TRPA1 mediates aromatase inhibitor-evoked pain by the aromatase substrate androstenedione

Aromatase inhibitors (AI) induce painful musculoskeletal symptoms (AIMSS), which are dependent upon the pain transducing receptor TRPA1. However, as the AI concentrations required to engage TRPA1 in mice are higher than those found in the plasma of patients, we hypothesized that additional factors may cooperate to induce AIMSS. Here we report that the aromatase substrate androstenedione, unique among several steroid hormones, targeted TRPA1 in peptidergic primary sensory neurons in rodent and human cells expressing the native or recombinant channel. Androstenedione dramatically lowered the concentration of letrozole required to engage TRPA1. Notably, addition of a minimal dose of androstenedione to physiologically ineffective doses of letrozole and oxidative stress byproducts produces AIMSS-like behaviors and neurogenic inflammatory responses in mice. Elevated androstenedione levels cooperated with low letrozole concentrations and inflammatory mediators were sufficient to provoke AIMSS-like behaviors. The generation of such painful conditions by small quantities of simultaneously administered TRPA1 agonists justifies previous failure to identify a precise link between AIs and AIMSS, underscoring the potential of channel antagonists to treat AIMSS

TRPA1/NOX in the soma of trigeminal ganglion neurons mediates migraine-related pain of glyceryl trinitrate in mice

Glyceryl trinitrate is administered as a provocative test for migraine pain. Glyceryl trinitrate causes prolonged mechanical allodynia in rodents, which temporally correlates with delayed glyceryl trinitrate-evoked migraine attacks in patients. However, the underlying mechanism of the allodynia evoked by glyceryl trinitrate is unknown. The proalgesic transient receptor potential ankyrin 1 (TRPA1) channel, expressed by trigeminal nociceptors, is sensitive to oxidative stress and is targeted by nitric oxide or its by-products. Herein, we explored the role of TRPA1 in glyceryl trinitrate-evoked allodynia. Systemic administration of glyceryl trinitrate elicited in the mouse periorbital area an early and transient vasodilatation and a delayed and prolonged mechanical allodynia. The systemic, intrathecal or local administration of selective enzyme inhibitors revealed that nitric oxide, liberated from the parent drug by aldehyde dehydrogenase 2 (ALDH2), initiates but does not maintain allodynia. The central and the final phases of allodynia were respectively associated with generation of reactive oxygen and carbonyl species within the trigeminal ganglion. Allodynia was absent in TRPA1-deficient mice and was reversed by TRPA1 antagonists. Knockdown of neuronal TRPA1 by intrathecally administered antisense oligonucleotide and selective deletion of TRPA1 from sensory neurons in Advillin-Cre; Trpa1fl/fl mice revealed that nitric oxide-dependent oxidative and carbonylic stress generation is due to TRPA1 stimulation, and resultant NADPH oxidase 1 (NOX1) and NOX2 activation in the soma of trigeminal ganglion neurons. Early periorbital vasodilatation evoked by glyceryl trinitrate was attenuated by ALDH2 inhibition but was unaffected by TRPA1 blockade. Antagonists of the calcitonin gene-related peptide receptor did not affect the vasodilatation but partially inhibited allodynia. Thus, although both periorbital allodynia and vasodilatation evoked by glyceryl trinitrate are initiated by nitric oxide, they are temporally and mechanistically distinct. While vasodilatation is due to a direct nitric oxide action in the vascular smooth muscle, allodynia is a neuronal phenomenon mediated by TRPA1 activation and ensuing oxidative stress. The autocrine pathway, sustained by TRPA1 and NOX1/2 within neuronal cell bodies of trigeminal ganglia, may sensitize meningeal nociceptors and second order trigeminal neurons to elicit periorbital allodynia, and could be of relevance for migraine-like headaches evoked by glyceryl trinitrate in humans

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD)

Destruction of Lymphoid Organ Architecture and Hepatitis Caused by CD4+ T Cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8+ T cells. However, we now show that during LCMV infection CD4+ T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4+ T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4+ T cells reduced B cells with an IgMhighIgDlow phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4+ T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4+ T cells in the induction of immunopathology in liver and spleen. This includes the CD4+ T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses

The ErbB2 receptor and breast carcinoma cell migration : memo is a novel mediator of cell motility

The ErbB family of receptor tyrosine kinases play important role in normal physiological processes occurring during development; moreover, their deregulated expression has been implicated in human cancer. Cancer patients, whose tumors have alterations in ErbB1 or ErbB2, tend to have a more aggressive disease associated with parameters predicting a poor clinical outcome, including tumor metastases. For tumors to metastasize, the cells have to possess specific characteristics, including the ability to migrate and to invade the surrounding basal membrane. The role of the Neu/ErbB2 receptor in cancer cell migration is the major topic of this thesis. The Neu/ErbB2 receptor is often overexpressed in different human tumors, including breast and ovarian tumors. Clinical and in vitro studies revealed that Neu/ErbB2 plays important functions in tumor cell motility. Upon ErbB receptors activation via ligand-induced dimerization, receptors autophosphorylate specific tyrosines in the carboxy domain leading to activation of downstream signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K) pathways. These pathways, which are known to be important for cell migration, are involved in actin cytoskeleton remodeling, leading to formation of lamellipodia and actin stress fibers. In this work we used T47D breast carcinoma cells expressing Neu/ErbB2 add-back mutants harboring none or only one of the five major autophosphorylation sites, to study the contribution of individual Neu/ErbB2 tyrosine autophosphorylation sites in cell migration. We showed that activation of MAPK and PI3K in T47D cell failed to induce efficient cell motility in the absence of the Neu/ErbB2 tyrosines 1201 or 1227 phosphorylation. Moreover, we present evidence that efficient, long-term cell migration depends upon ongoing transcription and translation. Signaling downstream of tyrosine 1201 and 1227 is required for de novo synthesis of RNA and protein involved in cell migration. Further investigation of the function of these two tyrosines led to the identification of a novel protein that specifically interacts with the phosphorylated tyrosine 1227. We called this new protein Memo for mediator of ErbB2-driven cell motility. Memo does not bind directly to the phosphorylated tyrosine 1227 of the Neu/ErbB2 receptor, but very likely via the adaptor molecule Shc. Memo is required for ErbB2-driven breast carcinoma cell migration, because its downregulation leads to decreased motility of cells expressing the receptor with the tyrosine 1227. Interestingly, we found that Memo is not only required for migration downstream of the ErbB receptors, but it may be a general mediator of growth factor-induced breast carcinoma cell migration. Cell migration is a multistep process and we further defined at which step Memo is required. We found that upon Neu/ErbB2 activation, wild type cells, but interestingly also Memo-deficient cells form actin stress fibers and extend lamellipodia. However, Memo-deficient cells are not able to extend microtubules toward the cell cortex. There is increasing evidence that not only the actin cytoskeleton but also the microtubule cytoskeleton plays a crucial role for cell migration. For instance, microtubules are required for the polarization of the cells and also for the transport of proteins required for motility to the cell leading edge. Further studies have to be done to understand the exact role of Memo in microtubule outgrowth and its contribution to cell motility. In summary, the work presented in the thesis shows the identification of a novel protein, Memo, which is required for breast carcinoma cell migration. We propose that Memo controls cell motility by transmitting extracellular chemotactic signals to the microtuble cytoskeleton

PI3Kgamma adaptor subunits define coupling to degranulation and cell motility by distinct PtdIns(3,4,5)P3 pools in mast cells

Phosphoinositide 3-kinase gamma (PI3Kgamma) plays a major role in chronic inflammation and allergy. It is a heterodimer of a catalytic p110gamma subunit and an adaptor protein, either p101 or the p101 homolog p84 (p87(PIKAP)). It is unclear whether both PI3Kgamma complexes specifically modulate responses such as chemotaxis and degranulation. In mast cells, the p84:p110gamma complex synergizes with immunoglobulin E (IgE)- and antigen-clustered FcepsilonRI receptor signaling and is required to achieve maximal degranulation. During this process, PI3Kgamma is activated by ligands of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), in particular adenosine receptors, through autocrine and paracrine pathways. Here, we show that p110gamma needs p84 to relay signals from GPCRs to formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], phosphorylation of Akt, migration of cells, and synergistic adenosine-enforced degranulation. Furthermore, the absence of adaptor subunits could not be compensated for by increased p110gamma abundance. Differentiated, p110gamma null cells also lost adaptor proteins. Complementation of p110gamma null mast cells with p101 and p110gamma restored the activation of Akt and cell migration, but failed to support degranulation. Lack of degranulation was attributed to a change in the spatiotemporal localization of PI3Kgamma-derived PtdIns(3,4,5)P(3); although both p84:p110gamma and p101:p110gamma complexes initially deposited PtdIns(3,4,5)P(3) at the plasma membrane, p101:p110gamma-derived PtdIns(3,4,5)P(3) was rapidly endocytosed to motile, microtubule-associated vesicles. In addition, p84:p110gamma, but not p101:p110gamma signaling was sensitive to disruption of lipid rafts. Our results demonstrate a nonredundant function for the p101 and p84 PI3Kgamma adaptor proteins and show that distinct pools of PtdIns(3,4,5)P(3) at the plasma membrane can elicit specific cell responses

Prevalencia de la infección por el VIH y de Treponema pallidum en mujeres trabajadoras sexuales de Argentina Prevalence of HIV infection and Treponema pallidum in Argentine female sex workers

OBJETIVO: Estimar la prevalencia de infección por el virus de la inmunodeficiencia humana (VIH) y de Treponema pallidum en mujeres trabajadoras sexuales (MTS) de Argentina, y describir las características sociodemográficas de este grupo de población asociadas a la transmisión del VIH. MÉTODOS: Entre octubre de 2006 y diciembre de 2009 se realizó un estudio de corte transversal y un muestreo por conveniencia en MTS mayores de 18 años de nueve ciudades de Argentina. Un total de 1 255 mujeres participaron en este estudio. Se capacitó especialmente a un grupo de MTS para convocar a las otras mujeres a participar en talleres y a realizarse el diagnóstico del VIH y T. pallidum. RESULTADOS: Las características socio-epidemiológicas más destacadas de las MTS incluyeron un alto porcentaje de abuso sexual, escaso uso de preservativos con parejas sexuales no comerciales y un alto porcentaje de situaciones de violencia. La prevalencia del VIH fue de 2% (25/1 255, intervalo de confianza de 95% [IC95%]:1,2-2,8) y la de T. pallidum, de 22,4% (245/ 1 094, IC95%: 19,9-24,9). En las MTS de la ciudad de La Plata, la prevalencia del VIH fue significativamente más baja comparada con las participantes del resto del país (0,3% vs 2,7%, P < 0,05, respectivamente). CONCLUSIONES: La menor prevalencia del VIH y la mayor tasa de uso de preservativos informadas por las mujeres encuestadas de La Plata sugieren que la presencia de un lugar de atención sanitaria para MTS influye directamente en la disminución de sus conductas de riesgo y, por ende, en la infección por el VIH. La creación de centros de salud "amigables", como el que ya hay en dicha ciudad, contribuye a dar una respuesta integral al problema que enfrentan estas mujeres; asimismo, favorece su acercamiento al sistema de salud y contribuye así a revertir su situación de mayor vulnerabilidad y mayor riesgo frente al VIH y otras infecciones de transmisión sexual.<br>OBJECTIVE: Estimate the prevalence of human immunodeficiency virus (HIV) infection and Treponema pallidum in Argentine female sex workers (FSW), and describe the sociodemographic characteristics of this population group associated with HIV transmission. METHODS: A cross-sectional study and convenience sampling were conducted in FSW over 18 years of age in nine Argentine cities from October 2006 to December 2009. A total of 1 255 women participated in this study. A group of FSW was especially trained to invite other women to participate in the workshops and undergo screening for HIV and T. pallidum. RESULTS: The most noteworthy socioepidemiological characteristics of the FSW included a high percentage of sexual abuse, limited condom use with noncommercial sex partners, and a high percentage of violence. HIV prevalence was 2% (25/1 255, 95% confidence interval [95% CI]:1.2-2.8) and T. pallidum prevalence was 22.4% (245/1 094; 95% CI: 19.9-24.9). In the city of La Plata, HIV prevalence in FSW was significantly lower compared to that of other regions of the country (0.3% vs. 2.7%, P < 0.05, respectively). CONCLUSIONS: The lower HIV prevalence and the higher rate of condom use reported by the women from La Plata surveyed suggest that the presence of a health center for FSW has a direct influence on reducing risky behavior and, consequently, HIV infection. The creation of "friendly" health centers like the one already in this city contributes to providing a comprehensive response to the problems faced by these women and encourages use of the health system. It therefore helps reverse their vulnerability and higher risk of contracting HIV and other sexually transmitted infections

PI3K gamma activity in leukocytes promotes adipose tissue inflammation and early-onset insulin resistance during obesity

The phosphoinositide 3-kinase g (PI3K gamma) plays a major role in leukocyte recruitment during acute inflammation and has been proposed to inhibit classical macrophage activation by driving immunosuppressive gene expression. PI3K gamma plays an important role in diet-induced obesity and insulin resistance. In seeking to determine the underlying molecular mechanisms, we showed that PI3K gamma action in high-fat diet-induced inflammation and insulin resistance depended largely on its role in the control of adiposity, which was due to PI3K gamma activity in a nonhematopoietic cell type. However, PI3K gamma activity in leukocytes was required for efficient neutrophil recruitment to adipose tissue. Neutrophil recruitment was correlated with proinflammatory gene expression in macrophages in adipose tissue, which triggered insulin resistance early during the development of obesity. Our data challenge the concept that PI3K gamma is a general suppressor of classical macrophage activation and indicate that PI3K gamma controls macrophage gene expression by non-cell-autonomous mechanisms, the outcome of which is context-dependent

Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures.

The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200μM concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress