CD69 controls the uptake of L-tryptophan through LAT1-CD98 and AhR-dependent secretion of IL-22 in psoriasis.

The activation marker CD69 is expressed by skin γδ T cells. Here we found that CD69 controlled the aryl hydrocarbon receptor (AhR)-dependent secretion of interleukin 22 (IL-22) by γδ T cells, which contributed to the development of psoriasis induced by IL-23. CD69 associated with the aromatic-amino-acid-transporter complex LAT1-CD98 and regulated its surface expression and uptake of L-tryptophan (L-Trp) and the intracellular quantity of L-Trp-derived activators of AhR. In vivo administration of L-Trp, an inhibitor of AhR or IL-22 abrogated the differences between CD69-deficient mice and wild-type mice in skin inflammation. We also observed LAT1-mediated regulation of AhR activation and IL-22 secretion in circulating Vγ9(+) γδ T cells of psoriatic patients. Thus, CD69 serves as a key mediator of the pathogenesis of psoriasis by controlling LAT1-CD98-mediated metabolic cues.

Plasmolipin regulates basolateral-to-apical transcytosis of ICAM-1 and leukocyte adhesion in polarized hepatic epithelial cells.

Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response.

Opposing Actions of TLR2 and TLR4 in Adipocyte Differentiation and Mature-Onset Obesity.

Understanding the signaling cascades that govern adipocyte metabolism and differentiation is necessary for the development of therapies for obesity. Toll-like receptors (TLRs) are key mediators in adipogenesis, but their specific role is not completely understood. In this study, siRNA knockdown of Tlr2 in 3T3-L1 cells allowed them to differentiate more efficiently into adipocytes, whereas the opposite was observed for the knockdown of Tlr4. At the same time, we show that TLR2 knock-out mice spontaneously developed mature-onset obesity and insulin resistance. Besides a higher incidence of hyperplasia and hypertrophy in white adipose tissue (WAT), we found a significantly increased number of adipocyte precursor cells in TLR2-/- mice compared to TLR4-/- mice. Interestingly, genetic inactivation of Tlr4 in TLR2-/- mice reverted their increased adiposity, insulin resistance, and restored normal levels of adipocyte precursor cells. These findings provide evidence that TLR2 and TLR4 play opposing roles in WAT homeostasis and point to the existence of cross-regulation among TLR2 and TLR4 during adipocyte differentiation both in vitro and in vivo.

Regulation of Cyclooxygenase-2 Expression in Human T Cells by Glucocorticoid Receptor-Mediated Transrepression of Nuclear Factor of Activated T Cells.

Cyclooxygenase (COX) is the key enzyme in prostanoid synthesis from arachidonic acid (AA). Two isoforms, named COX-1 and COX-2, are expressed in mammalian tissues. The expression of COX-2 isoform is induced by several stimuli including cytokines and mitogens, and this induction is inhibited by glucocorticoids (GCs). We have previously shown that the transcriptional induction of COX-2 occurs early after T cell receptor (TCR) triggering, suggesting functional implications of this enzyme in T cell activation. Here, we show that dexamethasone (Dex) inhibits nuclear factor of activated T cells (NFAT)-mediated COX-2 transcriptional induction upon T cell activation. This effect is dependent on the presence of the GC receptor (GR), but independent of a functional DNA binding domain, as the activation-deficient GRLS7 mutant was as effective as the wild-type GR in the repression of NFAT-dependent transcription. Dex treatment did not disturb NFAT dephosphorylation, but interfered with activation mediated by the N-terminal transactivation domain (TAD) of NFAT, thus pointing to a negative cross-talk between GR and NFAT at the nuclear level. These results unveil the ability of GCs to interfere with NFAT activation and the induction of pro-inflammatory genes such as COX-2, and explain some of their immunomodulatory properties in activated human T cells.

T cell-intrinsic role of Nod2 in promoting type 1 immunity to Toxoplasma gondii.

Nod2 belongs to the nucleotide-binding oligomerization domain receptor (NLR) family of proteins, which function as intracellular pathogen sensors in innate immune cells. Nod2 deficiency results in an impaired immune response to bacterial pathogens. However, how this protein promotes host defense against intracellular parasites is unknown. Here we found that Nod2(-/-) mice had less clearance of Toxoplasma gondii and lower interferon-gamma (IFN-gamma) production. Reconstitution of T cell-deficient mice with Nod2(-/-) T cells followed by T. gondii infection demonstrated a T cell-intrinsic defect. Nod2(-/-) CD4(+) T cells had poor helper T cell differentiation, which was associated with impaired production of interleukin 2 (IL-2) and nuclear accumulation of the transcription factor subunit c-Rel. Our data demonstrate a T cell-intrinsic role for Nod2 signaling that is critical for host defense against T. gondii.

GRK2 levels in myeloid cells modulate adipose-liver crosstalk in high fat diet-induced obesity.

Macrophages are key effector cells in obesity-associated inflammation. G protein-coupled receptor kinase 2 (GRK2) is highly expressed in different immune cell types. Using LysM-GRK2+/- mice, we uncover that a reduction of GRK2 levels in myeloid cells prevents the development of glucose intolerance and hyperglycemia after a high fat diet (HFD) through modulation of the macrophage pro-inflammatory profile. Low levels of myeloid GRK2 confer protection against hepatic insulin resistance, steatosis and inflammation. In adipose tissue, pro-inflammatory cytokines are reduced and insulin signaling is preserved. Macrophages from LysM-GRK2+/- mice secrete less pro-inflammatory cytokines when stimulated with lipopolysaccharide (LPS) and their conditioned media has a reduced pathological influence in cultured adipocytes or naïve bone marrow-derived macrophages. Our data indicate that reducing GRK2 levels in myeloid cells, by attenuating pro-inflammatory features of macrophages, has a relevant impact in adipose-liver crosstalk, thus preventing high fat diet-induced metabolic alterations.

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation.

BACKGROUND: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1ß, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. OBJECTIVE: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. METHODS: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). RESULTS: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1ß, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1ß stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1ß-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. CONCLUSION: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1ß/IL-17 axis.

The Dual-Specificity Phosphatase 10 (DUSP10): Its Role in Cancer, Inflammation, and Immunity.

Cancer is one of the most diagnosed diseases in developed countries. Inflammation is a common response to different stress situations including cancer and infection. In those processes, the family of mitogen-activated protein kinases (MAPKs) has an important role regulating cytokine secretion, proliferation, survival, and apoptosis, among others. MAPKs regulate a large number of extracellular signals upon a variety of physiological as well as pathological conditions. MAPKs activation is tightly regulated by phosphorylation/dephosphorylation events. In this regard, the dual-specificity phosphatase 10 (DUSP10) has been described as a MAPK phosphatase that negatively regulates p38 MAPK and c-Jun N-terminal kinase (JNK) in several cellular types and tissues. Several studies have proposed that extracellular signal-regulated kinase (ERK) can be also modulated by DUSP10. This suggests a complex role of DUSP10 on MAPKs regulation and, in consequence, its impact in a wide variety of responses involved in both cancer and inflammation. Here, we review DUSP10 function in cancerous and immune cells and studies in both mouse models and patients that establish a clear role of DUSP10 in different processes such as inflammation, immunity, and cancer.

Mitogen-Activated Protein Kinase 14 Promotes AKI.

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry-based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity-deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target.

Dose-dependent effects of prostaglandin E2 in macrophage adhesion and migration.

Macrophage migration to the focus of infection is a hallmark of the innate immune response. Macrophage spreading, adhesion, and migration through the extracellular matrix require dynamic remodeling of the actin cytoskeleton associated to integrin clustering in podosomes and focal adhesions. Here, we show that prostaglandin E2 (PGE2 ), the main prostaglandin produced by macrophages during inflammation, promote the distinctive dose-dependent formation of podosomes or focal adhesions in macrophages. Low concentrations of PGE2 increased p110γ PI3K expression, phosphorylation of actin-related protein 2, and formation of podosomes, which enhanced macrophage migration in response to chemokines. However, high doses of PGE2 increased phosphorylation of paxillin and focal adhesion kinase, the expression of serine/threonine protein kinase 1, and promoted focal adhesion formation and macrophage adhesion, reducing macrophage chemotaxis. In summary, we describe the dual role of PGE2 as a promoter of macrophage chemotaxis and adhesion, proposing a new model of macrophage migration to the inflammatory focus in the presence of a gradient of PGE2 .

Differential cytokine profiling in Chagasic patients according to their arrhythmogenic-status.

BACKGROUND: Chagas disease is caused by the protozoan Trypanosoma cruzi and is characterized by heart failure and sudden death. Identifying which factors are involved in evolution and treatment response is actually challenging. Thus, the aim of this work was to determine the Th1/Th17 (IL-6, IL-2, TNF, IL-17 and IFN-γ) and Th2 (IL-4 and IL-10) serum profile in Venezuelan Chagasic patients stratified according amiodarone treatment, hypertension and arrhythmias. METHODS: Sera from 38 chagasic patients were analyzed to determine the level of cytokines by Multiplexed Bead-Based Immunoassays. ANOVA test was applied to determine differences for each group. Additionally, a Linear Discriminant Analysis (LDA) was applied to observe the accuracy of different cytokines to discriminate between the groups. RESULTS: The levels of several cytokines were significantly higher in the high-risk of sudden death and untreated group. LDA showed that IL-2, IFN-γ and IL-10 were the best cytokines for discriminating between high-risk of sudden death and untreated patients versus low-risk of sudden death, treated and control groups. CONCLUSIONS: High IL-2 levels seem to identify patients with high-risk of sudden death and seems adequate as treatment efficacy marker. To our knowledge, this is the first report about the anti-inflammatory role of the amiodarone in Chagas disease, suggesting an inmunomodulatory effect that may be exploited as coadjutant therapy in chronic Chagas disease.

Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors.

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

The NFKB Inducing Kinase Modulates Hematopoiesis During Stress.

The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here, we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFκB pathway (the NFκB inducing kinase, NIK, and the downstream molecule NFκB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy, and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation); however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild-type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, that is, either NFκB2 or c-Rel, only NFκB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFκB pathway for the recovery of normal levels of hematopoiesis after stress.

Modulation of endothelial function by Toll like receptors.

Endothelial cells (EC) are able to actively control vascular permeability, coagulation, blood pressure and angiogenesis. Most recently, a role for endothelial cells in the immune response has been described. Therefore, the endothelium has a dual role controlling homeostasis but also being the first line for host defence and tissue damage repair thanks to its ability to mount an inflammatory response. Endothelial cells have been shown to express pattern-recognition receptors (PRR) including Toll-like receptors (TLR) that are activated in response to stimuli within the bloodstream including pathogens and damage signals. TLRs are strategic mediators of the immune response in endothelial cells but they also regulate the angiogenic process critical for tissue repair. Nevertheless, endothelial activation and angiogenesis can contribute to some pathologies. Thus, inappropriate endothelial activation, also known as endothelial dysfunction, through TLRs contributes to tissue damage during autoimmune and inflammatory diseases such as atherosclerosis, hypertension, ischemia and diabetes associated cardiovascular diseases. Also TLR induced angiogenesis is required for the growth of some tumors, atherosclerosis and rheumatoid arthritis, among others. In this review we discuss the importance of various TLRs in modulating the activation of endothelial cells and their importance in immunity to infection and vascular disease as well as their potential as therapeutic targets.

NFκB2/p100 is a key factor for endotoxin tolerance in human monocytes: a demonstration using primary human monocytes from patients with sepsis.

Endotoxin tolerance (ET) is a state of reduced responsiveness to endotoxin stimulation after a primary bacterial insult. This phenomenon has been described in several pathologies, including sepsis, in which an endotoxin challenge results in reduced cytokine production. In this study, we show that the NFκ L chain enhancer of activated B cells 2 (NFκB2)/p100 was overexpressed and accumulated in a well-established in vitro human monocyte model of ET. The p100 accumulation in these cells inversely correlated with the inflammatory response after LPS stimulation. Knocking down NFκB2/p100 using small interfering RNA in human monocytes further indicated that p100 expression is a crucial factor in the progression of ET. The monocytes derived from patients with sepsis had high levels of p100, and a downregulation of NFκB2/p100 in these septic monocytes reversed their ET status.

Coffee Silverskin Extract Protects against Accelerated Aging Caused by Oxidative Agents.

Nowadays, coffee beans are almost exclusively used for the preparation of the beverage. The sustainability of coffee production can be achieved introducing new applications for the valorization of coffee by-products. Coffee silverskin is the by-product generated during roasting, and because of its powerful antioxidant capacity, coffee silverskin aqueous extract (CSE) may be used for other applications, such as antiaging cosmetics and dermaceutics. This study aims to contribute to the coffee sector's sustainability through the application of CSE to preserve skin health. Preclinical data regarding the antiaging properties of CSE employing human keratinocytes and Caenorhabditis elegans are collected during the present study. Accelerated aging was induced by tert-butyl hydroperoxide (t-BOOH) in HaCaT cells and by ultraviolet radiation C (UVC) in C. elegans. Results suggest that the tested concentrations of coffee extracts were not cytotoxic, and CSE 1 mg/mL gave resistance to skin cells when oxidative damage was induced by t-BOOH. On the other hand, nematodes treated with CSE (1 mg/mL) showed a significant increased longevity compared to those cultured on a standard diet. In conclusion, our results support the antiaging properties of the CSE and its great potential for improving skin health due to its antioxidant character associated with phenols among other bioactive compounds present in the botanical material.

Lack of Galectin-3 Prevents Cardiac Fibrosis and Effective Immune Responses in a Murine Model of Trypanosoma cruzi Infection.

BACKGROUND: Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. One of the major causes of mortality in the disease is the cardiomyopathy observed in chronic patients, despite the low number of parasites detected in cardiac tissue. Galectin-3, a carbohydrate-binding protein with affinity for ß-galactoside-containing glycoconjugates, is upregulated upon infection, and it has been recently involved in the pathophysiology of heart failure. METHODS: We investigated the role of galectin-3 in systemic and local responses in a murine model of T. cruzi infection, using knockout animals. Molecular mechanisms underlying galectin-3-dependent inflammatory responses were further assessed in cultured dendritic cells in vitro. RESULTS: Mice deficient for galectin-3 have elevated blood parasitemia levels and impaired cytokine production during infection. Remarkably, galectin-3 promotes cellular infiltration in the heart of infected mice and subsequent collagen deposition and cardiac fibrosis. Furthermore, we show that an unbalanced Toll-like receptor expression on antigen-presenting cells may be the cause of the impaired immune response observed in galectin-3-deficient mice in vivo. CONCLUSIONS: These results suggest that galectin-3 is strongly involved in Chagas disease, not only in the immune response against T. cruzi, but also in mediating cardiac tissue damage.

Cyclooxygenase-2 deficiency in macrophages leads to defective p110γ PI3K signaling and impairs cell adhesion and migration.

Cyclooxygenase (Cox)-2 dependent PGs modulate several functions in many pathophysiological processes, including migration of immune cells. In this study, we addressed the role of Cox-2 in macrophage migration by using in vivo and in vitro models. Upon thioglycolate challenge, CD11b(+) F4/80(+) macrophages showed a diminished ability to migrate to the peritoneal cavity in cox-2(-/-) mice. In vivo migration of cox-2(-/-) macrophages from the peritoneal cavity to lymph nodes, as well as cell adhesion to the mesothelium, was reduced in response to LPS. In vitro migration of cox-2(-/-) macrophages toward MCP-1, RANTES, MIP-1α, or MIP-1ß, as well as cell adhesion to ICAM-1 or fibronectin, was impaired. Defects in cell migration were not due to changes in chemokine receptor expression. Remarkably, cox-2(-/-) macrophages showed a deficiency in focal adhesion formation, with reduced phosphorylation of paxillin (Tyr(188)). Interestingly, expression of the p110γ catalytic subunit of PI3K was severely reduced in the absence of Cox-2, leading to defective Akt phosphorylation, as well as cdc42 and Rac-1 activation. Our results indicate that the paxillin/p110γ-PI3K/Cdc42/Rac1 axis is defective in cox-2(-/-) macrophages, which results in impaired cell adhesion and migration.

Time trends in the prevalence of HPV in oropharyngeal squamous cell carcinomas in northern Spain (1990-2009).

Recent studies support an important role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (OPSCC), although the incidence varies widely depending on the geographic location and time period studied. The aim of this study was to determine the proportion of HPV in a large cohort of OPSCC in northern Spain in the years 1990-2009. Clinical records and paraffin embedded tumor specimens of 248 consecutive patients surgically treated for OPSCC (140 tonsillar and 108 base of tongue) between 1990 and 2009 were retrieved. OPSCC cases were histomorphologically evaluated, and protein expression of p16 and p53 was analyzed by immunohistochemistry. Detection of high-risk HPV DNA was performed by GP5+/6+-PCR and in situ hybridization (ISH). Thirty cases (12%) were positive for p16 immunostaining, of which eight (3.2% of the total series) were found positive for HPV type 16 by genotyping of GP5+6+-PCR products. All HPV GP5+/6+-PCR-positive tumors were p53-immunonegative, seven had a basaloid morphology and seven were also positive by HPV ISH. Presence of HPV correlated inversely with tobacco and alcohol consumption (p < 0.001), but not with age of onset of OPSCC. Overall survival was better in the HPV-positive group, although not statistically significant (p = 0.175). OPSCC patients in northern Spain demonstrated a low involvement of HPV, increasing (although not significantly, p = 0.120) from 1.8% in 1990-1999 to 6.1% of cases in 2000-2009.