Phase I study of α-galactosylceramide-pulsed antigen presenting cells administration to the nasal submucosa in unresectable or recurrent head and neck cancer

Background Human Vα24 natural killer T (NKT) cells are activated by the specific ligand, α-galactosylceramide (α-GalCer), in a CD1d-dependent manner. Potent anti-tumor activity of activated NKT cells has been previously demonstrated. Methods We conducted a phase I study with α-GalCer-pulsed antigen presenting cells (APCs) administered in the nasal submucosa of patients with head and neck cancer, and evaluated the safety and feasibility of such a treatment. Nine patients with unresectable or recurrent head and neck cancer received two treatments 1 week apart, of 1 × 10⁸ of α-GalCer-pulsed autologous APCs into the nasal submucosa. Results During the clinical study period, no serious adverse events (Common Terminology Criteria for Adverse Events version 3.0 greater than grade 3) were observed. After the first and the second administration of α-GalCer-pulsed APCs, an increased number of NKT cells was observed in four patients and enhanced natural killer activity was detected in the peripheral blood of eight patients. Conclusion The administration of α-GalCer-pulsed APCs into the nasal submucosa was found to be safe and induce anti-tumor activity in some patients.     

Exposure or cancer predisposition? Cytogenetic examination of head and neck squamous cancer patients

Search of different biomarkers is one of the most important demands of the national cancer prevention programme. We examined the usefulness of bleomycin sensitivity assay, whether it serves as a biomarker of individual sensitivity and risk for head and neck cancer under our environmental conditions. The test is based on the measurement of the means of chromatid breaks induced by bleomycin in vitro in a single lymphocyte (break/cell=b/c). 156 head and neck cancer patients were matched not only with 295 healthy controls (146 non-smokers and 149 smokers), but also with 51 strong alcoholic and smoking patients with liver disease whose lifestyle did not differ from that of the cancer patients. The aberrant cell frequency of cancer patients (2.85%), alcoholics (2.82%) and healthy smokers (2.81%) was similar and higher (p<0.03) than the values of non-smoker controls (2.25%). Thus, the results of conventional chromosome analysis indicate the effect of exposure to mutagens, derived mainly from smoking. Mutagen sensitivity measured by the bleomycin assay was significantly higher in both the cancer- (1.13 b/c) and the alcoholic patients (1.29 b/c) compared with smoker (1.04 b/c) and non-smoker controls (0.98 b/c). The bleomycin sensitivity assay, therefore, seems to be the biomarker not only for the cancer, but also for a disease of the same aetiology such as alcohol-related liver disease. However, the method is not suitable for the assessment of individual cancer risk due to overlapping of b/c values with those of controls. The proportion of mutagen sensitive persons in the group of Hungarian controls is 42-49%, which is two-fold of those in the US and Western Europe. When we estimate the cancer risk, the results of bleomycin sensitivity assay are equivocal under our experimental conditions, and they must be applied cautiously even in combination with the results of chromosome analysis.     

How many copies of GSTM1 and GSTT1 are associated with head and neck cancer risk?

Purpose: GSTM1 and GSTT1 present a polymorphism that can drive complete gene deletions. The current methodology can powerfully determine GSTM1 and GSTT1 copy number variations (CNVs), which may clarify the real contribution of each gene copies to the cellular detoxification process and tumour risk. However, only analysing the presence/absence of these genes yielded controversial results for several disorders, including cancer. Because head and neck cancer (HNC) is becoming a serious global health problem, this study determined the CNVs of GSTM1 and GSTT1 in an HNC case-control population and investigated the possible association between gene copy numbers and tumour risk.

Methods: CNV was evaluated by (Ct) 2^?ΔΔCt qPCR methodology in 619 HNC patients and 448 patients with no tumour diagnosis.

Results: The genes copy number range was 0–3. The CNV of GSTM1 and GSTT1 frequencies were similar between the cases and control. Thus, none copy of GSTM1 and GSTT1 were associated with HNC risk. Notwithstanding, one copy of both genes had higher frequencies among individuals who carried GSTM1 and GSTT1.

Conclusions: One copy number of GSTM1 and GSTT1 presented a higher frequency among carrier genes, but the CNV of GSTM1 and GSTT1 was not associated with HNC risk.     

Activation of p53-p21 is closely associated with the acquisition of resistance to apoptosis caused by β1-integrin silencing in head and neck cancer cells

The issue of whether aberrant expression of β1-integrin is associated with cancer progression and development of resistance to cytotoxic therapy is of considerable interest. Studies to date have shown that the anchorage-independent survival of cancer is attributed, in part, to epithelial-to-mesenchymal transition (EMT). Here, we have reported a novel alternative mechanism of anchorage-independent survival of cancer cells. Cell lines derived from head and neck cancer patients (AMC-HN-3 and AMC-HN-9) and the well-known EMT cancer cell line, MDA-MB231, were examined. The EMT features of AMC-HN-9 cells were comparable to those of MDA-MB231, whereas AMC-HN-3 cells showed no EMT characteristics. Although the pattern and degree of β1-integrin expression were similar in all three cell lines, sensitivities of the cells to β1-integrin knockdown with small interfering RNA (siRNA) were different. Cancer cells with no EMT features underwent cell death to a more significant extent following β1-integrin silencing than those with EMT. Intriguingly, we observed reactive activation of the p53-p21 pathway after β1-integrin silencing in AMC-HN-9 cells lacking an apparent cell death response. Simultaneous knockdown of wild-type p53 and β1-integrin in this cell line promoted cell death. Our data collectively indicate that β1-integrin-related cell death is closely associated with EMT phenotypes and activation of the p53-p21 pathway is partly involved in the acquisition of resistance to apoptosis induced by β1-integrin silencing. Further clarification of the mechanisms underlying p53 integration with β1-integrin signaling may facilitate the development of novel anti-cancer strategies.     

Immunological modulation by 1α,25-dihydroxyvitamin D3 in patients with squamous cell carcinoma of the head and neck

Prior studies showing that treatment of head and neck squamous cell carcinoma (HNSCC) patients with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] stimulated intratumoral immune infiltration were extended to analysis of cytokine profiles in the periphery and in oral tissues. Most prominent was the disparity between cytokine levels in plasma and in either pathologically normal oral tissue or HNSCC tissue from patients that were untreated or treated with 1,25(OH)(2)D(3). Levels of IL-6 and IL-10, but not IL-2, IFN-γ or TNF-α, tended to be increased in the plasma of HNSCC patients and 1,25(OH)(2)D(3) further increased plasma levels of all of these cytokines. While these cytokines tended to be increased in HNSCC tissue, 1,25(OH)(2)D(3) resulted in variable cytokine responses that showed a general tendency toward further increased levels. Levels of IL-8 and VEGF were increased in plasma and tissue of untreated HNSCC patients, and were further increased in plasma, but not in tissues, of patients treated with 1,25(OH)(2)D(3). Levels of IL-1α and IL-1β were similar in plasma of controls and HNSCC patients, but were increased in HNSCC tissues. In contrast to that seen in plasma where 1,25(OH)(2)D(3) increased levels of IL-1α and IL-1β, this was not seen in tissue following 1,25(OH)(2)D(3) treatment. These results show a discordant relationship between systemic and intratumoral cytokine profiles and suggest a tendency of 1,25(OH)(2)D(3)to increase a multitude of cytokines within tumor tissue.     

Comparison of retroviral p15E-related factors and interferon α in head and neck cancer

Head and neck squamous cell carcinomas (HNscc) produce low-molecular-mass factors (low-M _r factors,M _r25,000), which are antigenically related to the immunosuppressive retroviral transmembrane envelope protein p15E. These P15E-related tumour factors are thought to be responsible for some immunological impairments found in these patients (particularly the defective monocyte chemotaxis). A sequential and functional homology has been reported to exist between a bioactive fragment of interferon (IFN) and the putative immunosuppressive region of retroviral p15E (CKS-17). In this study we investigated (a) a possible functional and structural relationship between p15E and IFN, and (b) the presence of and the relationship between p15E-related low-M _r factors and IFN in HNscc patients. We report the following results. (a) Recombinant human (rhu) IFN was able to inhibit monocyte chemotaxis. (b) The anti-p15E antibodies crossreacted with rhuIFN in a dot-blot technique, however, the anti-IFN antibodies did not crossreact with disrupted murine leukaemia virus (p15E source). (c) Low-M _r factors (n=8–11) prepared from the sera of HNscc patients, which inhibit the monocyte chemotactic responsiveness, could be adsorbed by the anti-p15E antibodies as well as by the anti-IFN antibodies. However, the abilities of the factors to adsorb to the two categories of antibodies (namely, anti-p15E and anti-IFN) did not correlate. (d) Immunohistochemically we found IFN-related epitopes, in almost all HNscc specimens studied (17/18), in locations distinctive from those of p15E-related factors. The anti-IFN antibodies used in this study mainly reacted with basal epithelial cells close to the basal membrane, the prickle and granular cells of the squamous cell carcinomas. The anti-p15E antibodies mainly reacted with corneal layers, the granular and prickle cells, and did not react with basal epithelial cells. Our findings suggest that the immunosuppressive factors produced by HNscc cells are heterogeneous and p15E- and/or IFN-related.     

Time dependent behaviour of the cross-linking reaction of α-chymotrypsin with glutaraldehyde

Summary Immobilization of -chymotrypsin was done by cross-linking the enzyme with glutaraldehyde. Studies were done in a batch reactor to find out the effects of the concentrations of -chymotrypsin and glutaraldehyde on the rate of particle formation, the final yield of the crosslinked particles, and the residual activity of the cross-linked enzyme. Both intra- and inter-molecular cross-linking play important role at different time course of the reaction. At the begining of the cross-linking reaction, the large drop in the activity is because of the intra-molecular cross-linking and later on the large increase in the particle formation is because of the inter-molecular cross-linking.     

Molecular characterization of head and neck cancer: how close to personalized targeted therapy?

Molecular targeted therapy in head and neck squamous cell carcinoma (HNSCC) continues to make strides, and holds much promise. Cetuximab remains the sole US FDA-approved molecular targeted therapy available for HNSCC, though several new biologic agents targeting the epidermal growth factor receptor (EGFR) and other pathways are currently in the regulatory approval pipeline. While targeted therapies have the potential to be personalized, their current use in HNSCC is not personalized. This is illustrated for EGFR-targeted drugs, where EGFR as a molecular target has yet to be individualized for HNSCC. Future research needs to identify factors that correlate with response (or lack of one) and the underlying genotype-phenotype relationship that dictates this response. Comprehensive exploration of genetic and epigenetic landscapes in HNSCC is opening new frontiers to further enlighten and mechanistically inform newer as well as existing molecular targets, and to set a course for eventually translating these discoveries into therapies for patients. This opinion offers a snapshot of the evolution of molecular subtyping in HNSCC and its current clinical applicability, as well as new emergent paradigms with implications for controlling this disease in the future.     

Conversion of pancreatic α cells into insulin-producing cells modulated by β-cell insufficiency and supplemental insulin administration

The emergence of bihormonal (BH) cells expressing insulin and glucagon has been reported under diabetic conditions in humans and mice. Whereas lineage tracing studies demonstrated that glucagon-producing α cells can be reprogrammed into BH cells, the underlying dynamics of the conversion process remain poorly understood. In the present study, we investigated the identities of pancreatic endocrine cells by genetic lineage tracing under diabetic conditions. When β-cell ablation was induced by alloxan (ALX), a time-dependent increase in BH cells was subsequently observed. Lineage tracing experiments demonstrated that BH cells originate from α cells, but not from β cells, in ALX-induced diabetic mice. Notably, supplemental insulin administration into diabetic mice resulted in a significant increase in α-cell-derived insulin-producing cells that did not express glucagon. Furthermore, lineage tracing in Ins2^Akita diabetic mice demonstrated a significant induction of α-to-β conversion. Thus, adult α cells have plasticity, which enables them to be reprogrammed into insulin-producing cells under diabetic conditions, and this can be modulated by supplemental insulin administration.     

Intravesical administration of γδ T cells successfully prevents the growth of bladder cancer in the murine model

Background  Superficial bladder cancers are usually managed with transurethral resection followed by the intravesical administration of Bacillus Calmette-Guerin which requires major histocompatibility complex (MHC) class I expression on cancer cells. Since cancer cells often loose MHC expression, a novel immunotherapy such as MHC-unrestricted γδ T cell therapy is desired. Objective  To clarify the relationship between the expression of MHC class I and clinicopathological features in bladder cancer patients, and investigate the effects of the administration of intravesical γδ T cells on bladder cancer. Methods  Samples from 123 patients who had undergone either transurethral resection or radical cystectomies were examined for MHC expression and the relationship between this and the clinicopathological features was analyzed statistically. The in vitro and in vivo effects of γδ T cells expanded by zoledronic acid (ZOL) against several types of cancer cell line and an orthotopic bladder cancer murine model which was pretreated with ZOL were investigated. Results  MHC-diminished superficial bladder cancer was significantly more progressive than MHC-conservative bladder cancer (P = 0.047). In addition, there was a significant association between diminished MHC expression and poor disease free survival (P = 0.041) and overall survival (P = 0.018) after radical cystectomy. In vitro, all of the cell lines pretreated with 5-μM ZOL showed a marked increase in sensitivity to lysis by γδ T cells. Moreover, intravesical administration of γδ T cells with 5-μM ZOL significantly demonstrated antitumor activity against bladder cancer cells in the orthotopic murine model (P < 0.001), resulting in prolonged survival. Conclusion  The present murine model provides a potentially interesting option to develop immunotherapy using γδ T cells for bladder cancer in human. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Yuasa and K. Sato contributed equally to the study.     

Disorders of the spatiotemporal organization of the brain’s bioelectrical activity in patients with different depressions of consciousness after severe head injury

Specific changes in the bioelectrical activity of the brain have been found in 27 patients with different levels of posttraumatic consciousness depression (stupor, spoor-coma I, coma II) by the methods of cross correlation, and coherence and factor EEG analysis. The changes in activity of the morphofunctional systems of intracerebral integrations were expressed partly in a decrease in the nonspecific activating effects from brainstem structures, which was reflected in an increase in the slow wave activity along with a considerable decrease in the level of EEG coherence in the α and β ranges. The observed depression of the system’s organization of the interrelations of the bioelectrical brain activity in the frontal and occipital regions of both hemispheres could be due to a decreased activity of the associative systems of intercortical and thalamocortical integration. The results suggest a certain facilitation of the activity of the system providing direct interhemispheric connections through the corpus calossum and other commissural tracts of the telencephalon as a consequence of the depression of the mesodiencephalon structures (which normally largely contribute to the synergistic interhemispheric interaction via synchronous ascending effects on the cortex of both hemispheres). This results in steady, reciprocal, and almost antiphase relations of slow wave activity in symmetrical areas of the hemispheres.     

Locoregional immunotherapy of head and neck cancers utilizing allogeneic spleen cells — a report of 2 cases

Frozen-stored human spleen cells (SC) cultured with streptococcus preparation OK-432 acquired direct cytotoxicity to autologous as well as allogeneic tumor cells. The activated cells started to produce cytocidal cytokine TGIF, which is distinct from previously known cytokines.We examined the possibility of allogeneic adoptive immunotherapy (AIT) using these OK-432-stimulated spleen cells (OK-SC) in two cancer patients. Rapid necrosis of cancer tissue and remarkable decreases of tumor markers in tumor effusion were observed. There were no severe side effects.     

Is volumetric modulated arc therapy with constant dose rate a valid option in radiation therapy for head and neck cancer patients?

Background

Intensity-modulated radiotherapy (IMRT) improves dose distribution in head and neck (HN) radiation therapy. Volumetric-modulated arc therapy (VMAT), a new form of IMRT, delivers radiation in single or multiple arcs, varying dose rates (VDR-VMAT) and gantry speeds, has gained considerable attention. Constant dose rate VMAT (CDR-VMAT) associated with a fixed gantry speed does not require a dedicated linear accelerator like VDR-VMAT. The present study explored the feasibility, efficiency and delivery accuracy of CDR-VMAT, by comparing it with IMRT and VDR-VMAT in treatment planning for HN cancer.

High expression of integrin αvβ3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors

The cell line OVCAR-4 was recently ranked as one of the most representative cell lines for high grade serous ovarian cancer (HGSOC). However, little work has been done to assess the susceptibility of OVCAR-4 cells and tumors to the more common types of molecular targeting. Proteome profiles suggest OVCAR-4 express high levels of integrin αvβ3 receptors. Using flow cytometry with fluorescent antibodies we determined that OVCAR-4 cells have high number of integrin αvβ3 receptors ([9.8?±?2.5]?×?10⁴/cell) compared to the well-characterized cell line U87-MG ([5.2?±?1.4]?×?10⁴/cell). However, OVCAR-4 cells also have roughly three times the surface area of U87-MG cells, so the average αvβ3 receptor density is actually lower (11?±?3 versus 18?±?6?receptors/µm²). A series of new fluorescent molecular probes was prepared with structures comprised of a deep-red squaraine fluorophore (～680?nm emission) covalently attached to zero, one, or two cyclic pentapeptide cRGD sequences for integrin targeting. Microscopy studies showed that uptake of the divalent probe into cultured OVCAR-4 cells was 2.2?±?0.4 higher than the monovalent probe, which in turn was 2.2?±?0.4 higher than the untargeted probe. This probe targeting trend was also seen in OVCAR-4 mouse tumor models. The results suggest that clinically relevant OVCAR-4 cells can be targeted using molecular probes based on αvβ3 integrin receptor antagonists such as the cRGD peptide. Furthermore, deep-red fluorescent cRGD-squaraine probes have potential as targeted stains of cancerous tissue associated with HGSOC in surgery and pathology settings.     

γδ T cells cultured with artificial antigen-presenting cells and IL-2 show long-term proliferation and enhanced effector functions compared with γδ T cells cultured with only IL-2 after stimulation with zoledronic acid

Background aimsImmunotherapeutic approaches using γδ T cells have emerged as the function of γδ T cells in tumor surveillance and clearance has been discovered. In vitro expansion methods of γ9δ2 T cells have been based on phosphoantigens and cytokines, but expansion methods using feeder cells to generate larger numbers of γδ T cells have also been studied recently. However, there are no studies that directly compare γδ T cells cultured with phosphoantigens with those cultured with feeder cells. Therefore, this study aimed to compare the expansion, characteristics and effector functions of γδ T cells stimulated with K562-based artificial antigen-presenting cells (aAPCs) (aAPC-γδ T cells) and γδ T cells stimulated with only zoledronic acid (ZA) (ZA-γδ T cells).MethodsPeripheral blood mononuclear cells were stimulated with ZA for 7 days, and aAPC-γδ T cells were stimulated weekly with K562-based aAPCs expressing CD32, CD80, CD83, 4-1BBL, CD40L and CD70, whereas ZA-γδ T cells were stimulated with only IL-2. Cultured γδ T cells were analyzed by flow cytometry for the expression of co-stimulatory molecules, activating receptors and checkpoint inhibitors. Differentially expressed gene (DEG) analysis was also performed to determine the difference in gene expression between aAPC-γδ T cells and ZA-γδ T cells. In vitro cytotoxicity assay was performed with calcein AM release assay, and in vivo anti-tumor effect was compared using a U937 xenograft model.ResultsFold expansion on day 21 was 690.7 ± 413.1 for ZA-γδ T cells and 1415.2 ± 1016.8 for aAPC- γδ T cells. Moreover, aAPC-γδ T cells showed continuous growth, whereas ZA-γδ T cells showed a decline in growth after day 21. The T-cell receptor Vγ9⁺δ2⁺ percentages (mean ± standard deviation) on day 21 were 90.0 ± 2.7% and 87.0 ± 4.5% for ZA-γδ T cells and aAPC-γδ T cells, respectively. CD25 and CD86 expression was significantly higher in aAPC-γδ T cells. In DEG analysis, aAPC-γδ T cells and ZA-γδ T cells formed distinct clusters, and aAPC-γδ T cells showed upregulation of genes associated with metabolism and cytokine pathways. In vitro cytotoxicity revealed superior anti-tumor effects of aAPC-γδ T cells compared with ZA-γδ T cells on Daudi, Raji and U937 cell lines. In addition, in the U937 xenograft model, aAPC-γδ T-cell treatment increased survival, and a higher frequency of aAPC-γδ T cells was shown in bone marrow compared with ZA-γδ T cells.ConclusionsOverall, this study demonstrates that aAPC-γδ T cells show long-term proliferation, enhanced activation and anti-tumor effects compared with ZA-γδ T cells and provides a basis for using aAPC-γδ T cells in further studies, including clinical applications and genetic engineering of γδ T cells.     

Presence of activated lymphocytes in the urine of patients with superficial bladder cancer after intravesical immunotherapy with bacillus Calmette-Guérin

Summary To study the mode of action of intravesical bacillus Calmette-Guérin (BCG) immunotherapy in the prevention and cure of superficial bladder cancer, flow-cytofluorometric analysis of the cellular immunological reaction in the urine of patients was performed. Fresh urinederived leucocytes were obtained from eight patients before (t ₀) and 24 h (t ₂₄) and 48 h (t ₄₈) after repeated intravesical BCG instillations (at least 5 instillations). For two patients urine-derived leucocytes were investigated at the first BCG instillation. The number of leucocytes in the urine was markedly increased 24 h after repeated BCG instillations, indicating a local cellular immunological reaction induced by BCG. The mean number of cells per milliliter of urine at that time was 2.9×10⁶±3.6×10⁶ (n = 8). These leucocytes consisted mainly of granulocytes (75±11%,n = 8). In addition monocytes/macrophages (4±2%,n = 8) and T lymphocytes were present (1±1%,n = 5). The relative increase of monocytes/macrophages in the urine after BCG application tended to be higher compared to the other leucocyte subtypes. As T lymphocytes may play an important role in the BCG-mediated antitumour activity, subsets of lymphocytes were further characterized att ₀,t ₂₄, andt ₄₈ after repeated BCG instillations. The lymphocyte population consisted mainly of T cells (86% CD3⁺,t ₀). Most of the T cells were CD4⁺ (helper/inducer) and were significantly decreased at 48 h (62±9% att ₀ vs 49±6% att ₄₈). Lymphocytes partly expressed HLA-DR antigens (44%,t ₀). The percentage of lymphocytes with interleukin-2 (IL-2) receptors (CD25⁺) was significantly increased at 24 h and 48 h, compared to pre-instillation values (19±11% and 10±4% vs 3±3% respectively). Natural killer cells (CD 16⁺ and/or CD56⁺) and B cells (CD 19⁺) were less numerous (10% and 19% att ₀ respectively). After the first BCG instillation the increase in the number of leucocytes in urine seemed to be less compared to the numbers after repeated BCG instillations. Lymphocytes could not be detected in the urine collected before or after the first BCG instillation. In conclusion, we demonstrated the presence of considerable numbers of leucocytes in the urine 24 h after repeated BCG instillations, i.e. shortly after immunological activation. The antigen expression of the lymphocytes suggested that they may represent the lymphocytes in the bladder wall. Expression of HLA-DR and IL-2 receptors on lymphocytes indicated activation of T cells by the intravesical BCG treatment. These leucocytes may be useful for functional studies, which are essential to elucidate the actual effector mechanism(s) in the mode of action of BCG against superficial bladder cancer in man.     

Effectiveness of the No action level protocol for head & neck patients – Time considerations

BackgroundThe No Action Protocol (NAL) was used to diminish the systematic set-up error. Recently, owing to the development of image registration technologies, the on-line positioning control is more often used. This method significantly reduces the CTV–PTV margin at the expense of the lengthening of a treatment session. The efficiency of NAL in decreasing the total treatment time for Head&Neck patients was investigated.MethodsResults of set-up control of 30 patients were analyzed. The set-up control was carried out on-line. For each patient and each fraction, the set-error and the time needed for making the set-up control procedure were measured. Next, retrospectively, the NAL was applied to this data. The number of initial errors (without interventions) and after NAL protocol were compared in terms of errors larger than 3 and 4 mm. The average and total time used for portal control was calculated and compared.ResultsThe number of setup errors in the posterior-anterior, inferior-superior, and right–left directions ≥3 mm and ≥4 mm were 98, 79, and 91 sessions and 44, 38 and 30 sessions out of 884 sessions. After NAL protocol the number of errors ≥3 mm and ≥4 mm decreased to 84, 57, and 39 sessions and 31, 15 and 10 sessions, respectively. The average time needed for one set-up control was 5.1 min. NAL protocol allows saving 4049 min for the whole group.ConclusionsFor locations where the random set-up errors are small, the NAL enables a very precise treatment of patients. Implementation of this protocol significantly decreases the total treatment time.     

Verrucous carcinoma of the head and neck – not a human papillomavirus-related tumour?

Association between verrucous carcinoma (VC) of the head and neck and human papillomaviruses (HPV) is highly controversial. Previous prevalence studies focused mostly on α‐PV, while little is known about other PV genera. Our aim was to investigate the prevalence of a broad spectrum of HPV in VC of the head and neck using sensitive and specific molecular assays. Formalin‐fixed, paraffin‐embedded samples of 30 VC and 30 location‐matched normal tissue samples were analysed, by using six different polymerase chain reaction‐based methods targeting DNA of at least 87 HPV types from α‐PV, β‐PV, γ‐PV and μ‐PV genera, and immunohistochemistry against p16 protein. α‐PV, γ‐PV and μ‐PV were not detected. β‐PV DNA was detected in 5/30 VC (16.7%) and in 18/30 normal tissue samples (60.0%): HPV‐19, ‐24 and ‐36 were identified in VC, and HPV‐5, ‐9, ‐12, ‐23, ‐24, ‐38, ‐47, ‐49 and ‐96 in normal tissue, whereas HPV type was not determined in 2/5 cases of VC and in 6/18 normal tissue samples. p16 expression was detected in a subset of samples and was higher in VC than in normal tissue. However, the reaction was predominantly cytoplasmic and only occasionally nuclear, and the extent of staining did not exceed 75%. Our results indicate that α‐PV, γ‐PV and μ‐PV are not associated with aetiopathogenesis of VC of the head and neck. β‐PV DNA in a subset of VC and normal tissue might reflect incidental colonization, but its potential biological significance needs further investigation.     

Effects on antigen-presenting cells of short-term interaction with the human host defence peptide β-defensin 2

β-Defensins are antimicrobial peptides that exert their host-defence functions at the interface between the host and microbial biota. They display a direct, salt- and medium-sensitive cidal activity, in vitro, against a broad spectrum of bacteria and fungi, and there is increasing evidence that they also play a role in alerting and enhancing cellular components of innate and adaptive immunity. Their interaction with biological membranes plays a central role in both of these types of activities. In the present study, we have investigated the interaction of fluorescently labelled hBD2 (human β-defensin 2) with monocytes, macrophages and iDCs (immature dendritic cells), observing a differential capacity to be rapidly internalized into these cells. Complementary microscopy techniques [TEM (transmission electron microscopy), optical microscopy and IR microspectroscopy] were used to explore the functional and biological implications of these interactions on iDCs. Short-term exposure to the peptide resulted in significant alterations in membrane composition and re-organization of the endomembrane system, with the induction of degranulation. These events may be associated with the antigen-presenting activities or the chemotaxis of iDCs, which appears to occur via both CCR6 (CC chemokine receptor 6)-dependent and -independent mechanisms.