Immune System Efficiency in Cancer and the Microbiota Influence.

The immune system plays a critical role in preventing cancer development and progression. However, the complex network of cells and soluble factor that form the tumor microenvironment (TME) can dictate the differentiation of tumor-infiltrating leukocytes and shift the antitumor immune response into promoting tumor growth. With the advent of cancer immunotherapy, there has been a reinvigorated interest in defining how the TME shapes the antitumor immune response. This interest brought to light the microbiome as a novel player in shaping cancer immunosurveillance. Indeed, accumulating evidence now suggests that the microbiome may confer susceptibility or resistance to certain cancers and may influence response to therapeutics, particularly immune checkpoint inhibitors. As we move forward into the age of precision medicine, it is vital that we define the factors that influence the interplay between the triad immune system-microbiota-cancer. This knowledge will contribute to improve the therapeutic response to current approaches and will unravel novel targets for immunotherapy.

Myeloid HIF-1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection.

The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator of the response and function of myeloid cells in hypoxic and inflammatory microenvironments. To define the role of HIF-1α in tuberculosis, the progression of aerosol Mycobacterium tuberculosis infection was analysed in mice deficient in HIF-1α in the myeloid lineage (mHIF-1α-/- ). We show that myeloid HIF-1α is not required for the containment of the infection, as both wild-type (WT) and mHIF-1α-/- mice mounted normal Th1 responses and maintained control of bacterial growth throughout infection. However, during chronic infection mHIF-1α-/- mice developed extensive lymphocytic inflammatory involvement of the interstitial lung tissue and died earlier than WT mice. These data support the hypothesis that HIF-1α activity coordinates the response of myeloid cells during M. tuberculosis infection to prevent excessive leucocyte recruitment and immunopathological consequences to the host.

Differential post-transcriptional regulation of IL-10 by TLR2 and TLR4-activated macrophages.

The activation of TLRs by microbial molecules triggers intracellular-signaling cascades and the expression of cytokines such as IL-10. Il10 expression is tightly controlled to ensure effective immune responses, while preventing pathology. Maximal TLR-induction of Il10 transcription in macrophages requires signaling through the MAPKs, ERK, and p38. Signals via p38 downstream of TLR4 activation also regulate IL-10 at the post-transcriptional level, but whether this mechanism operates downstream of other TLRs is not clear. We compared the regulation of IL-10 production in TLR2 and TLR4-stimulated BM-derived macrophages and found different stability profiles for the Il10 mRNA. TLR2 signals promoted a rapid induction and degradation of Il10 mRNA, whereas TLR4 signals protected Il10 mRNA from rapid degradation, due to the activation of Toll/IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) and enhanced p38 signaling. This differential post-transcriptional mechanism contributes to a stronger induction of IL-10 secretion via TLR4. Our study provides a molecular mechanism for the differential IL-10 production by TLR2- or TLR4-stimulated BMMs, showing that p38-induced stability is not common to all TLR-signaling pathways. This mechanism is also observed upon bacterial activation of TLR2 or TLR4 in BMMs, contributing to IL-10 modulation in these cells in an infection setting.

Analysis of a local HIV-1 epidemic in portugal highlights established transmission of non-B and non-G subtypes.

The existing data support Portugal as the western European country with the highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1-infected individuals followed at the reference hospital of the province of Minho, Portugal, at which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%) and the C (14.5%), F1 (7.3%), and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with the A1 and F1 subtypes increased over the years compared with those with B, G, or C subtypes or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of the B and G subtypes were found. However, transmission clusters of non-B and non-G viruses among native individuals were also found, with the dates of the most recent common ancestor estimated to be in the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and are mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route.

Evidence for diversifying selection in a set of Mycobacterium tuberculosis genes in response to antibiotic- and nonantibiotic-related pressure.

Tuberculosis (TB) is a global health problem estimated to kill 1.4 million people per year. Recent advances in the genomics of the causative agents of TB, bacteria known as the Mycobacterium tuberculosis complex (MTBC), have allowed a better comprehension of its population structure and provided the foundation for molecular evolution analyses. These studies are crucial for a better understanding of TB, including the variation of vaccine efficacy and disease outcome, together with the emergence of drug resistance. Starting from the analysis of 73 publicly available genomes from all the main MTBC lineages, we have screened for evidences of positive selection, a set of 576 genes previously associated with drug resistance or encoding membrane proteins. As expected, because antibiotics constitute strong selective pressure, some of the codons identified correspond to the position of confirmed drug-resistance-associated substitutions in the genes embB, rpoB, and katG. Furthermore, we identified diversifying selection in specific codons of the genes Rv0176 and Rv1872c coding for MCE1-associated transmembrane protein and a putative l-lactate dehydrogenase, respectively. Amino acid sequence analyses showed that in Rv0176, sites undergoing diversifying selection were in a predicted antigen region that varies between "modern" lineages and "ancient" MTBC/BCG strains. In Rv1872c, some of the sites under selection are predicted to impact protein function and thus might result from metabolic adaptation. These results illustrate that diversifying selection in MTBC is happening as a consequence of both antibiotic treatment and other evolutionary pressures.

Synthesis and electrochemical study of a hybrid structure based on PDMS-TEOS and titania nanotubes for biomedical applications.

Metallic implants and devices are widely used in the orthopedic and orthodontic clinical areas. However, several problems regarding their adhesion with the living tissues and inflammatory responses due to the release of metallic ions to the medium have been reported. The modification of the metallic surfaces and the use of biocompatible protective coatings are two approaches to solve such issues. In this study, in order to improve the adhesion properties and to increase the corrosion resistance of metallic Ti substrates we have obtained a hybrid structure based on TiO2 nanotubular arrays and PDMS-TEOS films. TiO2 nanotubes have been prepared with two different diameters by means of electrochemical anodization. PDMS-TEOS films have been prepared by the sol-gel method. The morphological and the elemental analysis of the structures have been investigated by scanning electron microscopy and energy dispersive spectroscopy (EDS). Electrochemical impedance spectroscopy (EIS) and polarization curves have been performed during immersion of the samples in Kokubo's simulated body fluid (SBF) at 37 °C to study the effect of structure layers and tube diameter on the protective properties. The obtained results show that the modification of the surface structure of TiO2 and the application of PDMS-TEOS film is a promising strategy for the development of implant materials.

Differential arabinan capping of lipoarabinomannan modulates innate immune responses and impacts T helper cell differentiation.

Toll-like receptors (TLRs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM). Such structures are present in several pathogens, including Mycobacterium tuberculosis, being important for the initiation of immune responses. It is well established that the interaction of LM and LAM with TLR2 is a process dependent on the structure of the ligands. However, the implications of structural variations on TLR2 ligands for the development of T helper (Th) cell responses or in the context of in vivo responses are less studied. Herein, we used Corynebacterium glutamicum as a source of lipoglycan intermediates for host interaction studies. In this study, we have deleted a putative glycosyltransferase, NCgl2096, from C. glutamicum and found that it encodes for a novel α(1→2)arabinofuranosyltransferase, AftE. Biochemical analysis of the lipoglycans obtained in the presence (wild type) or absence of NCgl2096 showed that AftE is involved in the biosynthesis of singular arabinans of LAM. In its absence, the resulting molecule is a hypermannosylated (hLM) form of LAM. Both LAM and hLM were recognized by dendritic cells, mainly via TLR2, and triggered the production of several cytokines. hLM was a stronger stimulus for in vitro cytokine production and, as a result, a more potent inducer of Th17 responses. In vivo data confirmed hLM as a stronger inducer of cytokine responses and suggested the involvement of pattern recognition receptors other than TLR2 as sensors for lipoglycans.

TLR2 deficiency by compromising p19 (IL-23) expression limits Th 17 cell responses to Mycobacterium tuberculosis.

CD4(+) T(h)1 cells producing IFN-γ are of extreme importance in controlling infections by Mycobacterium tuberculosis both in mice and in men. In addition to IFN-γ-producing T cells, IL-17-producing T cells (T(h)17) have been observed during mycobacterial infections. Nevertheless, their contribution for the host immune response to mycobacteria as well as the signals triggering M. tuberculosis -specific T(h)17 cell differentiation and maintenance are not fully understood. We show that signaling via Toll-like receptor (TLR) 2 has a major impact on the regulation of p19 (IL-23) expression in response to M. tuberculosis and therefore on the establishment of T(h)17 cell responses to M. tuberculosis infection. Diminished T(h)17 responses in the lung of M. tuberculosis -infected TLR2-deficient animals were not caused by defective cell differentiation in the draining lymph node (LN) but rather by reduced maintenance at the site of infection. Consistent with the decreased numbers of T(h)17 cells in the lungs of infected TLR2-deficient animals, we observed reduced expression of CXCL9, CXCL10 and CXCL11, chemokines involved in recall responses to M. tuberculosis. Our data provides insights into the TLR2 role in infection with M. tuberculosis, with implications in pathophysiology of the disease and vaccine design.

IFN-gamma-dependent activation of macrophages during experimental infections by Mycobacterium ulcerans is impaired by the toxin mycolactone.

Buruli ulcer, caused by Mycobacterium ulcerans infections, is a necrotizing skin disease whose pathogenesis is associated with the exotoxin mycolactone. Despite the relevance of this emergent disease, little is known on the immune response against the pathogen. Following the recent demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of IFN-gamma and the antimycobacterial mechanisms activated by this cytokine in M. ulcerans-infected macrophages. Three M. ulcerans strains were tested: 5114 (mutant mycolactone-negative, avirulent strain); 94-1327 (intermediate virulence); and 98-912 (high virulence). We show in this study that IFN-gamma is expressed in mouse-infected tissues and that IFN-gamma-deficient mice display increased susceptibility to infection with strains 5114 and, to a lesser extent, 94-1327, but not with the highly virulent strain. Accordingly, IFN-gamma-activated cultured macrophages controlled the proliferation of the avirulent and the intermediate virulent strains. Addition of mycolactone purified from strain 98-912 to cultures of IFN-gamma-activated macrophages infected with the mycolactone-negative strain led to a dose-dependent inhibition of the IFN-gamma-induced protective mechanisms, involving phagosome maturation/acidification and increased NO production, therefore resulting in increased bacterial burdens. Our findings suggest that the protection mediated by IFN-gamma in M. ulcerans-infected macrophages is impaired by the local buildup of mycolactone.

Strategies for use of IL-10 or its antagonists in human disease.

SUMMARY: Interleukin-10 (IL-10) is a cytokine with broad anti-inflammatory properties by its suppression of both macrophage and dendritic cell function, including antigen-presenting cell function and the production of proinflammatory cytokines. This can result subsequently in the feedback regulation of both T-helper 1 (Th1)-type and Th2-type responses. This review discusses the potential use of IL-10 or agents that induce IL-10 as potential anti-inflammatory therapies in inflammatory diseases. Although IL-10-deficient mice develop colitis in the presence of normal gut flora and clear certain intracellular pathogens more efficiently, this is often accompanied by immunopathology, which can be lethal to the host. This reinforces the anti-inflammatory properties of IL-10, although it should be noted that as discussed below, IL-10 can also promote B-cell and other immune responses under particular settings. A penalty of its role to limit the immune and inflammatory responses to pathogens and prevent damage to the host is that high or dysregulated levels of IL-10 may result in chronic infection. Thus, antagonists of IL-10 show great potential as adjuvants in preventative or therapeutic vaccines against chronic infection or cancer. This article reviews basic published studies on IL-10, which may lead to potential uses of IL-10 or its antagonists in human disease.

Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems.

BACKGROUND: T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. METHODS: GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. RESULTS: Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. CONCLUSIONS: Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.

Mycobacterium ulcerans triggers T-cell immunity followed by local and regional but not systemic immunosuppression.

Buruli ulcer is a neglected infectious disease caused by Mycobacterium ulcerans and is characterized by necrotic cutaneous lesions induced by the exotoxin mycolactone. Despite evidence of Th1-mediated protective immunity, M. ulcerans infection has been associated with systemic immunosuppression. We show that early during mouse infection with either mycolactone-positive or negative strains, pathogen-specific gamma interferon (IFN-γ)-producing T cells developed in the draining lymph node (DLN). CD4(+) cells migrated to the infection foci, but progressive infection with virulent M. ulcerans led to the local depletion of recruited cells. Moreover, dissemination of virulent M. ulcerans to the DLN was accompanied by extensive DLN apoptotic cytopathology, leading to depletion of CD4(+) T cells and abrogation of IFN-γ expression. Advanced footpad infection with virulent M. ulcerans did not induce increased susceptibility to systemic coinfection by Listeria monocytogenes. These results show that infection with M. ulcerans efficiently triggers a mycobacterium-specific T-cell response in the DLN and that progression of infection with highly virulent M. ulcerans leads to a local and regional suppression of that immune response, but without induction of systemic immunosuppression. These results suggest that prophylactic and/or therapeutic interventions to prevent dissemination of M. ulcerans to DLN during the early phase of infection would contribute for the maintenance of protective immunity and disease control.

Towards the Development of a Female Animal Model of T1DM Using Hyaluronic Acid Nanocoated Cell Transplantation: Refinements and Considerations for Future Protocols.

Female mice (Black 6 strain) (C57BL/6) aged 6 weeks were subject to low dose streptozotocin (STZ) treatment for five consecutive days to mimic type 1 diabetes mellitus (T1DM) with insulitis. At two weeks after STZ injections, evaluation of the elevated glucose levels was used to confirm diabetes. The diabetic mice were then subject to the transplantation of pancreatic ß-cells (MIN-6 line). Four groups of mice were studied. The first group was injected with saline-only acting as the placebo surgery control, also known as SHAM group, the second and third groups were injected with MIN-6 single cells and polyethylene glycol-modified dipalmitoyl-glycerol-phosphatidyl ethanolamine (PEG-DPPE) modified MIN-6 single cells (500 µg per 1.106 cells), respectively, while the fourth group was injected with hyaluronic acid (HA)-coated MIN-6 single cells (5 bilayers). At seven- and fourteen-days following transplantation, the mice were euthanised. The renal and pancreatic tissues were then collected and histologically analysed. The induction of diabetes in female mice, through five-consecutive daily STZ injections resulted in inconsistent glycaemic levels. Interestingly, this shows an incomplete diabetes induction in female mice, of which we attribute to sex dimorphism and hormonal interferences. Transplantation failure of free-floating encapsulated cells was unable to decrease blood glucose hyperglycaemia to physiological ranges. The result is attributed to deprived cell-cell interactions, leading to decreased ß-cells functionality. Overall, we highlight the necessity of refining T1DM disease models in female subjects when using multiple low-dose STZ injections together with transplantation protocols. Considerations need to be made regarding the different developmental stages of female mice and oestrogen load interfering with pancreatic ß-cells susceptibility to STZ. The use of pseudo islets, cell aggregates and spheroids are sought to improve transplantation outcome in comparison to free-floating single cells.

Interleukin-10 induces interferon-γ-dependent emergency myelopoiesis.

In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production are observed and often mediated by the pro-inflammatory cytokine interferon gamma (IFN-γ). Interleukin-10 (IL-10) inhibits IFN-γ secretion, but paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work, we use different in vivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also find that IL-10, unexpectedly, reprograms CD4 and CD8 T cells toward an activation state that includes IFN-γ production by these T cell subsets in vivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening additional perspectives for the design of IL-10-based immunotherapies.

Early IL-10 promotes vasculature-associated CD4+ T cells unable to control Mycobacterium tuberculosis infection.

Cytokine-producing CD4+ T cells play a crucial role in the control of Mycobacterium tuberculosis infection; however, there is a delayed appearance of effector T cells in the lungs following aerosol infection. The immunomodulatory cytokine IL-10 antagonizes control of M. tuberculosis infection through mechanisms associated with reduced CD4+ T cell responses. Here, we show that IL-10 overexpression only before the onset of the T cell response impaired control of M. tuberculosis growth; during chronic infection, IL-10 overexpression reduced the CD4+ T cell response without affecting the outcome of infection. IL-10 overexpression early during infection did not, we found, significantly impair the kinetics of CD4+ T cell priming and effector differentiation. However, CD4+ T cells primed and differentiated in an IL-10-enriched environment displayed reduced expression of CXCR3 and, because they did not migrate into the lung parenchyma, their ability to control infection was limited. Importantly, these CD4+ T cells maintained their vasculature phenotype and were unable to control infection, even after adoptive transfer into low IL-10 settings. Together our data support a model wherein, during M. tuberculosis infection, IL-10 acts intrinsically on T cells, impairing their parenchymal migratory capacity and ability to engage with infected phagocytic cells, thereby impeding control of infection.

Increased CD3+, CD8+, or FoxP3+ T Lymphocyte Infiltrations Are Associated with the Pathogenesis of Colorectal Cancer but Not with the Overall Survival of Patients.

Tumor-infiltrating lymphocytes include heterogeneous populations of T lymphocytes that play crucial roles in the tumor immune response; importantly, their presence in the tumor tissue may predict clinical outcomes. Therefore, we herein studied the prognostic significance of the presence and location of CD3+, CD8+, and FoxP3+ T lymphocytes in colorectal cancer samples. In the intratumor analysis, our data did not reveal any association between lymphocyte infiltrations with clinical or pathological data. However, in the tumor margins, we found that the presence of high infiltrations of CD3+, CD8+, or FoxP3+ T lymphocytes were associated with TNM stages I-II (p = 0.021, p = 0.022, and p = 0.012, respectively) and absence of lymph node metastases (p = 0.010, p = 0.003, and p = 0.004, respectively). Despite these associations with good prognostic indicators, we were not able to find any statistically significant alterations in the overall survival of the patients, even though high infiltrations of FoxP3+ T lymphocytes in the tumor margins resulted in an increased overall survival of 14 months. Taken together, these data show that the presence of CD3+, CD8+, or FoxP3+T lymphocyte infiltrates in the tumor margins are associated with the pathogenesis of CRC, but only high Foxp3+ T lymphocyte infiltrations in the tumor invasive margins are inclined to indicate favorable prognosis.

The selective COX-2 inhibitor Etoricoxib reduces acute inflammatory markers in a model of neurogenic laryngitis but loses its efficacy with prolonged treatment.

OBJECTIVE: A randomised experimental study was used to evaluate the therapeutic effect of a selective cyclooxygenase-2 (COX-2) inhibitor in neurogenic laryngitis. MATERIALS AND METHODS: Male Wistar Han rats were subjected to the nasogastric intubation model (NGI) of laryngitis for 1 and 2 weeks. The NGI animals were divided into three groups: (1) treated with COX-2 inhibitor Etoricoxib, (2) vehicle and (3) non-intubated animals. A fourth group of animals was submitted to NGI only. Laryngeal sections were immunostained for substance P (SP) and calcitonin gene-related peptide (CGRP) fibre-immunoreactivity (IR) and quantification of COX-2 positive cells through stereological analysis. The expression of COX-2, interleukins IL-1beta, IL-6, IL-10 and tumour necrosis factor-alpha (TNF-alpha) was determined by quantitative real time QRT-PCR. TREATMENT: Etoricoxib (6 mg/kg/day) was prepared in 0.9% sterile saline with 5% glucose (vehicle) and administered daily during 1 or 2 weeks. RESULTS: Treatment for 1 week with Etoricoxib attenuated the CGRP-IR fibre depletion, the COX-2-IR increased cell number and the TNF-alpha and COX-2 mRNA increased levels induced by NGI. Two weeks of treatment had no beneficial effect. CONCLUSIONS: Etoricoxib is effective in neurogenic laryngitis for limited periods of administration, indicating that selective COX-2 inhibitors should be evaluated in the future.

Antimicrobial activity of Mycobacteriophage D29 Lysin B during Mycobacterium ulcerans infection.

Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans. The pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that induces extensive destruction of the skin and soft tissues. Currently, there are no effective measures to prevent the disease and, despite availability of antibiotherapy and surgical treatments, these therapeutic options are often associated with severe side effects. Therefore, it is important to develop alternative strategies for the treatment of BU. Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents against bacterial infections. However, mycobacteria have an unusual outer membrane composed of mycolylarabinogalactan-peptidoglycan. To overcome this complex barrier, some mycobacteriophages encode a lipolytic enzyme, Lysin B (LysB). In this study, we demonstrate for the first time that recombinant LysB displays lytic activity against M. ulcerans isolates. Moreover, using a mouse model of M. ulcerans footpad infection, we show that subcutaneous treatment with LysB prevented further bacterial proliferation, associated with IFN-γ and TNF production in the draining lymph node. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.

A new model of laryngitis: neuropeptide, cyclooxygenase, and cytokine profile.

OBJECTIVES/HYPOTHESIS: To develop and characterize a new model of laryngeal inflammation by analyzing the presence of neurogenic peptides and expression of cyclooxygenases (COX) and cytokines in the mucosa. STUDY DESIGN: Laryngitis induced by nasogastric intubation (NGI) was evaluated by histopathologic changes of the mucosa, alterations in calcitonin gene related peptide (CGRP) and substance P (SP) neuropeptides in sensory fibers, and COX-1,2, and cytokine (interleukin [IL]-1, IL-6, IL-10, tumor necrosis factor [TNF]-[alpha]) expression in the laryngeal mucosa. METHODS: Rats submitted to NGI for 1 to 5 weeks were compared with controls. Laryngeal sections were immunostained for stereologic analysis of SP and CGRP fiber density and number of mucosal cells expressing COX-2. Alterations in inflammatory mediators were evaluated by quantitative reverse-transcriptase polymerase chain reaction. RESULTS: NGI induced metaplasia of the epithelium and narrowing of the laryngeal lumen because of hypertrophy of laryngeal glandules and edema. An initial decrease in CGRP- and SP-immunoreactive fibers in the laryngeal mucosa (1-3 wk) was reverted with time (5 wk). COX-2 expression in mucosal cells increased progressively, reaching a maximum level at 5 weeks, and was observed in mononuclear immune cells, which is indicative of a chronic inflammatory process. In regard to mRNA expression levels of inflammatory mediators, TNF-[alpha] was increased during the 5 week NGI, and IL-10 decreased during the 5 weeks,whereas IL-1[beta], IL-6, and COX-2 increased in the first 1 to 2 weeks and returned to baseline at 5 weeks. CONCLUSIONS: This NGI model results in laryngeal chronic inflammation without direct mechanical aggression of the mucosa and may contribute to the study of future therapeutic approaches to this pathology.