses compared with E7. Furthermore, TC-1 tumor challenged mice were used to confirm the antitumor activity of the vaccines. As a result, mE7 generated complete antitumor activity against TC-1 tumors, while E7 only provided partial antitumor activity. Taken together, mE7 can be a promising immunotherapy for treating cervical cancer.

Immunotherapy of HPV-associated cancer: DNA/plant-derived vaccines and new orthotopic mouse models.

Under the optimistic assumption of high-prophylactic HPV vaccine coverage, a significant reduction of cancer incidence can only be expected after decades. Thus, immune therapeutic strategies are needed for persistently infected individuals who do not benefit from the prophylactic vaccines. However, the therapeutic strategies inducing immunity to the E6 and/or E7 oncoprotein of HPV16 are more effective for curing HPV-expressing tumours in animal models than for treating human cancers. New strategies/technologies have been developed to improve these therapeutic vaccines. Our studies focussed on preparing therapeutic vaccines with low-cost technologies by DNA preparation fused to either plant-virus or plant-toxin genes, such as saporin, and by plant-produced antigens. In particular, plant-derived antigens possess an intrinsic adjuvant activity that makes these preparations especially attractive for future development. Additionally, discrepancy in vaccine effectiveness between animals and humans may be due to non-orthotopic localization of animal models. Orthotopic transplantation leads to tumours giving a more accurate representation of the parent tumour. Since HPV can cause cancer in two main localizations, anogenital and oropharynx area, we developed two orthotopic tumour mouse models in these two sites. Both models are bioluminescent in order to follow up the tumour growth by imaging and are induced by cell injection without the need to intervene surgically. These models were utilized for immunotherapies with genetic or plant-derived therapeutic vaccines. In particular, the head/neck orthotopic model appears to be very promising for studies combining chemo-radio-immune therapy that seems to be very effective in patients.

Indolent T-lymphblastic proliferation: report of a case involving the upper aerodigestive tract.

T-lymphoblastic lymphoma (T-LBP) is a high-grade malignant lymphoma, which possesses the characteristic of high metastasis and high mortality without treatment. We are presenting a special T-lymphoblastic proliferation involving in the oropharynx, nasopharynx, sinus and trachea in a patient with local involved about 15-years without systemic dissemination. The immunophenotype of this case was similar to T-LBP. The proliferous cells were positive for terminal deoxynucleotidyl transferase (TdT), CD3, and appeared co-expression CD4 and CD8. No clonal rearrangements of TCRγ and/or TCRß gene were detected. Indolent T-lymphoblastic proliferations rarely occurred or unusually could not be diagnosed, combing with the relevant literature and clinically indolent manifestation, we interpreted this case as indolent T-lymphoblastic proliferation (iT-LBPs). So far, the mechanism of the T-lymphoblastic proliferations is still uncertain and requires further study.

Immune suppression in premalignant respiratory papillomas: enriched functional CD4+Foxp3+ regulatory T cells and PD-1/PD-L1/L2 expression.

PURPOSE: Respiratory papillomas, caused by human papillomaviruses types 6 and 11 (HPV6/11), are premalignant lesions with potential for malignant conversion. The cytokine and chemokine micromilieu of papillomas is T(H)2-like with a marked absence of IFN-γ expression. To illuminate why patients with recurrent respiratory papillomatosis (RRP) fail to effectively control their disease, we further investigated the suppressive cellular microenvironment in papillomas. EXPERIMENTAL DESIGN: CD4(+)CD25(+)CD127(low/-)Foxp3(+) regulatory T cells (Treg) and CD4(+)CD25(-)CD127(low/-)Foxp3(-) T cells within papillomas were characterized and isolated. Their suppressor function was measured by inhibition of peripheral blood mononuclear cell (PBMC) proliferation. Expression of PD-1, CD69, and Helios was identified on these T cells. PD-L1, PD-L2, CCL17, and CCL22 mRNA was also identified in papillomas by quantitative PCR. RESULTS: Functional Tregs were markedly enriched in papillomas and strongly inhibited anti-CD3 and anti-CD28 antibody activated PBMC proliferation. The natural Treg marker Helios was reduced on Tregs from papillomas, indicating that the majority of Tregs in papillomas are adaptive. The majority of the papilloma-derived CD4(+) T cells expressed the CD4(+)CD25(-)CD127(low/-)Foxp3(-)PD1(+)CD69(+) phenotype and failed to suppress PBMC proliferation, suggesting that they are chronically activated and exhausted. The Treg-attracting chemokine CCL22 was equally expressed by all laryngeal tissues examined. However, CCL17 was robustly expressed by papillomas compared with unaffected laryngeal tissues from RRP patients and individuals without RRP. PD-L1 was elevated in papillomas compared with control laryngeal tissues. CONCLUSIONS: Papilloma CD4(+) T cells are enriched with functional Tregs, and the adaptive Helios(-) Treg fraction was increased within the T(H)2-like papilloma micromilieu. CD4(+)CD25(-)CD127(low/-)Foxp3(-) T-cells failed to suppress PBMC proliferation and may be exhausted. The PD-1/PDL-1 pathway may represent an additional immunosuppressive mechanism that contributes to defective HPV6/11 clearance in RRP.

Association between toll-like receptor expression and human papillomavirus type 16 persistence.

The mechanisms involved in mucosal immune control of cervical human papillomavirus (HPV) infection remain ill defined. Because toll-like receptors (TLRs) are key players in innate immune responses, we investigated the association between TLR expression and viral persistence or clearance in young women with incident infections with oncogenic HPV types 16 or 51. Messenger RNA expression of TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8 and TLR9 was measured by quantitative reverse transcription-PCR using human endocervical specimens, collected before and after viral acquisition, in a cohort well characterized for HPV infections. Wilcoxon rank sum test was used to compare the change seen from preinfection to incident infection between women who subsequently cleared infection with those who did not. HPV 16 infections that cleared were significantly (p < 0.05) associated with an increase in expression of the four viral nucleic acid-sensing TLRs (TLR3, TLR7, TLR8 and TLR9) as well as TLR2 upon viral acquisition. Similar associations were not observed for HPV 51. In women who subsequently cleared their HPV 16 infection, changes in TLR1, TLR3, TLR7 and TLR8 expression levels between preincident and incident visits were significantly correlated with parallel changes in the levels of interferon-α2, measured by immunoassay in cervical lavage specimens. This study suggests that dampened TLR expression in the cervical mucosa is a type-specific mechanism by which HPV 16 interferes with innate immune responses, contributing to viral persistence, and that TLR upregulation and resultant cytokine induction is important in subsequent viral clearance.

Cytokine pattern in Kaposi's sarcoma associated with immune restoration disease in HIV and tuberculosis co-infected patients.

We analysed the evolution of different cytokines (IL-4, IL-6, tumour necrosis factor alpha and vascular endothelial growth factor; VEGF) involved in the development of Kaposi's sarcoma in two patients in whom HIV infection presented with disseminated Mycobacterium tuberculosis infection. They simultaneously developed tuberculosis-associated immune restoration disease and Kaposi's sarcoma shortly after the initiation of HAART. Analysis of VEGF and pro-inflammatory cytokines led us to hypothesize that Kaposi's sarcoma could be promoted by the tuberculosis immune response.

Immunocompetency in children with recurrent respiratory papillomatosis: prospective study.

OBJECTIVES: We sought to investigate the immunologic status of children with recurrent respiratory papillomatosis and to evaluate possible correlations between the patients' immunocompetency and the clinical course of the disease. METHODS: Twenty children with recurrent respiratory papillomatosis underwent immunologic evaluation every 6 months for determination of complete blood count, serum immunoglobulin levels, lymphocyte subpopulations, lymphocyte response to mitogen stimulation, and natural killer cell function. The patients were observed prospectively (42 to 56 months), and their clinical course was recorded. The findings were compared with those in healthy age-matched controls. RESULTS: The CD4/CD8 ratio and the lymphocyte response to mitogen stimulation were significantly reduced in the study children compared to normal controls. A reduction in lymphocyte response to mitogen stimulation was significantly correlated to a high number of papilloma sites and more frequent recurrences. Abnormal natural killer cell function was significantly correlated to more frequent recurrences. CONCLUSIONS: A compromised cell-mediated immune response may be associated with repeated or persistent human papillomavirus infections, leading to the development of recurrent respiratory papillomatosis. Patients with an aggressive clinical course may have underlying cell-mediated immunodeficiency. Long-term prospective investigations are needed to establish the role of the host immune system in the pathogenesis of recurrent respiratory papillomatosis in children.

HLA alleles, IFN-gamma responses to HPV-11 E6, and disease severity in patients with recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) remains an immunologic enigma. Human papillomavirus (HPV) types 6 and 11 are the predominant HPV viruses that cause papilloma development. However, it is unclear why only a very small fraction of HPV-exposed individuals develop RRP. We performed high-resolution HLA class I and II genotyping on 70 randomly selected patients (56 Caucasians and 14 African-Americans) with RRP. We report, for the first time, an increased frequency of HLA-DRB1*0102 in Caucasian patients with RRP, suggesting that this allele predisposes individuals to RRP. Additionally, HLA-DRB1*0301, DQB1*0201, and DQB1*0202 alleles were selectively enriched in Caucasians with severe disease, suggesting that these alleles may regulate disease severity. In contrast, HLA-DQB1*0602 was more frequent in controls than in Caucasians with severe disease, suggesting a severity-sparing effect of this allele. Furthermore, both DQB1*0201 and DQB1*0202 were enriched, whereas DQB1*0602 was absent, in African-Americans. Interestingly, HLA-DRB1*0301 and DQB1*0201 correlated with reduced interferon-gamma expression in patients with RRP. Larger studies are needed to identify other class II major histocompatibility complex alleles that may influence disease predisposition, disease severity, or both, especially in African-American patients, to ultimately illuminate the regulatory effects of these alleles in the predisposition and severity of RRP.

[The detection of cytokine and immunoglobulin in sera of children with respiratory papillomatosis].

OBJECTIVE: To investigate the levels and effects of cytokine and immunoglobulin on children with respiratory papillomatosis(RP). METHOD: The levels of cytokine and immunoglobulin in sera of 23 patients with RP were measured by ELISA and immunoassay and were compared with the normal data published by our department of Laboratory. RESULT: The levels of cytokine in RP group were significantly higher than that of normal group and the levels of immunoglobulin in RP group was significantly lower than that of normal group. CONCLUSION: The result shows that there are immune deficiency and abnormal immune reponse with cytokine in the RP patients.

Noninvasive methods to measure airway inflammation: future considerations.

This last contribution to the series focuses on open questions regarding: 1) methodological issues; and 2) the potential clinical application of the noninvasive methods such as induced sputum and the analysis of exhaled air for the assessment of airway inflammation. In addition their potential future role in occupational health and the early diagnosis of neoplastic lesions are briefly discussed. The future clinical application of noninvasive methods will depend on the progress made to improve their practicability, particularly in rendering them less time consuming and cheaper. To assess their clinical value, prospective studies are needed to establish whether patients actually benefit from the results obtained. This is also important to implement the methods into the healthcare system and to obtain adequate financial compensation. Therefore, it is necessary to know: 1) whether the assessment of airwav inflammation can aid in coming to an earlier and better defined diagnosis; 2) whether by repeated monitoring it is possible to avoid exacerbations through earlier interventions; and 3) whether the long-term outcome of patients is improved through knowledge of the type and degree of airway inflammation that is taken into account in selecting the appropriate treatment. In the meantime a wealth of data has become available, both for induced sputum and the analysis of exhaled air, which give these methods the potential to be incorporated into future clinical practice. This, however, will, amongst the other issues, depend on favourable cost-benefit ratios which should also be the subject of future prospective studies.

Recurrent respiratory papillomatosis: altered CD8(+) T-cell subsets and T(H)1/T(H)2 cytokine imbalance.

Human papillomaviruses (HPVs) cause benign papillomas and squamous cell carcinomas in the genital and respiratory tracts. Recurrent respiratory papillomas (RRP) generate a high level of morbidity and significant mortality because of their location, resistance to treatment, and relentless recurrence that can vary in frequency in a given patient and between patients. We have found that T-cells from these patients, when exposed to or isolated from autologous papilloma tissue, have an elevated percentage of CD8(+), CD28(-) T-cells, and that T-cells from many of these patients express an increase in T(H)2-like cytokine mRNA in response to autologous papilloma tissue. Furthermore, both of these immunologic findings correlate with disease severity. These observations suggest that patients with RRP, and possibly others with refractory HPV-induced lesions, are unable to manage their disease with an appropriate and effective HPV-specific, T-cell response. This immune imbalance may be responsible for the development and severity of HPV-induced respiratory papillomatosis.

Cyclosporin A enhances paclitaxel toxicity against leukemia and respiratory epithelial cancers.

Multidrug resistance is probably the single greatest obstacle to successful systemic therapy of human cancer. We have reported that cyclosporin A (CsA) can overcome multidrug resistance and improve the efficacy of etoposide in a murine model of drug-sensitive leukemia. The combination of CsA and paclitaxel (PCL) was also significantly superior to either drug alone against murine P388 (sensitive) and L1210 (resistant) leukemia. Lung cancer cells provide an ideal model system to study this phenomenon because both de novo and acquired drug resistance occur. Standard chemotherapy for advanced lung cancer is poorly effective, and although PCL is one of the most active new agents for this disease, responses occur in only 20% of patients. In vitro, CsA significantly enhanced the efficacy of PCL against lung (Lu-CSF-1 and 3LL) and oropharyngeal (CSCC-20) cancer cell lines. The combination also produced an increase in expression of interleukin 1beta, a cytokine known to inhibit the growth of Lu-CSF-1 cells. CsA alone had little or no antiproliferative activity in vitro and did not alter PCL transport. These results indicate that the activity of chemotherapy modulators may extend beyond mitigation of drug resistance to enhancement of therapeutic efficacy against drug-sensitive tumor cells in vitro and in vivo.

Inconsistent association of Epstein-Barr virus with CD56 (NCAM)-positive angiocentric lymphoma occuring in sites other than the upper and lower respiratory tract.

We previously described nine cases of angiocentric lymphoma of a possible natural killer (NK)-cell lineage with a surface CD3-CD56+ phenotype occurring in sites other than the upper and lower respiratory tract. This study was performed to investigate the association of Epstein-Barr virus (EBV) with these lymphomas, using the polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small RNAs (EBERs) and immunohistology for EBV-determined nuclear antigen-2 (EBNA-2) and latent membrane protein-1 (LMP-1) in paraffin sections. PCR and ISH produced almost identical results, and EBERs were identified in the nuclei of the lymphoma cells of three cases, two of which exhibited LMP-1 in the cytoplasm of tumour cells without EBNA-2 expression. Molecular genetic analysis revealed EBV to be incorporated into these three EBER-positive cases either clonally or biclonally. It was revealed by re-evaluation of their morphology with the established EBV status on each case that, in contrast to the rather variable and irregular cellular composition of the EBV-positive tumours, the EBV-negative tumours stood out because of their remarkably uniform 'blastoid' appearance, and could be grouped as blastic NK-cell lymphoma. The relationship of the EBV-positive cases with nasal NK-cell tumours has yet to be clarified.

Enriched HLA-DQ3 phenotype and decreased class I major histocompatibility complex antigen expression in recurrent respiratory papillomatosis.

Respiratory papillomas, caused by human papillomaviruses, are benign tumors that recur following removal. We evaluated immune function and major histocompatibility complex (MHC) phenotype and expression in these patients. MHC-independent immune function appeared normal. The frequency of peripheral blood MHC class II phenotypes was highly enriched for DQ3 and DR11, one split of DR5. Class I MHC antigen expression on papilloma tissue was markedly reduced. Together, these phenomena may facilitate papillomavirus evasion of the cellular immune response.

Antibody response to a synthetic peptide derived from the human papillomavirus type 6/11 L2 protein in recurrent respiratory papillomatosis: correlation between Southern blot hybridization, polymerase chain reaction, and serology.

Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age- and sex-matched controls were tested for the presence of HPV-specific antibodies using a synthetic peptide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV-specific antibody level and the number of surgery-necessitating recurrences.

Prevalence of serum IgG and IgM antibodies against Epstein-Barr virus capsid antigen in Indian patients with respiratory tract carcinomas.

Antibodies against Epstein-Barr virus capsid antigen (EBV-VCA) were measured in sera of Indian patients with respiratory tract carcinomas (lung, larynx, laryngopharynx, nasopharynx) using a commercial enzyme linked immunosorbent assay (ELISA) kit. Nineteen out of thirty-three (19/33) lung cancer, 12/22 laryngeal cancer, 11/16 laryngopharyngeal cancer, 6/9 nasopharyngeal carcinoma cases and 3/29 matched controls were positive for IgG antibody to VCA. None were positive for the IgM antibody to VCA. The antibody positivity was evaluated on the basis of immune status ratio (ISR) of the sera as described in the manual of the kit. Prevalence of the antibody was more pronounced among smokers with laryngeal and laryngopharyngeal carcinoma than among lung cancer cases. The results indicated that the antibody positivity rate among the patients declined with the advanced stages of the diseases. Serum IgG and IgM levels were raised in the patients who were tested positive for the viral antibody.

Antibodies in patients with recurrent respiratory papillomatosis treated with lymphoblastoid interferon.

Serum specimens from 53 evaluable patients enrolled in a clinical trial of lymphoblastoid interferon in recurrent respiratory papillomatosis were screened for the presence of interferon-binding antibodies by an indirect enzyme immunoassay and evaluated for neutralizing antibody measured as the inhibition of antiviral activity. Immunoglobulin G antibodies that specifically bound lymphoblastoid interferon were detected in 66% (35 of 53) of patients; neutralizing antibody was detected in 11 of the 35 patients having binding antibody (and in none of the patients who were negative for binding antibody). The incidence of detectable neutralizing antibody in this study population was 20.8% (11 of 53), which is markedly higher than in previous reports of lymphoblastoid interferon in patients with other diseases (i.e., less than 1% incidence). The cumulative dose received at the time of detection of neutralizing antibody ranged from 163 to 385 MU per square meter of body surface. Neutralizing antibody was detectable at a median time of 120 days after initiation of interferon therapy, and binding antibody appeared earlier in those patients (median 59 days) than in patients in whom only binding antibody was produced (median 116 days). Despite the tendency of binding antibody to appear either in patients in whom neutralizing antibody was eventually formed, the detection of binding antibody was not necessarily predictive of the subsequent development of neutralizing antibodies. Binding antibody persisted after neutralizing antibodies had become undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of exogenous interferon on the in vitro production of alpha- and gamma-interferon by the lymphocytes of patients with respiratory papillomatosis]. / Vliianie ékzogennogo interferona na produktsiiu al'fa- i gamma-interferona in vitro limfotsitami bol'nykh respiratornym papillomatozom.

The influence of various interferon concentrations in vitro on alpha- and gamma-interferon production by lymphocytes of children suffering from respiratory papillomatosis was studied for optimization of interferon therapy. Most of the children with clinical improvement after interferon treatment showed proportional dependence of alpha-interferon production upon exogenous interferon concentrations and stability of gamma-interferon production. In children without clinical improvement, initial production of both interferons was reduced significantly in the absence of IF in the medium, but no changes occurred when exogenous interferon was present in the medium. The test proposed here may be used for the determination of patients' sensitivity to interferon and for individualization of interferon treatment schedules in other long-lasting recurrent diseases.

[Natural killer activity in respiratory papillomatosis and its changes after interferon treatment]. / Aktivnost' estestvennykh killerov pri respiratornom papillomatoze i ee izmeneniia posle lecheniia interferonom.

A method to determine changes in the activity of natural killers (NK) in treatment of juvenile respiratory papillomatosis in children and correlation with the clinical course of the disease was developed and studies with whole blood specimens were carried out before interferon (IF) treatment and after it. Initially high levels of NK activity and their subsequent decline were observed in the cases where IF treatment had no effect, whereas initially low levels with subsequent increase after treatment or normal levels remaining unchanged in the course of treatment were typical of the patients manifesting some clinical effect of the treatment. These studies confirm the possibility of the modulating effect of IF, and the method may be used in mass surveys and working with children.