Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Background: Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results: TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions: Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Tetraploidization in Wilms tumor in an infant

Genetic instability is frequent in human cancer. Unscheduled tetraploidization can trigger cell transformation and tumorigenesis. We made a cytogenetic analysis by Giemsa-trypsin banding of a stage I, biphasic Wilms tumor diagnosed in a 10-month-old male. An evident karyotypic heterogeneity was found. Four different subclones of tumor cells were observed, with DNA content varying from diploid to near-tetraploid complements. The genetic events involved in the acquisition of aneuploidy in Wilms tumor remain unclear. We hypothesize that initial tetraploidization caused aberrant cell division, leading to abnormal chromosomal segregation, cell transformation and tumorigenesis.

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background: In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro. Methods: Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 mu M for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples >= 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors. Results: Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction = 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed. Conclusion: Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

Observations on Antricola ticks: Small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks

Ticks use bloodmeals as a Source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood ill a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts: and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccesful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed oil rabbits. and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure. consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections Of gut.

Glut1 and Glut3 as Potential Prognostic Markers for Oral Squamous Cell Carcinoma

We associated clinical-pathological features of 142 OSCC with the expression pattern of GLUT1 and GLUT3 in order to estimate their prognostic value. Methods: Clinical-pathological features and overall survival data of 142 patients with Oral Squamous Cell Carcinoma (OSCC) were retrospectively reviewed from A. C. Camargo hospital records. A tissue microarray (TMA) was built for the immunohistochemical (IHC) analysis of GLUT 1 and GLUT 3. IHC results were evaluated according to the staining pattern and number of positive cells. Results: GLUT 1 was over expressed in 50.3% of OSSC cases showing membrane staining pattern. However, nuclear expression was observed in 49.7% of the analyzed cases. GLUT 3 over expression was detected in 21.1% of OSCC cases. The pattern of GLUT 1 expression showed significant association with alcohol consumption (p = 0.004). Positive cell membrane GLUT 3 protein expression was associated with advanced clinic-staging of tumours (p = 0.005) as well as with vascular embolization (p = 0.005). Positive expression of GLUT 3 was associated with unfavorable free-disease survival (p = 0.021). Conclusion: GLUT1 and GLUT3 protein expression evaluated by immunohistochemistry are, significantly, indicators of poor prognosis outcome in oral squamous cell carcinoma, probably due to the enhanced glycolytic metabolism of more aggressive neoplastic cells.

Cross talk Initiated by Endothelial Cells Enhances Migration and Inhibits Anoikis of Squamous Cell Carcinoma Cells through STAT3/Akt/ERK Signaling

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL-6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

High Glucose-Mediated Oxidative Stress Impairs Cell Migration

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.

Exogenous Cx43 expression decrease cell proliferation rate in rat hepatocarcinoma cells independently of functional gap junction

Background: Gap junction intercellular communication (GJIC) is considered to play a role in the regulation of homeostasis because it regulates important processes, such as cell proliferation and cell differentiation. A reduced or lost GJIC capacity has been observed in solid tumors and studies have demonstrated that GJIC restoration in tumor cells contribute to reversion of the transformed phenotype. This observation supports the idea that restoration of the functional channel is essential in this process. However, in the last years, reports have proposed that just the increase in the expression of specific connexins can contribute to reversion of the malign phenotype in some tumor cells. In the present work, we studied the effects of exogenous Connexin 43 (Cx43) expression on the proliferative behavior and phenotype of rat hepatocarcinoma cells. Results: The exogenous Cx43 did not increase GJIC capacity of transfected cells, but it was critical to decrease the cell proliferation rate as well as reorganization of the actin filaments and cell flattening. We also observed more adhesion capacity to substrate after Cx43 transfection. Conclusion: Cx43 expression leads to a decrease of the growth of the rat hepatocellular carcinoma cells and it contributes to the reversion of the transformed phenotype. These effects were independent of the GJIC and were probably associated with the phosphorylation pattern changes and redistribution of the Cx43 protein.

Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

Background: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

Background: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. Methods: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. Results: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. Conclusions: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

Cellular Renewal and Improvement of Local Cell Effector Activity in Peritoneal Cavity in Response to Infectious Stimuli

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (Mempty set) subsets, namely small peritoneal Mempty set (SPM) and large peritoneal Mempty set (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for beta-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-gamma stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal Mempty set subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

The Spleen CD4(+) T Cell Response to Blood-Stage Plasmodium chabaudi Malaria Develops in Two Phases Characterized by Different Properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-Ab(-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-alpha and IFN-gamma production depend mostly on conventional CD4(+) T cells. IFN-gamma is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-gamma and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

Theory of small-signal ac response of a dielectric liquid containing two groups of ions

The analysis of Macdonald for electrolytes is generalized to the case in which two groups of ions are present. We assume that the electrolyte can be considered as a dispersion of ions in a dielectric liquid, and that the ionic recombination can be neglected. We present the differential equations governing the ionic redistribution when the liquid is subjected to an external electric field, describing the simultaneous diffusion of the two groups of ions in the presence of their own space charge fields. We investigate the influence of the ions on the impedance spectroscopy of an electrolytic cell. In the analysis, we assume that each group of ions have equal mobility, the electrodes perfectly block and that the adsorption phenomena can be neglected. In this framework, it is shown that the real part of the electrical impedance of the cell has a frequency dependence presenting two plateaux, related to a type of ambipolar and free diffusion coefficients. The importance of the considered problem on the ionic characterization performed by means of the impedance spectroscopy technique was discussed. (c) 2008 American Institute of Physics.