Regulatory networks of Neisseria meningitidis and their implications for pathogenesis

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.

Funktionelle Charakterisierung der Protein-Kinase CK2 in der Suppression Th2-vermittelter Immunantworten durch regulatorische T-Zellen

Regulatorische T-Zellen (Tregs) leisten durch ihre suppressiven Eigenschaften einen essenziellen Beitrag zur Aufrechterhaltung der immunologischen Toleranz. Sie verhindern schädliche Immunreaktionen gegen Autoantigene, kommensale Bakterien, sowie harmlose Nahrungsmittel-bestandteile. Gleichzeitig gewährleisten sie die Entwicklung effektiver Immunantworten gegen eindringende Pathogene, wie z.B. Parasiten, Bakterien und Viren. Damit haben Tregs direkten Einfluss auf das Gleichgewicht zwischen Immunität und Toleranz. Fehler in der suppressiven Funktionsweise von Tregs begünstigen daher auf der einen Seite die Entstehung zahlreicher autoimmuner Erkrankungen und Allergien. Auf der anderen Seite können Tregs Immunreaktionen bei chronischen Infektionen reduzieren, sowie die Entstehung effektiver Immunantworten gegen Tumore hemmen. Ihre Beteiligung an der Ätiologie all dieser Krankheiten macht Tregs zu einem bedeutenden potenziellen Zielobjekt, um diese Krankheiten effektiv zu therapieren. Die Erweiterung des Grundwissens um die molekularen Mechanismen der Treg-vermittelten Suppression ist daher ein notwendiger Schritt bei der Entwicklung Treg-basierter Theraphieansätze. 2003 konnte mit Foxp3 ein Transkriptionsfaktor identifiziert werden, der maßgeblich die suppressiven Funktionen von Tregs steuert. Um weiteren Einblick in die der Suppression zugrundeliegenden Signalwege zu erhalten, wurde im Institut für Immunologie ein komparativer Kinomarray durchgeführt, anhand dessen die Casein Kinase 2 (CK2) als eine der aktivsten Kinasen in Tregs identifiziert wurde (Daten freundlicherweise von Prof. Dr. Tobias Bopp bereitgestellt). rnBasierend auf den Ergebnissen des Kinomarrays wurde in dieser Arbeit die Funktion der CK2 in Tregs untersucht. Dabei konnte in in vitro Experimenten die Treg-vermittelte Suppression durch den pharmakologische CK2 Inhibitor DMAT aufgehoben werden. Weil derartige Inhibitoren jedoch nicht absolut spezifisch die Aktivität nur einer Kinase supprimieren, wurden außerdem Mäuse mit konditionalem „knockout“ der CK2β Untereinheit spezifisch in Tregs gekreuzt (CK2βTreg-/- Mäuse). Die Analyse dieser Tiere offenbarte eine essenzielle Beteiligung der CK2 an den suppressiven Funktionen von Tregs. So entwickeln CK2βTreg-/- Mäuse mit zunehmendem Alter Splenomegalien und Lymphadenopathien, von denen in besonderem Maße die Mukosa-assoziierten Lymphknoten betroffen sind. Eine Analyse des Aktivierungsstatus der T-Zellen in den Tieren konnte zudem einen erhöhten Anteil sogenannter Effektor-Gedächtnis T-Zellen aufdecken, die charakteristische Merkmale eines Th2 Phänotyps zeigten. Erhöhte Titer des Antikörperisotyps IgE in den Seren von CK2βTreg-/- Mäusen suggerieren zusätzlich eine fehlerhafte Suppression speziell Th2-vermittelter Immunantworten durch CK2β-defiziente Tregs. In Th2-vermittelten Asthma Experimenten in vivo konnte der Verdacht der fehlerhaften Kontrolle von Th2-Antwort bestätigt werden, wobei zusätzlich aufgedeckt wurde, dass bereits unbehandelte CK2βTreg-/- Mäuse Zeichen einer Entzündungsreaktion in der Lunge aufweisen. Bei der Suche nach den molekularen Ursachen der fehlerhaften Suppression Th2-vermittelter Immunantworten durch CK2β-defiziente Tregs konnten zwei mögliche Erklärungsansätze gefunden werden. Zum einen zeigen CK2β-defiziente Tregs eine verringerte Expression von Foxp3, was, in Analogie zu Ergebnissen der Gruppe von R. Flavell (Wang Y.Y. Nature. 445, 766-770 (2007)), zu einer Konversion von Tregs zu Th2 Zellen und damit zur Entstehung eines Th2-basierten, autoimmunen Phänotyps führt. Des Weiteren weisen CK2β-defiziente Tregs eine reduzierte Expression des Transkriptionsfaktors IRF4 auf, der in Tregs entscheidend für die Kontrolle Th2-basierter Immunreaktionen ist (Zheng Y. Nature. 19; 351-356 (2009)). Die dargelegten Ergebnisse identifizieren die CK2 damit als Kinase, die entscheidend an der Treg-vermittelten Suppression speziell Th2-basierter Immunantworten beteiligt ist. Demnach könnten pharmakologische CK2 Inhibitoren beispielsweise dazu eingesetzt werden, um die Treg-vermittelte Suppression im Rahmen chronischer Parasiten-Infektionen aufzuheben. Die in CK2βTreg-/- Mäusen beobachtete Prävalenz der Funktion der CK2 für Mukosa-assoziierte Organe stellt dabei einen zusätzlichen Vorteil dar, weil systemische Nebenwirkungen, die durch die Blockade der Treg-vermittelte Suppression entstehen, zumindest in nicht-Mukosa-assoziierten Geweben nicht zu erwarten sind.rn

Loss of the CBX7 protein expression correlates with a more aggressive phenotype in pancreatic cancer

Polycomb group (PcG) proteins function as multiprotein complexes and are part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. Several studies have implicated the deregulation of different PcG proteins in neoplastic progression. Pancreatic ductal adenocarcinoma is an aggressive neoplasm that follows a multistep model of progression through precursor lesions called pancreatic intraepithelial neoplasia (PanIN). Aim of this study was to investigate the role of PcG protein CBX7 in pancreatic carcinogenesis and to evaluate its possible diagnostic and prognostic significance. We analysed by immunohistochemistry the expression of CBX7 in 210 ductal pancreatic adenocarcinomas from resection specimens, combined on a tissue microarray (TMA) including additional 40 PanIN cases and 40 normal controls. The results were evaluated by using receiver operating characteristic (ROC) curve analysis for the selection of cut-off scores and correlated to the clinicopathological parameters of the tumours and the outcome of the patients. Expression of E-cadherin, a protein positively regulated by CBX7, was also assessed. A significantly differential, and progressively decreasing CBX7 protein expression was found between normal pancreatic tissue, PanINs and invasive ductal adenocarcinoma. Loss of CBX7 expression was associated with increasing malignancy grade in pancreatic adenocarcinoma, whereas the maintenance of CBX7 expression showed a trend toward a longer survival. Moreover, loss of E-cadherin expression was associated with loss of CBX7 and with a trend towards worse patient survival. These results suggest that CBX7 plays a role in pancreatic carcinogenesis and that its loss of expression correlates to a more aggressive phenotype.

Lysosomal degradation of RhoH protein upon antigen receptor activation in T but not B cells

RhoH is a member of the Rho (ras homologous) GTPase family, yet it lacks GTPase activity and thus remains in its active conformation. Unlike other Rho GTPases, the RhoH gene transcript is restricted to hematopoietic cells and RhoH was shown to be required for adequate T-cell activation through the TCR. Here, we demonstrate that both blood T and B cells, but not neutrophils or monocytes, express RhoH protein under physiological conditions. Upon TCR complex activation, RhoH was degraded in lysosomes of primary and Jurkat T cells. Pharmacologic activation of T cells distal to the TCR complex had no effect on RhoH protein levels suggesting that early events during T-cell activation are required for RhoH protein degradation. In contrast to T cells, activation of the BCR in blood B cells was not associated with changes in RhoH levels. These data suggest that RhoH function might be regulated by lysosomal degradation of RhoH protein following TCR complex but not BCR activation. This newly discovered regulatory pathway of RhoH expression might limit TCR signaling and subsequent T-cell activation upon Ag contact.

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy

The vascular-stromal compartment of lymph nodes is important for lymph node function, and high endothelial venules (HEVs) play a critical role in controlling the entry of recirculating lymphocytes. In autoimmune and autoinflammatory diseases, lymph node swelling is often accompanied by apparent HEV expansion and, potentially, targeting HEV expansion could be used therapeutically to limit autoimmunity. In previous studies using mostly flow cytometry analysis, we defined three differentially regulated phases of lymph node vascular-stromal growth: initiation, expansion, and the re-establishment of vascular quiescence and stabilization. In this study, we use optical projection tomography to better understand the morphologic aspects of HEV growth upon immunization with ovalbumin/CFA (OVA/CFA). We find HEV elongation as well as modest arborization during the initiation phase, increased arborization during the expansion phase, and, finally, vessel narrowing during the re-establishment of vascular quiescence and stabilization. We also examine acutely enlarged autoinflammatory lymph nodes induced by regulatory T cell depletion and show that HEVs are expanded and morphologically similar to the expanded HEVs in OVA/CFA-stimulated lymph nodes. These results reinforce the idea of differentially regulated, distinct phases of vascular-stromal growth after immunization and suggest that insights gained from studying immunization-induced lymph node vascular growth may help to understand how the lymph node vascular-stromal compartment could be therapeutically targeted in autoimmune and autoinflammatory diseases.

Regulatory mechanism of the light-activable allosteric switch LOV-TAP for the control of DNA binding: a computer simulation study

The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multiprotein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct light-oxygen-voltage (LOV)-tryptophan-activated protein (TAP), in which the LOV-2-Jα photoswitch of phototropin1 from Avena sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor (TrpR) protein from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region interlinking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA.

Characterization of the bovine IkappaB kinases (IKK)alpha and IKKbeta, the regulatory subunit NEMO and their substrate IkappaBalpha

The Nuclear factor (NF)-kappaB signalling pathway plays a critical role in the regulation and coordination of a wide range of cellular events such as cell growth, apoptosis and cell differentiation. Activation of the IKK (inhibitor of NF-kappaB kinase) complex is a crucial step and a point of convergence of all known NF-kappaB signalling pathways. To analyse bovine IKKalpha (IKK1), IKKbeta (IKK2) and IKKgamma (or NF-kappaB Essential MOdulator, NEMO) and their substrate IkappaBalpha (Inhibitor of NF-kappaB), the corresponding cDNAs of these molecules were isolated, sequenced and characterized. A comparison of the amino acid sequences with those of their orthologues in other species showed a very high degree of identity, suggesting that the IKK complex and its substrate IkappaBalpha are evolutionarily highly conserved components of the NF-kappaB pathway. Bovine IKKalpha and IKKbeta are related protein kinases showing 50% identity which is especially prominent in the kinase and leucine zipper domains. Co-immunoprecipitation assays and GST-pull-down experiments were carried out to determine the composition of bovine IKK complexes compared to that in human Jurkat T cells. Using these approaches, the presence of bovine IKK complexes harbouring IKKalpha, IKKbeta, NEMO and the interaction of IKK with its substrate IkappaBalpha could be demonstrated. Parallel experiments using human Jurkat T cells confirmed the high degree of conservation also at the level of protein-protein interactions. Finally, a yeast two-hybrid analysis showed that bovine NEMO molecules, in addition to the binding to IKKalpha and IKKbeta, also strongly interact with each other.

Assessing The Role Of Multi-protein Complexes In Determining Phenotype

Understanding regulatory mechanisms in complex biological systems is an important challenge, in particular to understand disease mechanisms, and to discover new therapies and drugs. In this paper, we consider the important question of cellular regulation of phenotype. Using single gene deletion data, we address the problem of linking a phenotype to underlying functional roles in the organism and provide a sound computational and statistical paradigm that can be extended to address more complex experimental settings such as multiple deletions. We apply the proposed approaches to publicly available data sets to demonstrate strong evidence for the involvement of multi-protein complexes in the phenotypes studied.

A Mitogen-activated protein kinase controls differentiation of bloodstream forms of Trypanosoma brucei

African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.

Proteomic alterations in heat shock protein 27 and identification of phosphoproteins in ascending aortic aneurysm associated with bicuspid and tricuspid aortic valve

Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. We have performed 2-dimensional gel electrophoresis and mass spectrometry coupled with dephosphorylation and phosphostaining experiments to reveal and define protein alterations and the high abundant structural phosphoproteins in BAV compared to TAV aortic aneurysm samples. 2-D gel patterns showed a high correlation in protein expression between BAV and TAV specimens (n=10). Few proteins showed significant differences, among those a phosphorylated form of heat shock protein (HSP) 27 with significantly lower expression in BAV compared to TAV aortic samples (p=0.02). The phosphoprotein tracing revealed four different phosphoproteins including Rho GDP dissociation inhibitor 1, calponin 3, myosin regulatory light chain 2 and four differentially phosphorylated forms of HSP27. Levels of total HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients (p=0.07). Immunohistochemical analysis revealed that differences in HSP27 occur in the cytoplasma of VSMC's and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated.

MicroRNAs: a new class of regulatory genes affecting metabolism

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by binding to target mRNAs, which leads to reduced protein synthesis and sometimes decreased steady-state mRNA levels. Although hundreds of miRNAs have been identified, much less is known about their biological function. Several studies have provided evidence that miRNAs affect pathways that are fundamental for metabolic control in higher organisms such as adipocyte and skeletal muscle differentiation. Furthermore, some miRNAs have been implicated in lipid, amino acid, and glucose homeostasis. These studies open the possibility that miRNAs may contribute to common metabolic diseases and point to novel therapeutic opportunities based on targeting of miRNAs.

High protein intake reduces intrahepatocellular lipid deposition in humans

BACKGROUND: High sugar and fat intakes are known to increase intrahepatocellular lipids (IHCLs) and to cause insulin resistance. High protein intake may facilitate weight loss and improve glucose homeostasis in insulin-resistant patients, but its effects on IHCLs remain unknown. OBJECTIVE: The aim was to assess the effect of high protein intake on high-fat diet-induced IHCL accumulation and insulin sensitivity in healthy young men. DESIGN: Ten volunteers were studied in a crossover design after 4 d of either a hypercaloric high-fat (HF) diet; a hypercaloric high-fat, high-protein (HFHP) diet; or a control, isocaloric (control) diet. IHCLs were measured by (1)H-magnetic resonance spectroscopy, fasting metabolism was measured by indirect calorimetry, insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and plasma concentrations were measured by enzyme-linked immunosorbent assay and gas chromatography-mass spectrometry; expression of key lipogenic genes was assessed in subcutaneous adipose tissue biopsy specimens. RESULTS: The HF diet increased IHCLs by 90 +/- 26% and plasma tissue-type plasminogen activator inhibitor-1 (tPAI-1) by 54 +/- 11% (P < 0.02 for both) and inhibited plasma free fatty acids by 26 +/- 11% and beta-hydroxybutyrate by 61 +/- 27% (P < 0.05 for both). The HFHP diet blunted the increase in IHCLs and normalized plasma beta-hydroxybutyrate and tPAI-1 concentrations. Insulin sensitivity was not altered, whereas the expression of sterol regulatory element-binding protein-1c and key lipogenic genes increased with the HF and HFHP diets (P < 0.02). Bile acid concentrations remained unchanged after the HF diet but increased by 50 +/- 24% after the HFHP diet (P = 0.14). CONCLUSIONS: Protein intake significantly blunts the effects of an HF diet on IHCLs and tPAI-1 through effects presumably exerted at the level of the liver. Protein-induced increases in bile acid concentrations may be involved. This trial was registered at www.clinicaltrials.gov as NCT00523562.

Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.