Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia.

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.

Innovative robust basophil activation test using a novel gating strategy reliably diagnosing allergy with full automation.

BACKGROUND: Allergy is one of the most common chronic diseases in Europe. Therefore, an increased need for specific and sensitive diagnostic tests that truly detect allergy exists. This study aimed at establishing a highly specific high-throughput and automated basophil activation test (BAT) that proves the existence of an allergy with utmost probability. METHODS: BAT from 1104 samples was analyzed; a novel gating strategy with three antibodies (FcεRIα, CD203c, CD63) was established and compared with our published protocol (12 antibodies). Based on the novel gating strategy, storage conditions, automated measurement, and analyses using R (1376 samples out of 1389) were optimized to set up a high-throughput BAT. RESULTS: No differences in sensitivity and specificity were found between the novel three antibody (FcεRIα, CD203c, CD63) and the 12 antibody gating strategy or between automated and manually analyzed samples (saving up to 90% of labor time). The time frame for basophil activation measurement after blood donation has been extended considerably (whole blood storage ≤7 days (RT) and 17 days (4°C) prior to BAT preparation and measurement). Respective storage conditions were optimized for samples after stimulation, staining, and preparation (≤7 days (RT) and 28 days (4°C)). These achievements were confirmed by a nationwide ring trial showing robustness and applicability of our BAT on a variety of flow cytometers. CONCLUSION: Our considerable optimizations overcame the hurdles that until now prevented the BAT from being used as high-throughput allergy diagnostic test in routine laboratories and shall allow for collaborative studies between clinics and research centers.

Novel insights into the role of S100A8/A9 in skin biology.

S100A8 and S100A9 belong to the damage-associated molecular pattern molecules. They are upregulated in a number of inflammatory skin disorders. Owing to their abundance in myeloid cells, the main function of S100A8/A9 has been attributed to their role in inflammatory cells. However, it is becoming increasingly clear that they also exert important roles in epithelial cells. In this review, we discuss the context-dependent function of S100A8/A9 in epithelial cells and their impact on wound healing, psoriasis and other skin diseases.

Interleukin-4-inducing principle from Schistosoma mansoni eggs contains a functional C-terminal nuclear localization signal necessary for nuclear translocation in mammalian cells but not for its uptake.

Interleukin-4-inducing principle from schistosome eggs (IPSE/alpha-1) is a protein produced exclusively by the eggs of the trematode Schistosoma mansoni. IPSE/alpha-1 is a secretory glycoprotein which activates human basophils via an IgE-dependent but non-antigen-specific mechanism. Sequence analyses revealed a potential nuclear localization signal (NLS) at the C terminus of IPSE/alpha-1. Here we show that this sequence (125-PKRRRTY-131) is both necessary and sufficient for nuclear localization of IPSE or IPSE-enhanced green fluorescent protein (EGFP) fusions. While transiently expressed EGFP-IPSE/alpha-1 was exclusively nuclear in the Huh7 and U-2 OS cell lines, a mutant lacking amino acids 125 to 134 showed both nuclear and cytoplasmic staining. Moreover, insertion of the IPSE/alpha-1 NLS into a tetra-EGFP construct rendered the protein nuclear. Alanine scanning mutagenesis revealed a requirement for the KRRR residues. Fluorescence microscopy depicted, and Western blotting further confirmed, that recombinant IPSE/alpha-1 protein added exogenously is rapidly internalized by CHO cells and accumulates in nuclei in an NLS-dependent manner. A mutant protein in which the NLS motif was disrupted by triple mutation (RRR to AAA) was able to penetrate CHO cells but did not translocate to the nucleus. Furthermore, the uptake of native glycosylated IPSE/alpha-1 was confirmed in human primary monocyte-derived dendritic cells and was found to be a calcium- and temperature-dependent process. Live-cell imaging showed that IPSE/alpha-1 is not targeted to lysosomes. In contrast, peripheral blood basophils do not take up IPSE/alpha-1 and do not require the presence of an intact NLS for activation. Taken together, our results suggest that IPSE/alpha-1 may have additional nuclear functions in host cells.

The cyclic AMP response element modulator {alpha} suppresses CD86 expression and APC function.

The cAMP response element modulator (CREM)alpha is a widely expressed transcriptional repressor that is important for the termination of the T cell immune response and contributes to the abnormal T cell function in patients with systemic lupus erythematosus. We present evidence that APCs of Crem(-/-) mice express increased amounts of the costimulatory molecule CD86 and induce enhanced Ag-dependent and Ag-independent T cell proliferation. Similarly, human APCs in which CREMalpha was selectively suppressed expressed more CD86 on the surface membrane. CREMalpha was found to bind to the CD86 promoter and suppressed its activity. Transfer of APCs from Crem(-/-) mice into naive mice facilitated a significantly stronger contact dermatitis response compared with mice into which APCs from Crem(+/+) mice had been transferred. We conclude that CREMalpha is an important negative regulator of costimulation and APC-dependent T cell function both in vitro and in vivo.

WNT6/ACC2-induced storage of triacylglycerols in macrophages is exploited by Mycobacterium tuberculosis.

In view of emerging drug-resistant tuberculosis (TB), host-directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One approach is to interfere with the formation of lipid-laden "foamy" macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase 2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and Mtb survival in macrophages. Moreover, treatment of Mtb-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights, compared with treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrate that Mtb exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding that opens new perspectives for host-directed adjunctive treatment of pulmonary TB.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia.

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Neutrophil Adhesion Is a Prerequisite for Antibody-Mediated Proteolytic Tissue Damage in Experimental Models of Epidermolysis Bullosa Acquisita.

Although uncontrolled proteolytic activity mediated by activated neutrophils is a major reason for tissue damage, therapeutic approaches using protease inhibitors are inefficient. Here, we investigated the role of the immune complex-induced neutrophil adhesion and protease release in tissue damage. We show both in vitro and in vivo that immune complex-mediated neutrophil adhesion to the target tissue depends on ß2 integrins. Without affecting elastase or reactive oxygen species release, blocking of adhesion drastically inhibited tissue damage in an experimental model of autoantibody-mediated skin blistering disease. By using a cell-bound fluorescent resonance energy transfer-based elastase sensor, we detected elastase enzyme activity on the surface of adherent cells resistant to protease inhibitors. Inhibitor resistance was lost by CD18 blockade or deficiency in vitro and in vivo. Immune complex-induced neutrophil adhesion created an enclosed protected space between the cell and its target structure where proteinases and reactive oxygen species can execute their tissue-damaging effect. Because immune complex-induced neutrophil adhesion represents an indispensable step for tissue damage of many diseases, our findings may facilitate the development of strategies for the treatment of such disorders.

Terminating the stress: peripheral peptidolysis of proopiomelanocortin-derived regulatory hormones by the dermal microvascular endothelial cell extracellular peptidases neprilysin and angiotensin-converting enzyme.

The skin including the microvascular endothelium is an established peripheral source and target of the immunomodulatory proopiomelanocortin (POMC) peptides ACTH and alpha-MSH. Whereas intracellular POMC peptide generation is well characterized, less is known on their extracellular processing in peripheral tissues by the neuropeptide-specific zinc metalloproteases neprilysin (NEP) and angiotensin-converting enzyme (ACE). This may locally control POMC peptide bioavailability and activation of ACTH/alpha-MSH-specific melanocortin receptors (MCs). In a cell-free system, endothelial cell (EC) membranes prepared from ACE(high)/NEP(low)-expressing primary human dermal microvascular ECs and the ACE(low)/NEP(high) expressing EC line HMEC-1 degraded ACTH(1-39) over time, resulting in temporary increased alpha-MSH immunoreactivity. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy peptide mapping and electrospray ionization-mass spectroscopy sequencing identified several stable fragments generated from ACTH(1-39), ACTH(1-24), and alpha-MSH by EC membranes or recombinant NEP and ACE. Whereas some fragments could be assigned to a cell-specific NEP or ACE activity, other degradation products require additional enzyme activity. Pharmacological NEP inhibition enhanced the ACTH and alpha-MSH-mediated activation of EC ectopically expressing MC(1). Likewise, selected peptides such as alpha-MSH(2-12) generated from ACTH(1-39) and alpha-MSH by recombinant NEP displayed equipotent MC(1)-activating properties in vitro and antiinflammatory activity in murine allergic contact dermatitis in vivo as compared with the parental peptides. Thus, NEP and ACE significantly contribute to the EC processing of stress hormones (ACTH) and antiinflammatory peptides (alpha-MSH), which modulates MC(1) activation but does not completely inactivate the peptide ligand. Because NEP and ACE are regulated by inflammatory mediators and UV light, this may be important for ACTH/MSH-modulated skin inflammation.

Neuropeptide control mechanisms in cutaneous biology: physiological and clinical significance.

The skin as a barrier and immune organ is exposed to omnipresent environmental challenges such as irradiation or chemical and biologic hazards. Neuropeptides released from cutaneous nerves or skin and immune cells in response to noxious stimuli are mandatory for a fine-tuned regulation of cutaneous immune responses and tissue maintenance and repair. They initialize host immune responses, but are equally important for counter regulation of proinflammatory events. Interaction of the nervous and immune systems occurs both locally - at the level of neurogenic inflammation and immunocyte activation - and centrally - by controlling inflammatory pathways such as mononuclear activation or lymphocyte cytokine secretion. Consequently, a deregulated neurogenic immune control results in disease manifestation and frequently accompanies chronic development of cutaneous disorders. The current understanding, therapeutic options, and open questions of the role that neuropeptides such as substance P, calcitonin gene-related peptide, vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide, neuropeptide Y, or others play in these events are discussed. Progress in this field will likely result in novel therapies for the management of diseases characterized by deregulated inflammation, tissue remodeling, angiogenesis, and neoplasm.

Identification of potential antigenic proteins of Theileria lestoquardi.

A PCR strategy was used to identify potential antigenic proteins of T. lestoquardi suitable for the development of an ELISA by searching for homologous proteins previously identified in other theilierial parasites to be antigenic.

A light-controlled switch after dual targeting of proliferating tumor cells via the membrane receptor EGFR and the nuclear protein Ki-67.

Using nanotechnology for optical manipulation of molecular processes in cells with high spatial and temporal precision promises new therapeutic options. Especially tumor therapy may profit as it requires a combination of both selectivity and an effective cell killing mechanism. Here we show a dual targeting approach for selective and efficient light-controlled killing of cells which are positive for epidermal growth factor receptor (EGFR) and Ki-67. Liposomes with the covalently linked EGFR antibody Erbitux enabled selective uptake of FITC-labeled Ki-67 antibody TuBB-9 in EGFR-positive cells pre-loaded with the photoactive dye BPD. After irradiation at 690 nm, BPD disrupted the endosomal membranes and delivered the antibodies to the nucleoli of the cells. The second irradiation at 490 nm activated the FITC-labeled TuBB-9, which caused inactivation of the Ki-67 protein and subsequent cell death via apoptosis. Efficient cell killing was possible at nanomolar concentrations of TuBB-9 due to the effective transport by immune liposomes and the high efficacy of the Ki-67 light-inactivation. Delivery of the liposomal constructs and cell destruction correlated well with the EGFR expression pattern of different cell lines (HeLa, OVCAR-5, MCF-7, and human fibroblasts), demonstrating an excellent selectivity.

Cutaneous allergic contact dermatitis responses are diminished in mice deficient in neurokinin 1 receptors and augmented by neurokinin 2 receptor blockage.

Sensory neuropeptides such as neurokinin A (NKA) or particularly substance P (SP) by neurokinin receptor (NK-R) activation modulate skin and immune cells functions during neurogenic inflammation. In this study, we examined the relative importance of SP/NK-1Rs or NKA/NK-2Rs in a murine model for allergic contact dermatitis (ACD) and tested if the functional absence of NK-Rs will impair inflammatory response in vivo. Mice lacking NK-1Rs (C57BL/6J-NK-1R-/-) displayed a significantly reduced ACD inflammatory ear swelling response to dinitrofluorobenzene (DNFB) with histological less edema and 50% fewer infiltrating leukocytes compared with the ACD response in wild-type (+/+) animals. In NK-1R+/+ mice, transient NK-1R inhibition impaired ACD sensitization. In vitro haptenized bone marrow-derived dendritic cells from NK-1R+/+ mice matured in the presence of an NK-1R antagonist displayed a reduced capability to induce T cell proliferation in vitro and ACD after adoptive transfer into naïve wild-type mice in vivo. By contrast, NK-2R inhibition significantly enhanced the ACD response in NK-1R null or in wild-type mice, whereas epicutaneous application of NK-2R agonists diminished the ACD inflammation. In conclusion, NK-1R and SP are required for antigen sensitization and a full inflammatory response to cutaneous allergens and NKA and the NK-2R mediate a contrasting anti-inflammatory role in ACD. Thus, SP, NKA, NK-1R, and NK-2R have important but differential roles in the regulation of cutaneous inflammatory responses.

Solar-simulated ultraviolet radiation-induced upregulation of the melanocortin-1 receptor, proopiomelanocortin, and alpha-melanocyte-stimulating hormone in human epidermis in vivo.

Ultraviolet light is one of the most crucial environmental factors with regard to its capacity to induce skin cancer, premature aging of the skin, and immunosuppression. Although ultraviolet directly affects the function of epidermal cells, many of these effects are mediated by induction of cytokines, growth factors, and neuropeptides, such as alpha-melanocyte-stimulating hormone. Recently, in addition to its well-known pigmentation inducing activity, a strong anti-inflammatory as well as an immunomodulatory potential of alpha-melanocyte-stimulating hormone has been recognized. The aim of this study was to determine, whether ultraviolet irradiation affects the expression of both alpha-melanocyte-stimulating hormone and the melanocortin-1 receptor in human epidermis in vivo. The volar aspects of the forearms were exposed to twice the minimal erythema dose of solar-simulating radiation. Three, 6, and 24 h after irradiation, the proopiomelanocortin and interleukin-10 mRNA levels in suction blister induced epidermal sheets were considerably upregulated as detected by semiquantitative reverse transcription-polymerase chain reaction. Furthermore, alpha-melanocyte-stimulating hormone and interleukin-10 protein levels in blister fluids were significantly increased 24 h after ultraviolet irradiation, an effect that could be abolished by application of the broad-spectrum sunscreen Anthélios XL prior to ultraviolet (solar-simulating radiation) exposure. In addition, enhanced melanocortin-1 receptor mRNA and receptor protein expression upon solar-simulating radiation was ascertained by reverse transcription-polymerase chain reaction and immunohistochemistry of the epidermal sheets, respectively. Proopiomelanocortin-derived neuropeptides, such as alpha-melanocyte-stimulating hormone may therefore play an important part in modulating ultraviolet-induced inflammation.

L-FABP is exclusively expressed in alveolar macrophages within the myeloid lineage: evidence for a PPARalpha-independent expression.

Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particular, PPARgamma is involved in monocyte/macrophage differentiation. Members of the fatty acid-binding protein (FABP) family have been reported to function as transactivators for PPARs. Therefore, the expression of PPARs and FABPs in the myeloid lineage was investigated by real-time PCR and immunofluorescence analysis. We found adipocyte-, epidermal-, and heart-type FABP to be ubiquitously expressed within the myeloid lineage. In contrast, liver-type FABP was exclusively detected in murine alveolar macrophages (AM), confirmed on protein level by double fluorescence analysis. The PPAR subtypes also showed a temporally and spatially regulated expression pattern in myeloid cells: the beta-subtype was expressed in bone marrow, peritoneal, and alveolar macrophages, whereas it was not detected in dendritic cells (DCs). The gamma1-isoform was present in all cells, however, at different levels, whereas the gamma2-isoform was expressed in alveolar macrophages and dendritic cells. A low level PPARalpha mRNA could be detected in peritoneal macrophages and immature dendritic cells but not in mature dendritic cells and bone marrow macrophages. Interestingly, PPARalpha mRNA was also absent in the alveolar macrophages although liver-type FABP was expressed, indicating that gene expression of liver-type FABP was independent of PPARalpha. Since liver-type FABP is known as transactivator of PPARgamma the simultaneous expression of both proteins may have general implications for the activation of PPARgamma in alveolar macrophages.

New insights into the functions of alpha-MSH and related peptides in the immune system.

There is a substantial body of evidence that the tridecapeptide alpha-melanocyte-stimulating hormone (alpha-MSH) functions as a mediator of immunity and inflammation. The immunomodulating capacity of alpha-MSH is primarily because of its effects on melanocortin receptor (MC-1R)-expressing monocytes, macrophages, and dendritic cells (DCs). alpha-MSH down-regulates the production of proinflammatory and immunomodulating cytokines (IL-1, IL-6, TNF-alpha, IL-2, IFN-gamma, IL-4, IL-13) as well as the expression of costimulatory molecules (CD86, CD40, ICAM-1) on antigen-presenting DCs. In contrast, the production of the cytokine synthesis inhibitor IL-10 is up-regulated by alpha-MSH. At the molecular level, these effects of alpha-MSH are mediated via the inhibition of the activation of transcription factors such as NFkappaB. Not only alpha-MSH but also its C-terminal tripeptide (alpha-MSH 11-13, KPV) was able to bind to MC-1R and to modulate the function of APCs. In vivo, using a mouse model of contact hypersensitivity (CHS) systemic and topical application of alpha-MSH or KPV inhibited the sensitization and the elicitation phase of CHS and was able to induce hapten-specific tolerance. To investigate the underlying mechanisms of tolerance induction, we have performed in vivo transfer experiments. Treatment of naive mice with bone marrow-derived immature haptenized and alpha-MSH-pulsed DCs resulted in a significant inhibition of CHS. Furthermore, tolerance induction was found to be mediated by the generation of CTLA4(+) and IL-10-producing T lymphocytes. The potent capacity of alpha-MSH to modulate the function of antigen-presenting cells (APCs) has been further supported in another experimental approach. In vitro, by activating APCs, alpha-MSH has been shown to modulate IgE production by IL-4 and anti-CD40 stimulated B lymphocytes. Moreover, in a murine model of allergic airway inflammation, systemic treatment with alpha-MSH resulted in a significant reduction of allergen-specific IgE production, eosinophil influx, and IL-4 production. These effects were mediated via IL-10 production, because IL-10 knockout mice were resistant to alpha-MSH treatment. Therefore, therapeutic application of alpha-MSH or related peptides (KPVs) as well as alpha-MSH/KPV-pulsed DCs may be a useful approach for the treatment of inflammatory, autoimmune, and allergic diseases in the future.

Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor.

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

Monitoring neuropeptide-specific proteases: processing of the proopiomelanocortin peptides adrenocorticotropin and alpha-melanocyte-stimulating hormone in the skin.

The neuroendocrine precursor protein proopiomelanocortin (POMC) and its derived neuropeptides are involved in a number of important regulatory processes in the central nervous system as well as in peripheral tissues. Despite its important role in controlling the local activation of melanocortin (MC) receptors, the extracellular proteolytic processing of POMC peptides has received little attention. The mechanisms relevant for controlling the bioavailability of adrenocorticotropin and melanocyte-stimulating hormones for the corresponding MC receptors in the skin by specific peptidases such as neprilysin (neutral endopeptidase; NEP) or angiotensin-converting enzyme (ACE) have been addressed in a number of recent investigations. This review summarizes the current body of knowledge concerning the qualitative and quantitative POMC peptide processing with respect to the action and specificity of NEP and ACE and discusses relevant recent analytical methodologies.

Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells.

The nuclear Ki-67 protein (pKi-67) has previously been shown to be exclusively expressed in proliferating cells. As a result, antibodies against this protein are widely used as prognostic tools in cancer diagnostics. Here we show, that despite the strong downregulation of pKi-67 expression in non-proliferating cells, the protein can nevertheless be detected at sites linked to ribosomal RNA (rRNA) synthesis. Although this finding does not argue against the use of pKi-67 as a proliferation marker, it has wide ranging implications for the elucidation of pKi-67 function. Employing the novel antibody TuBB-9, we could further demonstrate that also in proliferating cells, a fraction of pKi-67 is found at sites linked to the rRNA transcription machinery during interphase and mitosis. Moreover, chromatin immunoprecipitation (ChIP) assays provided evidence for a physical association of pKi-67 with chromatin of the promoter and transcribed region of the rRNA gene cluster. These data strongly suggest a role for pKi-67 in the early steps of rRNA synthesis.