Biofilm-producing Bacteria and Quality of Life after Endoscopic Sinus Surgery in Patients with Chronic Rhinosinusitis with Nasal Polyposis.

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common health disorders in humans and has a major impact on health-related quality of life (HRQoL). Of the many factors contributing to the etiology of CRS, less is known about the correlation between CRS and bacterial biofilms and their impact on HRQoL. OBJECTIVE: The aim of this prospective study was to investigate the relationship between biofilm-producing bacteria and patients' objective findings and HRQoL. METHODS: Forty-eight patients with CRSwNP were enrolled in a 12-month prospective study. The Lund-Mackay (LM) CT and endoscopic Lund-Kennedy (LK) scores were obtained before endoscopic sinus surgery (ESS), and patients completed the HRQoL instruments: the 22-item Sinonasal Outcome Test (SNOT-22), the 36-item Short Questionnaire (SF-36), and the visual analog scale (VAS). A sinus culture was obtained at ESS, bacteria were isolated, and in vitro quantification of the biofilm was performed. The LK score and HRQoL were determined postoperatively at months 1, 3, 6, and 12. RESULTS: The most common bacterial isolates in patients with CRSwNP were Staphylococcus aureus (28%), coagulase-negative staphylococci (52%), and Pseudomonas aeruginosa (8%). Preoperatively, the highest LM and LK scores were found in patients with strong biofilm producers. Postoperative LK scores were significantly reduced in all patients. Postoperative VAS scores were significantly reduced from month 1 to month 12 postoperatively. Patients with strong biofilm producers had significantly worse nasal blockage, secretion, headache, facial pressure and pain, and loss of smell preoperatively, compared to patients with low biofilm producers. The most significant reduction in preoperative scores SNOT-22 and SF-36 (excluding physical functioning) was seen in patients with S. aureus and P. aeruginosa. CONCLUSIONS: Patients with strong biofilm producers had higher LK and LM scores preoperatively, and greater improvement in LK and HRQoL scores postoperatively. Microbiologic surveillance of all CRS patients is recommended.

In Vitro Antibiofilm Effect of N-Acetyl-L-cysteine/Dry Propolis Extract Combination on Bacterial Pathogens Isolated from Upper Respiratory Tract Infections.

Bacterial biofilms play an important role in the pathogenesis of chronic upper respiratory tract infections. In addition to conventional antimicrobial therapy, N-acetyl-L-cysteine (NAC) and propolis are dietary supplements that are often recommended as supportive therapy for upper respiratory tract infections. However, no data on the beneficial effect of their combination against bacterial biofilms can be found in the scientific literature. Therefore, the aim of our study was to investigate the in vitro effect of N-acetyl-L-cysteine (NAC) and dry propolis extract in fixed combinations (NAC/dry propolis extract fixed combination) on biofilm formation by bacterial species isolated from patients with chronic rhinosinusitis, chronic otitis media, and chronic adenoiditis. The prospective study included 48 adults with chronic rhinosinusitis, 29 adults with chronic otitis media, and 33 children with chronic adenoiditis. Bacteria were isolated from tissue samples obtained intraoperatively and identified using the MALDI-TOF Vitek MS System. The antimicrobial activity, synergism, and antibiofilm effect of NAC/dry propolis extract fixed combination were studied in vitro. A total of 116 different strains were isolated from the tissue samples, with staphylococci being the most frequently isolated in all patients (57.8%). MICs of the NAC/dry propolis extract fixed combination ranged from 1.25/0.125 to 20/2 mg NAC/mg propolis. A synergistic effect (FICI ≤ 0.5) was observed in 51.7% of strains. The majority of isolates from patients with chronic otitis media were moderate biofilm producers and in chronic adenoiditis they were weak biofilm producers, while the same number of isolates in patients with chronic rhinosinusitis were weak and moderate biofilm producers. Subinhibitory concentrations of the NAC/propolis combination ranging from 0.625-0.156 mg/mL to 10-2.5 mg/mL of NAC combined with 0.062-0.016 mg/mL to 1-0.25 mg/mL of propolis inhibited biofilm formation in all bacterial strains. Suprainhibitory concentrations ranging from 2.5-10 mg/mL to 40-160 mg/mL of NAC in combination with 0.25-1 mg/mL to 4-16 mg/mL of propolis completely eradicated the biofilm. In conclusion, the fixed combination of NAC and dry propolis extract has a synergistic effect on all stages of biofilm formation and eradication of the formed biofilm in bacteria isolated from upper respiratory tract infections.

Bulky Recurrent Juvenile Nasopharyngeal Angiofibroma.

Juvenile nasopharyngeal angiofibroma is a rare and highly vascularized tumor that accounts for 0.05 to 0.5% of all head and neck neoplasms. The aim of this work was to present a case of a large recurrent juvenile nasopharyngeal angiofibroma coexisting with a facial lipoma in a 16-year-old boy. The patient was referred to our institution because of frequent unilateral epistaxis. Computed tomography revealed a hypervascular tumor with ethmoidal cell destruction and spread to the nasopharynx. Operative treatment of nasal cavity tumors was carried out using a transpalatal approach. After 6 months, the recurrence of the angiofibroma was verified radiologically. Primary as well as secondary surgical procedures were assisted with an endoscopic procedure. Accurate preoperative assessment and staging are essential for choosing a surgical procedure. The primary treatment is surgical excision. Early diagnosis, accurate staging, adequate treatment, and regular postoperative follow-up are essential in the treatment of these lesions.

Savory, Oregano and Thyme Essential Oil Mixture (HerbELICO®) Counteracts Helicobacter pylori.

Fifty percent of the world's population is infected with Helicobacter pylori, which can trigger many gastrointestinal disorders. H. pylori eradication therapy consists of two to three antimicrobial medicinal products, but they exhibit limited efficacy and may cause adverse side effects. Alternative therapies are urgent. It was assumed that an essential oil mixture, obtained from species from genera Satureja L., Origanum L. and Thymus L. and called the HerbELICO® essential oil mixture, could be useful in H. pylori infection treatment. HerbELICO® was analyzed by GC-MS and assessed in vitro against twenty H. pylori clinical strains isolated from patients of different geographical origins and with different antimicrobial medicinal products resistance profiles, and for its ability to penetrate the artificial mucin barrier. A customer case study included 15 users of HerbELICO®liquid/HerbELICO®solid dietary supplements (capsulated HerbELICO® mixture in liquid/solid form). Carvacrol and thymol were the most dominant compounds (47.44% and 11.62%, respectively), together with p-cymene (13.35%) and γ-terpinene (18.20%). The minimum concentration required to inhibit in vitro H. pylori growth by HerbELICO® was 4-5% (v/v); 10 min exposure to HerbELICO® was enough to kill off the examined H. pylori strains, while HerbELICO® was able to penetrate through mucin. A high eradication rate (up to 90%) and acceptance by consumers was observed.

Immune Cell and Biochemical Biomarkers in Advanced Laryngeal Cancer.

Objective: The aim of this study was to evaluate cell and biochemical biomarkers and establish their prognostic value in patients with advanced laryngeal cancer. Material and Methods: A prospective study included 52 patients with advanced laryngeal carcinoma surgically treated at the tertiary referral center. Tumor tissue was immunohistochemically stained for T-cell markers (CD4 and CD8), and levels of cytokines (IL-6 and IL-8) and C-reactive protein were analyzed from blood samples. Results: Overall 3-year survival (OS) of patients included in the study was 69.2% and the disease specific survival (DSS) 72.5%. Higher expression of CD4+ and CD8+ were significant prognostic factors with positive impact on both OS and DSS in univariate analysis, but not in multivariate analysis. Levels of IL-8 were a significant predictor of 3-year OS and DSS survival in patients with advanced laryngeal cancer but not levels of IL-6 and CRP values. Conclusion: Though high expression of CD4 and CD8 were demonstrated in the tumor tissue, but their prognostic role was not established. Higher values of IL-8 proved to be significant negative predictor of DSS. This could further collaborate the inclusion of combination of biomarkers in assessment of favorable treatment choice in patients with advanced laryngeal carcinoma.

Otitis media with effusion as an initial manifestation of granulomatosis with polyangiitis.

Granulomatosis with polyangiitis is a systemic vasculitis of unknown etiology, characterized by necrotizing granulomas. It is an autoimmune disease affecting small- and medium-sized vessels of upper and lower respiratory tract, kidneys, and other organs. We described a case of a patient with otitis media with effusion as the first manifestations of granulomatosis with polyangiitis. A 54-year-old female presented as an urgent case with history of a severe otalgia, hearing loss, vertigo, and fever. The patient was treated with diagnosis of otitis media with effusion and acute rhinosinusitis, but without significant success. She developed an acute kidney dysfunction as a sign of glomerulonephritis with rapidly progressive renal failure. Diagnosis of granulomatosis with polyangiitis was confirmed after the histopathological analysis of kidney tissue, not by analysis of middle ear and paranasal sinus mucosa specimens. The patient was treated according to generally accepted protocol, and over time, there was an almost complete recovery.

Fecal microbiota composition associates with the capacity of human peripheral blood monocytes to differentiate into immunogenic dendritic cells in vitro.

Although promising for active immunization in cancer patients, dendritic cells (DCs) vaccines generated in vitro display high inter-individual variability in their immunogenicity, which mostly limits their therapeutic efficacy. Gut microbiota composition is a key emerging factor affecting individuals' immune responses, but it is unknown how it affects the variability of donors' precursor cells to differentiate into immunogenic DCs in vitro. By analyzing gut microbiota composition in 14 healthy donors, along with the phenotype and cytokines production by monocyte-derived DCs, we found significant correlations between immunogenic properties of DC and microbiota composition. Namely, donors who had higher α-diversity of gut microbiota and higher abundance of short-chain fatty acid (SCFAs) and SCFA-producing bacteria in feces, displayed lower expression of CD1a on immature (im)DC and higher expression of ILT-3, costimulatory molecules (CD86, CD40) proinflammatory cytokines (TNF-α, IL-6, IL-8) and IL-12p70/IL-10 ratio, all of which correlated with their lower maturation potential and immunogenicity upon stimulation with LPS/IFNγ, a well-known Th1 polarizing cocktail. In contrast, imDCs generated from donors with lower α-diversity and higher abundance of Bifidobacterium and Collinsella in feces displayed higher CD1a expression and higher potential to up-regulate CD86 and CD40, increase TNF-α, IL-6, IL-8 production, and IL-12p70/IL-10 ratio upon stimulation. These results emphasize the important role of gut microbiota on the capacity of donor precursor cells to differentiate into immunogenic DCs suitable for cancer therapy, which could be harnessed for improving the actual and future DC-based cancer therapies.

Antibiofilm effects of amoxicillin-clavulanic acid and levofloxacin in patients with chronic rhinosinusitis with nasal polyposis.

PURPOSE: Microbial biofilms have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). The aim of our study was to evaluate in vitro effects of amoxicillin-clavulanic acid and levofloxacin on biofilm formation by bacterial species isolated from sinus tissue in patients with CRSwNP. METHODS: The sinus mucosal specimens were harvested from the upper parts and roof of ethmoid cavity of 48 patients with CRSwNP. Each sample was washed thoroughly in three separate beakers of sterile saline to remove any planktonic bacteria and further subjected to microbiology analysis. The biofilm-forming capacity of isolated strains was detected by microtiter-plate method and the effects of subinhibitory (1/2× to 1/16× MIC) and suprainhibitory concentrations (4, 8, 16, 32, and 64 µg/ml) of amoxicillin-clavulanic acid and levofloxacin on biofilm production were investigated. RESULTS: Bacterial strains were isolated in 42 (87.5%) patients: one microorganism in 80.9% and two microorganisms in 19.1% of patients. The most prevalent bacteria in CRSwNP biofilms were Staphylococcus epidermidis (34%) and S. aureus (28%) followed by S. haemolyticus (12%), Pseudomonas aeruginosa (8%), Moraxella catarrhalis (6%), Streptococcus pneumoniae (6%), and other staphylococci (6%). Subinhibitory concentrations of amoxicillin-clavulanic acid and levofloxacin significantly reduced biofilm formation (p < 0.01 and p < 0.05, respectively), with better efficacy of amoxicillin-clavulanic acid (1/2-1/8× MIC) on staphylococci and levofloxacin (1/2- 1/4× MIC) on M. catarrhalis and P. aeruginosa biofilm formation. Suprainhibitory concentrations of both tested antibiotics (4-64 µg/ml) significantly eradicated mature biofilms of staphylococci (p < 0.01). The effect of levofloxacin on eradication of staphylococcal biofilms was more noticeable, compared to the effect of amoxicillin-clavulanic acid (p < 0.01). Suprainhibitory concentrations of both tested antibiotics had no effect on eradication of previously formed M. catarrhalis and P. aeruginosa biofilms (p > 0.05). CONCLUSIONS: The amoxicillin-clavulanic acid and levofloxacin are shown to be potent antibiofilm agents in patients with CRSwNP. The effects of tested compounds depend on bacterial species and the volume of formed biofilm.

Antibiofilm effects of topical corticosteroids and intranasal saline in patients with chronic rhinosinusitis with nasal polyps depend on bacterial species and their biofilm-forming capacity.

Microbial biofilms have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). Intranasal application of corticosteroids and saline is a reliable option for their management. The aim of our study was to evaluate in vitro antibiofilm effects of corticosteroids and isotonic and hypertonic nasal saline in CRSwNP patients. The sinus mucosal specimens were harvested from the ethmoid cavity of 48 patients with CRSwNP and further subjected to hematoxylin-eosin staining and microbiology analysis. The biofilm-forming capacity of isolated bacterial strains was detected by microtiter-plate method and the effects of therapeutic doses of mometasone, fluticasone, isotonic and hypertonic saline on biofilm production were investigated. Bacterial strains were isolated in 42 (87.5%) patients: one organism in 34 (80.9%) and two organisms in 8 (19.1%). Staphylococcus epidermidis (34%) and Staphylococcus aureus (28%) were the most prevalent bacteria in biofilms of CRSwNP patients. Corticosteroids and saline solutions significantly reduced biofilm formation (p < 0.01 and p < 0.05, respectively) with better efficacy of fluticasone and isotonic nasal saline. Treatment with fluticasone, mometasone, isotonic and hypertonic nasal saline completely prevented biofilm production in 66, 50, 84 and 38% of bacterial strains, respectively. The most significant density reduction was observed in biofilm formed by Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae compared to other bacterial species (p < 0.01, p < 0.05, p < 0.05, respectively). The antibiofilm effects of corticosteroids and saline solutions also greatly depended on bacterial biomass (p < 0.05), with the most significant effect on high compared to small amount of formed biofilm. The topical steroids and nasal saline are shown to be potent antibiofilm agents in patients with CRSwNP. The effects of tested compounds depend on bacterial species and volume of formed biofilm.

Quantification of biofilm formation on silicone intranasal splints: An in vitro study.

OBJECTIVES: Biofilms are associated with persistent infections and resistant to conventional therapeutic strategies. The aim of this study was to investigate the quantity of biofilm produced on silicone intranasal splints. METHODS: Quantity of biofilm formation on silicone splints (SS) was tested on 15 strains of Staphylococcus aureus and Moraxella catarrhalis, respectively. Antimicrobial susceptibility testing was performed in accordance with European Committee on Antimicrobial Susceptibility Testing recommendations. RESULTS: All tested strains formed different amounts of biofilm on SS: 66.7% S. aureus and 93.3% M. catarrhalis were weak biofilm producers and 33.3% S. aureus and 6.7% M. catarrhalis were moderate biofilm producers. S. aureus formed significantly higher quantity of biofilm compared with M. catarrhalis (p < 0.05). Multidrug resistant S. aureus produced significantly higher amount of biofilm compared with non-multidrug resistant strains (p < 0.05). CONCLUSION: Quantity of biofilm on SS is highly dependent on bacterial species and their resistance patterns. Future studies are needed to ascertain another therapeutic option for prophylaxis prior to SS placement.

Biofilm formation on tympanostomy tubes depends on methicillin-resistant Staphylococcus aureus genetic lineage.

Bacterial biofilm formation has been implicated in the high incidence of persistent otorrhoea after tympanostomy tube insertion. The aim of the study was to investigate whether biofilm formation on tympanostomy tubes depends on the genetic profile of methicillin-resistant Staphylococcus aureus (MRSA) strains. Capacity of biofilm formation on fluoroplastic tympanostomy tubes (TTs) was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on TTs. Tested MRSA strains were classified into SCCmec type I (36.7 %), III (23.3 %), IV (26.7 %) and V (13.3 %), agr type I (50 %), II (36.7 %) and III (13.3 %), and 5 clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on TTs was as following: 13.3 % of strains belonged to the category of no biofilm producers, 50 % to the category of weak biofilm producers and 36.7 % to moderate biofilm producers. There was a statistically significant difference between CC, SCCmec and agr types and the category of biofilm production on TTs tubes (p < 0.001): CC5, SCCmecI type and agrII type with a moderate amount of biofilm, and CC8 and agrI type with a low amount of biofilm. Biofilm formation by MRSA on TTs is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related post-tympanostomy tube otorrhea.

Fast dendritic cells matured with Poly (I:C) may acquire tolerogenic properties.

BACKGROUND AIMS: Because of the labor-intensive and time-consuming conventional protocols for the generation of dendritic cells (DCs) as the most promising tools for anti-cancer therapy that enable the induction of a T-helper (Th)1-mediated anti-tumor immune response, the use of short-term protocols has been proposed. However, data on the applicability of such protocols in cancer immunotherapy are quite limited. METHODS: We compared the phenotypic and functional capability of fast DCs (fDCs) differentiated for 24 h and then matured for 48 h with Poly (I:C), a strong Th1-promoting agent, with donor-matched conventional DCs (cDCs) differentiated for 5 days and matured likewise. RESULTS: Of 12 donors tested, we identified seven whose monocytes failed to develop into immunogenic DCs through the use of fDC protocol, on the basis of incomplete downregulation of CD14, low expression of CD1a and macrophage-like morphology. Such fDCs have significantly lower expression of CD83, CD86, CCR7 and CD40, weaker allo-stimulatory Th1- and Th17-polarizing capacity caused by poor production of interleukin (IL)-12p70 and IL-23 and high production of IL-10, and prominent Th2-polarizing capacity, compared with donor-matched cDCs. Furthermore, such fDCs had tolerogenic properties as judged by higher expression of indolamine dioxigenase-3, IDO-1 and IL-1ß and induction of a higher percentage of CD4(+)CD25(+)FoxP3(+) T cells. These findings correlated with increased transforming growth factor (TGF)-ß production by fDC-primed CD3(+)T cells and their stronger anti-proliferative capacity. CONCLUSIONS: We emphasize that although fDCs could probably be applied as an alternative to cDCs for cancer therapy, the fDC protocol should not be applied to donors whose DCs acquire tolerogenic capabilities.

Tumor necrosis factor-α promotes survival and phenotypic maturation of poly(I:C)-treated dendritic cells but impairs their Th1 and Th17 polarizing capability.

BACKGROUND AIMS: Toll-like receptor (TLR)-3 synthetic agonist polyinosinic-polycytidylic acid (poly(I:C)) is a promising agent for dendritic cell (DC)-based anti-tumor vaccines because of its ability to induce a strong maturation of DCs, but such an effect is followed by stimulation of DC apoptosis. Tumor necrosis factor (TNF)-α may promote the survival of poly(I:C)-stimulated DCs, but it is not known in detail how this combination affects the maturation and polarization capacity of monocyte-derived (Mo)DCs. METHODS: Immature MoDCs, generated from human monocytes, were treated with different concentrations of poly(I:C) combined with TNF-α, and the effect on survival, phenotype, production of cytokines, allostimulatory and Th polarization capacity was assessed after 24 and 48 h. RESULTS: We showed that TNF-α inhibited the dose-dependent pro-apoptotic effect of poly(I:C). However, TNF-α also decreased poly(I:C)-induced production of interleukin (IL)-12 and IL-23 by MoDCs, which correlated with their diminished capacity to stimulate cellular proliferation, interferon-γ and IL-17 production by allogeneic CD4(+)T cells in co-culture. Such an effect was more pronounced after 24 h and could not be restored by CD40 ligation. In the presence of CD40L, TNF-α even stimulated IL-10 production and immunoglobulin-like transcript 3 expression by poly(I:C)-matured DCs, which correlated with their increased capacity to induce IL-10 production by CD4(+)T cells. CONCLUSION: Even though TNF-α could promote the survival of poly(I:C)-matured MoDCs, it also suppresses key anti-tumor functions of these cells, which could have important implications when considering this, already suggested, protocol for the DC-based anti-tumor therapy.

Laryngeal Granuloma - Benefit in Treatment with Zinc Supplementation?

Laryngeal granulomas present as contact and postintubation ulcers and granulomas. Essentially, a contact granuloma is a pseudotumor of the lateral wall of the posterior glottis. The most common etiological factor is voice abuse, with predisponing factors such as reflux disease. Postintubation ulcers and granulomas, although of different etiology, according to all the other traits belong to this clinical entity. The therapy of choice is conservative treatment. Surgical laser excision is indicated for resistant cases and those whose size is causing respiratory distress. Treatment of laryngeal granulomas with zinc supplementation is reported in the literature as one of the forms of conservative treatment, and we wanted to consider it in this review. Zinc is an essential mineral that plays a vital role in many biochemical reactions and is considered very important for wound healing.

Oncological and functional outcome after surgical treatment of early glottic carcinoma without anterior commissure involvement.

INTRODUCTION: Glottic carcinoma can be successfully diagnosed in its early stages and treated with high percentage of success. Organ preservation and optimal functional outcomes could be achieved with wide array of surgical techniques for early glottic cancer, including endoscopic approaches or open laryngeal preserving procedures, making surgery the preferred method of treatment of early glottic carcinoma in the last few years. MATERIAL AND METHODS: Prospective study was done on 59 patients treated for Tis and T1a glottic carcinoma over a one-year time period in a tertiary medical center. Patients were treated with endoscopic laser cordectomy (types II-IV cordectomies according to European Laryngological Society classification of endoscopic cordectomies) and open cordectomy through laryngofissure. Follow-up period was 60 months. Clinical and oncological results were followed postoperatively. Voice quality after the treatment was assessed using multidimensional voice analysis 12 months after the treatment. RESULTS: There were no significant differences between oncological and functional results among two groups of patients, though complications were more frequent in patients treated with open cordectomy. CONCLUSION: Endoscopic laser surgery should be the first treatment of choice in treatment of early glottic carcinomas, though open approach through laryngofissure should be available for selected cases where anatomical factors present limiting adequate tumor removal.

Tick on the tympanic membrane.

INTRODUCTION: Different foreign bodies can reach the lumen of the external auditory canal. Clinical presence of the foreign bodies depends on the nature of the foreign body, localization, morphological features, and the presence of pathological process. CASE REPORT: This study gives a report on a rare foreign body--a tick on the eardrum, which is a very rare localization in European countries. CONCLUSION: Identification, determination of the nature of the foreign body and the way of extracting it depend on the application of adequate diagnostic and therapeutic approaches.

An anti-DEC-205 monoclonal antibody stimulates binding of thymocytes to rat thymic dendritic cells and promotes apoptosis of thymocytes.

DEC-205, a transmembrane receptor responsible for cross-presentation of apoptotic cell-derived antigens, is expressed by cortical thymic epithelial cells (TEC) and thymic dendritic cells (TDC) in humans and mice, but its function in T-cell development is still unclear. In this work we have studied for the first time the expression of DEC-205 in the rat thymus by HD83 monoclonal antibody (mAb) and immunohistochemistry, as well as the ability of this mAb to modulate thymocyte - TDC interactions in vitro. We showed the positivity of cortical TEC in situ, including thymic nurse cells (TNC) in suspension, and TDC, whereas subcapsular, perivascular and medullary TEC were negative. All examined DEC-205 positive and DEC-205 negative structures were MHC class II positive. HD83 mAb increased apoptosis of thymocytes in co-culture with TDC in vitro and the process was associated with increased binding of thymocytes to TDC in a rosette form. Since negative selection of thymocytes by clonal deletion (apoptosis) was mediated predominantly by TDC, our results suggest the possible indirect effect of the DEC-205 molecule in these mechanisms.

Morphometric analysis of Ki-67 and p16 expression in laryngeal precursor lesions.

Laryngeal precursor lesions represent areas of altered epithelium with an increased likelihood for progression to squamous cell carcinoma. The exact molecular mechanisms of malignant transformation of laryngeal mucosa are not completely clear, but are certainly due to deregulation of cell proliferation. To assess the potential value of the p16 and Ki-67 as markers of malignant progression, we undertook a retrospective immunohistochemical and morphometric analysis on biopsy specimens from patients with precancerous lesions in the larynx. Morphometric analysis of samples stained with p16 antibody showed epithelial cell positivity in 29 (100 %) of samples with simple hyperplasia, 31 (100 %) samples with basal/parabasal cell hyperplasia, 23 (88 %) samples with atypical hyperplasia and 20 (95 %) samples with in situ carcinoma. There was a significant difference in percentage of p16-positive cells between samples with simple hyperplasia and samples with in situ carcinoma. Morphometric analysis of samples stained with Ki-67 antibody showed epithelial cell positivity in 27 (93 %) of samples with simple hyperplasia, 30 (97 %) samples with basal/parabasal cell hyperplasia, 26 (100 %) samples with atypical hyperplasia and 18 (86 %) samples with in situ carcinoma. There was a significant difference not only in the percentage of Ki-67-positive cells between samples with simple hyperplasia and samples with in situ carcinoma, but also between samples with simple and basal/parabasal cell hyperplasia. Laryngeal epithelial precursor lesions show significantly opposite patterns in p16 and Ki-67 immunopositivity. Simple hyperplasia on average shows 12 % of Ki-67-positive cells and 46 % of p16-positive cells. In situ carcinoma on average shows 23 % of Ki-67-positive cells and 36 % of p16-positive cells.

[T lymphocyte function in laryngectomized patients].

AIM: Malignant tumors of the larynx account for 2.3% of all malignancies, while their frequency among tumors of the head and neck ranges between 12% and 20%. Research on the general immune competence in patients with malignant diseases has provided useful insight in the relationship between immune disorders on one side and the clinical course on the other. Unfortunately, only few complete studies have been published so far with this regard in patients with malignant tumors of the larynx, and therefore our study was essentially aimed at establishing of general immunocompetence, presence and levels of the possible immune disorders and their association with the malignant tumors. MATERIAL AND METHOD: The study included forty two patients with primary squamocellular laryngeal cancer. All the patients underwent surgery, out of whom fifteen were treated postoperatively with radiotherapy. We tested the immune competence prior to the operation and in the postoperative period nine months later. In the venous blood we examined T lymphocyte function, monocyte levels and mononuclear phagocyte function. RESULTS: Preoperative evaluation of the presence and levels of general immune competence in patients with laryngeal cancer, showed a distinct decrease in the proliferative response to the PHA mitogen in vitro, with a tendency to normalize in patients who do not develop a relapse of the disease or distant metastasis during the follow-up period. During the whole study period, the number of monocytes and mononuclear phagocyte activity was above the normal level. CONCLUSION: The patients with operable laryngeal carcinoma had considerable immune disorders at various levels, primarily at the level of T lymphocytes. Of all the disorders, reduced mitotic activity of T lymphocytes in response to mitogens showed the highest dependance on the presence of malignant tissue in the organism.

Comparison of two different protocols for the induction of maturation of human dendritic cells in vitro.

BACKGROUND: Dendritic cells (DC) have been used for immunotherapy of malignant tumors, different kinds of infections, and other clinical conditions. For that purpose, optimal conditions for the generation of functionally mature DC in vitro are required. Two different protocols for the induction of maturation of monocyte-derived DC (MDDC) were compared in this study. METHODS: MDDC were generated in vitro by cultivating adherent monocytes of healthy volunteers with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) during 6-days period. The immature DC thus prepared were induced to mature using two protocols. DC were stimulated for 2 days with lipopolysaccharide (LPS), or with a cocktail of proinflammatory mediators (PM) containing IL-1beta, IL-6, tumor necrosis factor alpha (TNFalpha), and prostaglandin E2 (PGE2), respectively. Phenotypic characteristics of MDDC and their endocytic activity were studied by flow cytometry. Allostimulatory activity of these cells was tested in the mixed leukocyte reaction (MLR), whereas the production of cytokines was determined by ELISA kits. RESULTS: MDDC matured with PM (PM-DC) were predominantly non-adherent cells, while about 30% of LPS-matured DC were adherent cells. In comparison with LPS-DC, PM-DC expressed higher levels of CD86 and CD83, had lower endocytic activity, produced higher levels of IL-10 and lower levels of IL-12, and more strongly stimulated proliferation of allogeneic lymphocytes. CONCLUSION: The protocol based on the combination of proinflammatory cytokines and PGE2 is better for the induction of maturation of human MDDC in vitro than the protocol using LPS alone.