Repetitive Exposure of IL-17 Into the Murine Air Pouch Favors the Recruitment of Inflammatory Monocytes and the Release of IL-16 and TREM-1 in the Inflammatory Fluids.

The infiltration of Th17 cells in tissues and organs during the development of many autoimmune diseases is considered a key step toward the establishment of chronic inflammation. Indeed, the localized and prolonged release of IL-17 in specific tissues has been associated with an increased severity of the inflammatory response that remains sustained over time. The cellular and molecular mechanisms behind these effects are far from being clear. In this study we investigated the effects of two repetitive administration of recombinant IL-17 into the murine air pouch to simulate a scenario where IL-17 is released over time in a pre-inflamed tissue. Consistent with our previous observations, mice receiving a single dose of IL-17 showed a transitory influx of neutrophils into the air pouch that peaked at 24 h and declined at 48 h. Conversely, mice receiving a double dose of the cytokine-one at time 0 and the second after 24 h-showed a more dramatic inflammatory response with almost 2-fold increase in the number of infiltrated leukocytes and significant higher levels of TNF-α and IL-6 in the inflammatory fluids. Further analysis of the exacerbated inflammatory response of double-injected IL-17 mice showed a unique cellular and biochemical profile with inflammatory monocytes as the second main population emigrating to the pouch and IL-16 and TREM-1 as the most upregulated cytokines found in the inflammatory fluids. Most interestingly, mice receiving a double injection of IL-1ß did not show any change in the cellular or biochemical inflammatory response compared to those receiving a single injection or just vehicle. Collectively these results shed some light on the function of IL-17 as pro-inflammatory cytokine and provide possible novel ways to target therapeutically the pathogenic effects of IL-17 in autoimmune conditions.

Effects of Exemestane and Tamoxifen treatment on bone texture analysis assessed by TBS in comparison with bone mineral density assessed by DXA in women with breast cancer.

We performed an analysis of a substudy of the randomized Tamoxifen Exemestane Adjuvant Multinational trial to determine the effects of exemestane (EXE) and tamoxifen (TAM) adjuvant treatment on bone mineral density (BMD) measured by dual-energy X-ray absorptiometry compared with the trabecular bone score, a novel grey-level texture measurement that correlates with 3-dimensional parameters of bone texture in postmenopausal women with hormone receptor-positive breast cancer for the first time. In total, 36 women were randomized to receive TAM (n = 17) or EXE (n = 19). Patients receiving TAM showed a mean increase of BMD in lumbar spine from baseline of 1.0%, 1.5%, and 1.9% and in trabecular bone score of 2.2%, 3.5%, and 3.3% at 6-, 12-, and 24-mo treatment, respectively. Conversely, patients receiving EXE showed a mean decrease from baseline in lumbar spine BMD of -2.3%, -3.6%, and -5.3% and in trabecular bone score of -0.9%, -1.7%, and -2.3% at 6-, 12-, and 24-mo treatment, respectively. Changes in trabecular bone score from baseline at spine were also significantly different between EXE and TAM: p = 0.05, 0.007, and 0.006 at 6, 12, and 24 mo, respectively. TAM induced an increase in BMD and bone texture analysis, whereas EXE resulted in decreases. The results were independent from each other.

Human CD4+CD25+ regulatory T cells are sensitive to low dose cyclophosphamide: implications for the immune response.

Regulatory T cells (Treg) play a pivotal role in the immune system since they inhibit the T cell response. It is well known that cyclophosphamide applied at low dose is able to stimulate the immune response while high dose cyclophosphamide exerts inhibitory activity. Data obtained in mice indicate that cyclophosphamide provokes a reduction in the number of Treg and impairs their suppressive activity, resulting in immune stimulation. Here, we addressed the question of the sensitivity of human Treg to cyclophosphamide, comparing Treg with cytotoxic T cells (CTL) and T helper cells (Th). We show that Treg are more sensitive than CTL and Th to mafosfamide, which is an active derivative of cyclophosphamide, which does not need metabolic activation. The high sensitivity of Treg was due to the induction of apoptosis. Treg compared to CTL and Th were not more sensitive to the alkylating drugs temozolomide and nimustine and also not to mitomycin C, indicating a specific Treg response to mafosfamide. The high sensitivity of Treg to mafosfamide resulted not only in enhanced cell death, but also in impaired Treg function as demonstrated by a decline in the suppressor activity of Treg in a co-culture model with Th and Helios positive Treg. Treatment of Treg with mafosfamide gave rise to a high level of DNA crosslinks, which were not repaired to the same extent as observed in Th and CTL. Also, Treg showed a low level of γH2AX foci up to 6 h and a high level 24 h after treatment, indicating alterations in the DNA damage response. Overall, this is the first demonstration that human Treg are, in comparison with Th and CTL, hypersensitive to cyclophosphamide, which is presumably due to a DNA repair defect.

Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant.

Immunodeficiency is a severe therapy-limiting side effect of anticancer chemotherapy resulting from sensitivity of immunocompetent cells to DNA damaging agents. A central role in the immune system is played by monocytes that differentiate into macrophages and dendritic cells (DCs). In this study we compared human monocytes isolated from peripheral blood and cytokine matured macrophages and DCs derived from them and assessed the mechanism of toxicity of the DNA methylating anticancer drug temozolomide (TMZ) in these cell populations. We observed that monocytes, but not DCs and macrophages, were highly sensitive to the killing effect of TMZ. Studies on DNA damage and repair revealed that the initial DNA incision was efficient in monocytes while the re-ligation step of base excision repair (BER) can not be accomplished, resulting in an accumulation of DNA single-strand breaks (SSBs). Furthermore, monocytes accumulated DNA double-strand breaks (DSBs) following TMZ treatment, while DCs and macrophages were able to repair DSBs. Monocytes lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 whose expression is restored during differentiation into macrophages and DCs following treatment with GM-CSF and GM-CSF plus IL-4, respectively. These proteins play a key role both in BER and DSB repair by B-NHEJ, which explains the accumulation of DNA breaks in monocytes following TMZ treatment. Although TMZ provoked an upregulation of XRCC1 and ligase IIIα, BER was not enhanced likely because PARP-1 was not upregulated. Accordingly, inhibition of PARP-1 did not sensitize monocytes, but monocyte-derived DCs in which strong PARP activation was observed. TMZ induced in monocytes the DNA damage response pathways ATM-Chk2 and ATR-Chk1 resulting in p53 activation. Finally, upon activation of the Fas-receptor and the mitochondrial pathway apoptosis was executed in a caspase-dependent manner. The downregulation of DNA repair in monocytes, resulting in their selective killing by TMZ, might impact on the immune response during cancer chemotherapy.

Managing arthralgia in a postmenopausal woman taking an aromatase inhibitor for hormonesensitive early breast cancer: a case study.

BACKGROUND: In order to reduce the risk of recurrence, adjuvant treatment with an aromatase inhibitor (AI) is recommended for postmenopausal women following surgery for hormone receptor-positive breast cancer. AIs are associated with improved disease-free survival compared with tamoxifen. The adverse events associated with AIs resemble those of menopause, such as bone density loss and musculoskeletal symptoms. PURPOSE: We examine the case of a postmenopausal woman who was prescribed anastrozole, a nonsteroidal AI, as adjuvant therapy following surgery for estrogen and progesterone receptor-positive (ER and PgR+) breast cancer. METHODS AND SAMPLE: A 58-year-old postmenopausal woman diagnosed with ER and PgR+ breast cancer was prescribed anastrozole as adjuvant therapy following a right-inferior quadrantectomy. After experiencing joint pain and stiffness, she was prescribed paracetamol and a topical nonsteroidal anti-inflammatory drug. She was also counseled on nonpharmacological interventions. However, she continued to experience symptoms, and reported that she was not taking anastrozole regularly. RESULTS: The case study patient ultimately found relief by switching to letrozole, another aromatase inhibitor. This approach is supported by recent studies examining the benefits of switching strategies between aromatase inhibitors in order to relieve symptoms of arthralgia/myalgia. CONCLUSIONS: Both adherence and strategies for managing aromatase inhibitor-associated arthralgia are key to deriving maximal clinical benefit from AI therapy. Switching from one aromatase inhibitor to another may provide a viable option in managing adverse events and enhancing adherence to medication.

Human monocytes are severely impaired in base and DNA double-strand break repair that renders them vulnerable to oxidative stress.

Monocytes are key players in the immune system. Crossing the blood barrier, they infiltrate tissues and differentiate into (i) macrophages that fight off pathogens and (ii) dendritic cells (DCs) that activate the immune response. A hallmark of monocyte/macrophage activation is the generation of reactive oxygen species (ROS) as a defense against invading microorganisms. How monocytes, macrophages, and DCs in particular respond to ROS is largely unknown. Here we studied the sensitivity of primary human monocytes isolated from peripheral blood and compared them with macrophages and DCs derived from them by cytokine maturation following DNA damage induced by ROS. We show that monocytes are hypersensitive to ROS, undergoing excessive apoptosis. These cells exhibited a high yield of ROS-induced DNA single- and double-strand breaks and activation of the ATR-Chk1-ATM-Chk2-p53 pathway that led to Fas and caspase-8, -3, and -7 activation, whereas macrophages and DCs derived from them were protected. Monocytes are also hypersensitive to ionizing radiation and oxidized low-density lipoprotein. The remarkable sensitivity of monocytes to oxidative stress is caused by a lack of expression of the DNA repair proteins XRCC1, ligase IIIα, poly(ADP-ribose) polymerase-1, and catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs)), causing a severe DNA repair defect that impacts base excision repair and double-strand break repair by nonhomologous end-joining. During maturation of monocytes into macrophages and DCs triggered by the cytokines GM-CSF and IL-4, these proteins become up-regulated, making macrophages and DCs repair-competent and ROS-resistant. We propose that impaired DNA repair in monocytes plays a role in the regulation of the monocyte/macrophage/DC system following ROS exposure.

Beta1 integrin-mediated adhesion signalling is essential for epidermal progenitor cell expansion.

BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate this discrepancy we generated hypomorphic mice expressing reduced beta1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.

Kindlin-3 is required for beta2 integrin-mediated leukocyte adhesion to endothelial cells.

Integrin activation is essential for the function of all blood cells, including platelets and leukocytes. The blood cell-specific FERM domain protein Kindlin-3 is required for the activation of the beta1 and beta3 integrins on platelets. Impaired activation of beta1, beta2 and beta3 integrins on platelets and leukocytes is the hallmark of a rare autosomal recessive leukocyte adhesion deficiency syndrome in humans called LAD-III, characterized by severe bleeding and impaired adhesion of leukocytes to inflamed endothelia. Here we show that Kindlin-3 also binds the beta2 integrin cytoplasmic domain and is essential for neutrophil binding and spreading on beta2 integrin-dependent ligands such as intercellular adhesion molecule-1 and the complement C3 activation product iC3b. Moreover, loss of Kindlin-3 expression abolished firm adhesion and arrest of neutrophils on activated endothelial cells in vitro and in vivo, whereas selectin-mediated rolling was unaffected. Thus, Kindlin-3 is essential to activate the beta1, beta2 and beta3 integrin classes, and loss of Kindlin-3 function is sufficient to cause a LAD-III-like phenotype in mice.

Beta1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity.

Inhibiting the alpha(4) subunit of the integrin heterodimers alpha(4)beta(1) and alpha(4)beta(7) with the monoclonal antibody natalizumab is an effective treatment for multiple sclerosis (MS). However, the pharmacological action of natalizumab is not understood conclusively. Previous studies suggested that natalizumab inhibits activation, proliferation, or extravasation of inflammatory cells. To specify which mechanisms, cell types, and alpha(4) heterodimers are affected by the antibody treatment, we studied MS-like experimental autoimmune encephalomyelitis (EAE) in mice lacking the beta(1)-integrin gene either in all hematopoietic cells or selectively in T lymphocytes. Our results show that T cells critically rely on beta(1) integrins to accumulate in the central nervous system (CNS) during EAE, whereas CNS infiltration of beta(1)-deficient myeloid cells remains unaffected, suggesting that T cells are the main target of anti-alpha(4)-antibody blockade. We demonstrate that beta(1)-integrin expression on encephalitogenic T cells is critical for EAE development, and we therefore exclude alpha(4)beta(7) as a target integrin of the antibody treatment. T cells lacking beta(1) integrin are unable to firmly adhere to CNS endothelium in vivo, whereas their priming and expansion remain unaffected. Collectively, these results suggest that the primary action of natalizumab is interference with T cell extravasation via inhibition of alpha(4)beta(1) integrins.

Executive functions and risky decision-making in patients with opiate dependence.

Recent evidence suggests that individuals with opiate dependence may have cognitive dysfunctions particularly within the spectrum of executive functioning and emotional processing. Such dysfunctions can also compromise daily decisions associated with risk-taking behaviors. However, it remains unclear whether patients addicted to opiates show impaired decision-making on gambling tasks that specify explicit rules for rewards and punishments and provide information about probabilities associated with different long-term outcomes. In this study, we examined 18 individuals with opiate dependence and 18 healthy comparison subjects, matched for age, gender, and education with the Game of Dice Task (GDT). The GDT is a gambling task with explicit rules for gains and losses and fix winning probabilities. In addition, all subjects completed a neuropsychological test battery that primarily focused on executive functions and a personality questionnaire. On the GDT, patients chose the risky alternatives more frequently than the control group. Patients' GDT performance was related to executive functioning but not to other neuropsychological constructs, personality or dependence specific variables with one exception that is the number of days of abstinence. Thus, patients with opiate dependence demonstrate abnormalities in decision-making that might be neuropsychologically associated with dysfunctional behavior in patients' daily lives. Decision-making and other neuropsychological functioning should be considered in the treatment of opiate dependence.

Myocardial ischaemia-reperfusion injury in haematopoietic cell-restricted beta1 integrin knockout mice.

Evidence indicates that the intercellular adhesion molecule-1 and its counter-receptor beta2 integrin are cardioprotective proteins during myocardial ischaemia-reperfusion, but no data are available concerning the role of blood cell beta1 integrins in this process. We studied the effects of temporary myocardial ischaemia and reperfusion in blood cell-restricted beta1 integrin knockout mice (beta1(-/-)). The left descending coronary artery in conditional beta1(-/-) integrin (beta1(-/-)), beta1 integrin +/+ (beta1(+/+)) and beta1 integrin -/- bone marrow chimeric (beta1(-/-) BM) mice was ligated for 30 min, followed by reperfusion of either 3 h or 3 weeks. Plasma levels of troponin T were evaluated as an index of cardiac cellular damage. The histological evaluation of tissue damage was performed with Haematoxylin and Eosin stained sections. Cell infiltrations in the ischaemic area were investigated by immunofluorescence studies. It was found that plasma troponin T was at a similar level in beta1(-/-), beta1(+/+) and beta1(-/-) BM mice treated with 30 min ischaemia and 3 h reperfusion. Histological analysis showed that ischaemia-reperfusion resulted in marked myocardial injury in all groups of animals, but the damage score of the hearts was not significantly different between beta1(-/-), beta1(+/+) and beta1(-/-) BM mice after 3 h of reperfusion following 30 min of ischaemia (2.8 +/- 0.5, 2.6 +/- 0.5 and 2.8 +/- 0.6, respectively, n.s.). Furthermore, no difference in scar sizes in ischaemia-injured hearts was found 3 weeks after ischaemia. Semi-quantification of cells demonstrated that, compared with beta1(+/+) mice, the number of infiltrating neutrophils was significantly reduced in beta1(-/-) and beta1(-/-) BM mice, whereas MAC-1(CD11b/CD18)-positive cells in the ischaemic regions were similar in myocardial tissues of all groups. We conclude that absence of beta1 integrin expression in haematopoietic cells results in reduced neutrophil infiltration in the ischaemic regions, but does not influence myocardial damage of ischaemic hearts.

Overlapping activator sequences determined for two oppositely oriented promoters in halophilic Archaea.

Transcription of the genomic region involved in gas vesicle formation in Halobacterium salinarum (p-vac) and Haloferax mediterranei (mc-vac) is driven by two divergent promoters, P(A) and P(D), separated by only 35 nt. Both promoters are activated by the transcription activator GvpE which in the case of P(mcA) requires a 20-nt sequence (UAS) consisting of two conserved 8-nt sequence portions located upstream of BRE. Here, we determined the two UAS elements in the promoter region of p-vac by scanning mutageneses using constructs containing P(pD) (without P(pA)) fused to the bgaH reporter gene encoding an enzyme with beta-galactosidase activity, or the dual reporter construct pApD with P(pD) fused to bgaH and P(pA) to an altered version of gvpA. The two UAS elements found exhibited a similar extension and distance to BRE as previously determined for the UAS in P(mcA). Their distal 8-nt portions almost completely overlapped in the centre of P(pD)-P(pA), and mutations in this region negatively affected the GvpE-mediated activation of both promoters. Any alteration of the distance between BRE and UAS resulted in the loss of the GvpE activation, as did a complete substitution of the proximal 8-nt portion, underlining that a close location of UAS and BRE was very important.

Analysis of integrin functions in peri-implantation embryos, hematopoietic system, and skin.

Integrins mediate cell adhesion, permit traction forces important for cell migration, and cross-talk with growth factor receptors to regulate cell proliferation, cell survival, and cell differentiation. The plethora of functions explains their central role for development and disease. The progress in mouse genetics and the ease with which the mouse genome can be manipulated enormously contributed to our understanding of how integrins exert their functions at the molecular level. In the present chapter, we describe tests that are routinely used in our laboratory to investigate embryos, organs, and cells (peri-implantation embryos, hematopoietic system, epidermis, and hair follicles) that lack the expression of integrins or integrin-associated proteins.

RhoH is important for positive thymocyte selection and T-cell receptor signaling.

RhoH is a small GTPase expressed only in the hematopoietic system. With the use of mice with targeted disruption of the RhoH gene, we demonstrated that RhoH is crucial for thymocyte maturation during DN3 to DN4 transition and during positive selection. Furthermore, the differentiation and expansion of DN3 and DN4 thymocytes in vitro were severely impaired. These defects corresponded to defective TCR signaling. Although RhoH is not required for TCR-induced activation of ZAP70 and ZAP70-mediated activation of p38, it is crucial for the tyrosine phosphorylation of LAT, PLCgamma1, and Vav1 and for the activation of Erk and calcium influx. These data suggest that RhoH is important for pre-TCR and TCR signaling because it allows the efficient interaction of ZAP70 with the LAT signalosome, thus regulating thymocyte development.

Beta1 integrins: zip codes and signaling relay for blood cells.

At least eight of the twelve known members of the beta1 integrin family are expressed on hematopoietic cells. Among these, the VCAM-1 receptor alpha4beta1 has received most attention as a main factor mediating firm adhesion to the endothelium during blood cell extravasation. Therapeutic trials are ongoing into the use of antibodies and small molecule inhibitors to target this interaction and hence obtain anti-inflammatory effects. However, extravasation is only one possible process that is mediated by beta1 integrins and there is evidence that they also mediate leukocyte retention and positioning in the tissue, lymphocyte activation and possibly migration within the interstitium. Genetic mouse models where integrins are selectively deleted on blood cells have been used to investigate these functions and further studies will be invaluable to critically evaluate therapeutic trials.

Adult murine hematopoiesis can proceed without beta1 and beta7 integrins.

The function of alpha4beta1 and alpha4beta7 integrins in hematopoiesis is controversial. While some experimental evidence suggests a crucial role for these integrins in retention and expansion of progenitor cells and lymphopoiesis, others report a less important role in hematopoiesis. Using mice with a deletion of the beta1 and the beta7 integrin genes restricted to the hematopoietic system we show here that alpha4beta1 and alpha4beta7 integrins are not essential for differentiation of lymphocytes or myelocytes. However, beta1beta7 mutant mice displayed a transient increase of colony-forming unit (CFU-C) progenitors in the bone marrow and, after phenylhydrazine-induced anemia, a decreased number of splenic erythroid colony-forming units in culture (CFUe's). Array gene expression analysis of CD4(+)CD8(+) double-positive (DP) and CD4(-)CD8(-) double-negative (DN) thymocytes and CD19(+) and CD4(+) splenocytes did not provide any evidence for a compensatory mechanism explaining the mild phenotype. These data show that alpha4beta1 and alpha4beta7 are not required for blood cell differentiation, although in their absence alterations in numbers and distribution of progenitor cells were observed.

Dysfunction of endocytic and autophagic pathways in a lysosomal storage disease.

OBJECTIVE: To understand the mechanisms of skeletal muscle destruction and resistance to enzyme replacement therapy in Pompe disease, a deficiency of lysosomal acid alpha-glucosidase (GAA), in which glycogen accumulates in lysosomes primarily in cardiac and skeletal muscles. METHODS: We have analyzed compartments of the lysosomal degradative pathway in GAA-deficient myoblasts and single type I and type II muscle fibers isolated from wild-type, untreated, and enzyme replacement therapy-treated GAA knock-out mice. RESULTS: Studies in myoblasts from GAA knock-out mice showed a dramatic expansion of vesicles of the endocytic/autophagic pathways, decreased vesicular movement in overcrowded cells, and an acidification defect in a subset of late endosomes/lysosomes. Analysis by confocal microscopy of isolated muscle fibers demonstrated that the consequences of the lysosomal glycogen accumulation are strikingly different in type I and II muscle fibers. Only type II fibers, which are the most resistant to therapy, contain large regions of autophagic buildup that span the entire length of the fibers. INTERPRETATION: The vastly increased autophagic buildup may be responsible for skeletal muscle damage and prevent efficient trafficking of replacement enzyme to lysosomes.