Prognostic value of integrin αV expression and localization pattern in invasive breast carcinomas.

Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.

Environmental control of mammary carcinoma cell expansion by acidification and spheroid formation in vitro.

Breast cancer is the leading cause of cancer death among women worldwide. Like other cancers, mammary carcinoma progression involves acidification of the tumor microenvironment, which is an important factor for cancer detection and treatment strategies. However, the effects of acidity on mammary carcinoma cell morphology and phenotype have not been thoroughly characterized. Here, we evaluated fundamental effects of environmental acidification on mammary carcinoma cells in standard two-dimensional cultures and three-dimensional spheroids. Acidification decreased overall mammary carcinoma cell viability, while increasing their resistance to the anthracycline doxorubicin. Environmental acidification also increased extracellular vesicle production by mammary carcinoma cells. Conditioned media containing these vesicles appeared to increase fibroblast motility. Acidification also increased mammary carcinoma cell motility when cultured with fibroblasts in spheroids. Taken together, results from this study suggest that environmental acidification induces drug resistance and extracellular vesicle production by mammary carcinoma cells that promote tumor expansion.

Combined p14ARF and Interferon-ß Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation.

Immune evasion is an important cancer hallmark and the understanding of its mechanisms has generated successful therapeutic approaches. Induction of immunogenic cell death (ICD) is expected to attract immune cell populations that promote innate and adaptive immune responses. Here, we present a critical advance for our adenovirus-mediated gene therapy approach, where the combined p14ARF and human interferon-ß (IFNß) gene transfer to human melanoma cells led to oncolysis, ICD and subsequent activation of immune cells. Our results indicate that IFNß alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. In situ gene therapy of s.c. SK-MEL-147 tumors in Nod-Scid mice revealed inhibition of tumor growth and increased survival in response to IFNß alone or in combination with p14ARF. Emission of critical markers of ICD (exposition of calreticulin, secretion of ATP and IFNß) was stronger when cells were treated with combined p14ARF and IFNß gene transfer. Co-culture of previously transduced SK-MEL-147 cells with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors resulted in increased levels of activation markers HLA-DR, CD80, and CD86. Activated Mo-DCs were able to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. Preliminary data showed that T cells primed by Mo-DCs activated with p14ARF+IFNß-transduced SK-MEL-147 cells were able to induce the loss of viability of fresh non-transduced SK-MEL-147 cells, suggesting the induction of a specific cytotoxic population that recognized and killed SK-MEL-147 cells. Collectively, our results indicate that p14ARF and IFNß delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

Trichomes related to an unusual method of water retention and protection of the stem apex in an arid zone perennial species.

It is well known that trichomes protect plant organs, and several studies have investigated their role in the adaptation of plants to harsh environments. Recent studies have shown that the production of hydrophilic substances by glandular trichomes and the deposition of this secretion on young organs may facilitate water retention, thus preventing desiccation and favouring organ growth until the plant develops other protective mechanisms. Lychnophora diamantinana is a species endemic to the Brazilian 'campos rupestres' (rocky fields), a region characterized by intense solar radiation and water deficits. This study sought to investigate trichomes and the origin of the substances observed on the stem apices of L. diamantinana. Samples of stem apices, young and expanded leaves were studied using standard techniques, including light microscopy and scanning and transmission electron microscopy. Histochemical tests were used to identify the major groups of metabolites present in the trichomes and the hyaline material deposited on the apices. Non-glandular trichomes and glandular trichomes were observed. The material deposited on the stem apices was hyaline, highly hydrophilic and viscous. This hyaline material primarily consists of carbohydrates that result from the partial degradation of the cell wall of uniseriate trichomes. This degradation occurs at the same time that glandular trichomes secrete terpenoids, phenolic compounds and proteins. These results suggest that the non-glandular trichomes on the leaves of L. diamantinana help protect the young organ, particularly against desiccation, by deposition of highly hydrated substances on the apices. Furthermore, the secretion of glandular trichomes probably repels herbivore and pathogen attacks.

Upregulation of innate antiviral restricting factor expression in the cord blood and decidual tissue of HIV-infected mothers.

Programs for the prevention of mother-to-child transmission of HIV have reduced the transmission rate of perinatal HIV infection and have thereby increased the number of HIV-exposed uninfected (HEU) infants. Natural immunity to HIV-1 infection in both mothers and newborns needs to be further explored. In this study, we compared the expression of antiviral restricting factors in HIV-infected pregnant mothers treated with antiretroviral therapy (ART) in pregnancy (n=23) and in cord blood (CB) (n=16), placental tissues (n=10-13) and colostrum (n=5-6) samples and compared them to expression in samples from uninfected (UN) pregnant mothers (n=21). Mononuclear cells (MNCs) were prepared from maternal and CB samples following deliveries by cesarean section. Maternal (decidua) and fetal (chorionic villus) placental tissues were obtained, and colostrum was collected 24 h after delivery. The mRNA and protein expression levels of antiviral factors were then evaluated. We observed a significant increase in the mRNA expression levels of antiviral factors in MNCs from HIV-infected mothers and CB, including the apolipoprotein B mRNA-editing enzyme 3G (A3G), A3F, tripartite motif family-5α (TRIM-5α), TRIM-22, myxovirus resistance protein A (MxA), stimulator of interferon (IFN) genes (STING) and IFN-ß, compared with the levels detected in uninfected (UN) mother-CB pairs. Moreover, A3G transcript and protein levels and α-defensin transcript levels were decreased in the decidua of HIV-infected mothers. Decreased TRIM-5α protein levels in the villi and increased STING mRNA expression in both placental tissues were also observed in HIV-infected mothers compared with uninfected (UN) mothers. Additionally, colostrum cells from infected mothers showed increased tetherin and IFN-ß mRNA levels and CXCL9 protein levels. The data presented here indicate that antiviral restricting factor expression can be induced in utero in HIV-infected mothers. Future studies are warranted to determine whether this upregulation of antiviral factors during the perinatal period has a protective effect against HIV-1 infection.

TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns.

Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway could function as an adjuvant to improve maternal-neonatal innate immunity.

Profile of autoantibodies against phosphorylcholine and cross-reactivity to oxidation-specific neoantigens in selective IgA deficiency with or without autoimmune diseases.

Immunoglobulin A deficiency (IgAD) is considered the most common form of primary immunodeficiency. The majority of IgA-deficient individuals are considered asymptomatic, even though IgAD has been associated with an increased frequency of recurrent infections, allergy, and autoimmune diseases. In this study we evaluate the Natural autoantibodies (NatAbs) reactivity to phosphorylcholine (PC) and to some pro-inflammatory molecules in IgAD with or without autoimmune disorders. We observed that in the absence of IgA there is an enhancement of IgG subclasses functioning as NatAbs against PC. Immunoglobulin G (IgG) against lipopolysaccharide, C-reactive protein, and IgA was found in IgAD, regardless of the autoimmune manifestations. Nonetheless, IgAD patients with autoimmune disease showed significantly higher IgG reactivity against pro-inflammatory molecules, such as cardiolipin, oxidized low-density lipoproteins, and phosphatidylserine, with positive correlation between them. In conclusion, the IgG NatAbs against PC may represent a compensatory defense mechanism against infections and control excess of inflammation, explaining the asymptomatic status in the IgA deficiency.