Expression of Toll-like receptors in oral squamous cell carcinoma.

Almost 380,000 new cases of oral cancer were reported worldwide in 2020. Oral squamous cell carcinoma (OSCC) accounts for 90% of all types of oral cancers. Emerging studies have shown association of Toll-like receptors (TLRs) in carcinogenesis. The present study aimed to investigate the expression levels and tissue localization of TRL1 to TRL10 and NF-κB between OSCC and healthy oral mucosa, as well as effect of Candida colonization in TRL expression in OSCC. Full thickness biopsies and microbial samples from 30 newly diagnosed primary OSCC patients and 26 health controls were collected. The expression of TLR1 to TLR10 and NF-κB was analyzed by immunohistochemistry. Microbial samples were collected from oral mucosa to detect Candida. OSCC epithelium showed lower staining intensity of TRL1, TRL2 TRL5, and TRL8 as compared to healthy controls. Similarly, staining intensity of TRL3, TRL4, TRL7, and TRL8 were significantly decreased in basement membrane (BM) zone. Likewise, OSCC endothelium showed lower staining intensity of TLR4, TLR7 and TLR8. Expression of NF-κB was significantly stronger in normal healthy tissue compared to OSCC sample. Positive correlation was found between the expression of NF-κB, TRL9 and TRL10 in basal layer of the infiltrative zone OSCC samples (P = 0.04 and P = 0.002, respectively). Significant increase in TRL4 was seen in BM zone of sample colonized with Candida (P = 0.01). According to the limited number of samples, our data indicates downregulation of TLRs and NF-κB in OSCC, and upregulation of TLR4 expression with presence of Candida.

Gut microbiome-derived bacterial extracellular vesicles in patients with solid tumours.

INTRODUCTION: Gut microbiome-derived nanoparticles, known as bacterial extracellular vesicles (bEVs), have garnered interest as promising tools for studying the link between the gut microbiome and human health. The diverse composition of bEVs, including their proteins, mRNAs, metabolites, and lipids, makes them useful for investigating diseases such as cancer. However, conventional approaches for studying gut microbiome composition alone may not be accurate in deciphering host-gut microbiome communication. In clinical microbiome research, there is a gap in the knowledge on the role of bEVs in solid tumor patients. OBJECTIVES: Analyzing the functionality of bEVs using (meta)genomics and proteomics could highlight the unique aspects of host-gut microbiome interactions in solid tumor patients. Therefore, we performed a comparative analysis of the proteome and microbiota composition of gut microbiome-derived bEVs isolated from patients with solid tumors and healthy controls. METHODS: After isolating bEVs from the feces of solid tumor patients and healthy controls, we performed spectrometry analysis of their proteomes and next-generation sequencing (NGS) of the 16S gene. We also investigated the gut microbiomes of feces from patients and controls using 16S sequencing and used machine learning to classify the samples into patients and controls based on their bEVs and fecal microbiomes. RESULTS: Solid tumor patients showed decreased microbiota richness and diversity in both the bEVs and feces. However, the bEV proteomes were more diverse in patients than in the controls and were enriched with proteins associated with the metabolism of amino acids and carbohydrates, nucleotide binding, and oxidoreductase activity. Metadata classification of samples was more accurate using fecal bEVs (100%) compared with fecal samples (93%). CONCLUSION: Our findings suggest that bEVs are unique functional entities. There is a need to explore bEVs together with conventional gut microbiome analysis in functional cancer research to decipher the potential of bEVs as cancer diagnostic or therapeutic biomarkers.

Delivery Mode and Perinatal Antibiotics Influence the Infant Gut Bacteriome and Mycobiome: A Network Analysis.

Both exposure to antibiotics at birth and delivery via Caesarean section influence the gut bacteriome's development in infants. Using 16S rRNA and internal transcribed spacer sequencing on the Ion Torrent platform, we employed network analysis to investigate the bacterial and fungal interkingdom relationships in the gut microbiome from birth to age 18 months in a prospective cohort study of 140 infants. The gut microbiome at ages six and 18 months revealed distinctive microbial interactions, including both positive and negative associations between bacterial and fungal genera in the gut ecosystem. Perinatal factors, delivery mode and intrapartum antibiotic exposure affected the associations between bacterial and fungal species. In infants exposed and unexposed to perinatal antibiotics, the gut microbiome formed distinct networks for the bacteriome and mycobiome. The fungi Saccharomyces, Trichosporon, Pezoloma, Cystofilobasidium, Rigidoporus and Fomitopsis were strongly associated with exposure to antibiotics at birth. Hyaloscypha, Trichosporon, Fomitopsis and Vishniacozyma were strongly associated with the control group that was not exposed to antibiotics. Five distinct networks were formed according to delivery mode. The present study confirms that bacteria and fungi clearly interact in the infant gut ecosystem. Furthermore, perinatal factors appear to influence the relationships between bacteria and fungi in the developing gut microbiome.

Development of gut mycobiome in infants and young children: a prospective cohort study.

BACKGROUND: The composition of the gut fungal microbiome, mycobiome, is likely associated with human health. Yet, the development of gut mycobiome is poorly understood in infants and children. Here we investigate how perinatal events influence the development of gut mycobiome. METHODS: In this prospective cohort study of 140 infants, we used ITS gene sequencing of fecal samples from birth to the age of 18 months. We compared gut mycobiome composition according to delivery mode and exposure to intrapartum antibiotics during vaginal delivery. RESULTS: At birth, gut mycobiome were dominated by the genus Candida, at 6-month stool samples by Malassezia and Cystofilobasidium, and the 18-month stool samples by Trichosporon and unidentified fungi. Perinatal factors altered mycobiome. At 18 months, gut mycobiome of infants born vaginally consisted mostly of Trichosporon (32%) and unidentified fungi (31%), while those born via Cesarean section delivery samples had mycobiome dominated by Saccharomyces (50%). At the age of 18 months, those exposed to intrapartum antibiotics had mycobiome dominated by Trichosporon (66%) not seen in those unexposed to antibiotics. CONCLUSIONS: Delivery mode and exposure to intrapartum antibiotic prophylaxis were markedly associated with gut mycobiome composition from birth to 18 months of age. IMPACT: The composition of the gut mycobiome is likely associated with human health. Yet, the development of gut mycobiome is poorly understood in infants and children. In this prospective cohort study, delivery mode and exposure to intrapartum antibiotic prophylaxis were markedly associated with gut mycobiome composition from birth to 18 months of age. The impact of intrapartum antibiotic prophylaxis on fungal microbiome in vaginally born infants, previously shown to influence gut bacteriome composition, may be explained by the interaction between bacteria and fungi. Gut mycobiome composition likely deserves further investigation in relation to gut microbiome and health in children.

Microbiota and Extracellular Vesicles in Anti-PD-1/PD-L1 Therapy.

Cancer is a deadly disease worldwide. In light of the requisite of convincing therapeutic methods for cancer, immune checkpoint inhibition methods such as anti-PD-1/PD-L1 therapy appear promising. Human microbiota have been exhibited to regulate susceptibility to cancer as well as the response to anti-PD-1/PD-L1 therapy. However, the probable contribution of bacterial extracellular vesicles (bEVs) in cancer pathophysiology and treatment has not been investigated much. bEVs illustrate the ability to cross physiological barriers, assemble around the tumor cells, and likely modify the tumor microenvironment (EVs). This systematic review emphasizes the correlation between cancer-associated extracellular vesicles, particularly bEVs and the efficacy of anti-PD-1/PD-L1 therapy. The clinical and pharmacological prospective of bEVs in revamping the contemporary treatments for cancer has been further discussed.

Comparison of Titanium and PEEK Medical Plastic Implant Materials for Their Bacterial Biofilm Formation Properties.

This study investigated two of the most commonly used CAD-CAM materials for patient-specific reconstruction in craniomaxillofacial surgery. The aim of this study was to access the biofilm formation of Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis, and Escherichia coli on titanium and PEEK medical implant materials. Two titanium specimens (titanium grade 2 tooled with a Planmeca CAD-CAM milling device and titanium grade 5 tooled with a computer-aided design direct metal laser sintering device (CAD-DMLS)) and one PEEK specimen tooled with a Planmeca CAD-CAM milling device were studied. Bacterial adhesion on implants was evaluated in two groups (saliva-treated group and non-saliva-treated group) to imitate intraoral and extraoral surgical routes for implant placement. The PEEK medical implant material showed higher bacterial adhesion by S. aureus, S. mutans, and E. coli than titanium grade 2 and titanium grade 5, whereas E. faecalis showed higher adhesion to titanium as compared to PEEK. Saliva contamination of implants also effected bacterial attachment. Salivary coating enhanced biofilm formation by S. aureus, S. mutans, and E. faecalis. In conclusion, our findings imply that regardless of the implant material type or tooling techniques used, salivary coating plays a vital role in bacterial adhesion. In addition, the majority of the bacterial strains showed higher adhesion to PEEK than titanium.

Bacterial Extracellular Vesicles in Gastrointestinal Tract Cancer: An Unexplored Territory.

Bacterial extracellular vesicles are membrane-enclosed, lipid bi-layer nanostructures that carry different classes of biomolecules, such as nucleic acids, lipids, proteins, and diverse types of small molecular metabolites, as their cargo. Almost all of the bacteria in the gut secrete extracellular vesicles to assist them in competition, survival, material exchange, host immune modulation, infection, and invasion. The role of gut microbiota in the development, progression, and pathogenesis of gastrointestinal tract (GIT) cancer has been well documented. However, the possible involvement of bacterial extracellular vesicles (bEVs) in GIT cancer pathophysiology has not been given due attention. Studies have illustrated the ability of bEVs to cross physiological barriers, selectively accumulate near tumor cells, and possibly alter the tumor microenvironment (TME). A systematic search of original published works related to bacterial extracellular vesicles on gastrointestinal cancer was performed for this review. The current systemic review outlines the possible impact of gut microbiota derived bEVs in GIT cancer in light of present-day understanding. The necessity of using advanced sequencing technologies, such as genetic, proteomic, and metabolomic investigation methodologies, to facilitate an understanding of the interrelationship between cancer-associated bacterial vesicles and gastrointestinal cancer is also emphasized. We further discuss the clinical and pharmaceutical potential of bEVs, along with future efforts needed to understand the mechanism of interaction of bEVs in GIT cancer pathogenesis.

No differences in infections between patient-specific implants and conventional mini-plates in mandibular bilateral sagittal split osteotomy - Up to 3-year follow-up.

The use of individually designed osteotomies, combined with individually manufactured osteosynthesis material, is rapidly becoming a standard for more challenging maxillofacial surgery. The benefits of patient-specific implants (PSI) in orthognathic surgery are clear in complex cases. PSIs can enhance precision and ease up the surgical protocol. We previously reported on the benefits of PSIs as reposition and fixation systems during Le Fort I osteotomy. The aim of this study was to evaluate a cohort of 28 patients, treated with bilateral sagittal split osteotomy (BSSO) and PSIs for fixation, with regard to healing for up to 3 years. A retrospective cohort of 48 patients with conventional mini-plate repositioned mandibles was also collected for statistical analysis. No statistically significant differences were found with regard to infection, soft tissue problems, or reoperations between these two groups.

A Bifidobacterial pilus-associated protein promotes colonic epithelial proliferation.

Development of the human gut microbiota commences at birth, with certain bifidobacterial species representing dominant and early colonisers of the newborn gastrointestinal tract. The molecular basis of Bifidobacterium colonisation, persistence and presumed communication with the host has remained obscure. We previously identified tight adherence (Tad) pili from Bifidobacterium breve UCC2003 as an essential colonisation factor. Here, we demonstrate that bifidobacterial Tad pili also promote in vivo colonic epithelial proliferation. A significant increase in cell proliferation was detectable 5 days postadministration of B. breve UCC2003. Using advanced functional genomic approaches, bacterial strains either (a) producing the Tad2003 pili or (b) lacking the TadE or TadF pseudopilins were created. Analysis of the ability of these mutant strains to promote epithelial cell proliferation in vivo demonstrated that the pilin subunit, TadE, is the bifidobacterial molecule responsible for this proliferation response. These findings were confirmed in vitro using purified TadE protein. Our data imply that bifidobacterial Tad pili may contribute to the maturation of the naïve gut in early life through the production of a specific scaffold of extracellular protein structures, which stimulate growth of the neonatal mucosa.

Comparison between patient specific implants and conventional mini-plates in Le Fort I osteotomy with regard to infections: No differences in up to 3-year follow-up.

Individually designed osteotomies and milled or printed patient-specific osteosynthesis materials are rapidly becoming a standard in maxillofacial reconstructive surgery. The benefits of using patient-specific implants (PSIs) in orthognathic surgery are especially clear in complex cases, and for this reason they are rapidly becoming common practice. We have earlier reported the benefits related to the use of PSIs as reposition and fixation system in Le Fort I osteotomy. The aim of this study was to compare complications associated with fixation with PSIs (31 patients) versus conventional mini-plates (37 patients) in Le Fort I osteotomy. No statistically significant differences in infection, reoperations or soft tissue problems were observed between the two systems used. Interestingly, three of the 37 patients in the mini-plate group underwent reoperation due to insufficient advancement or malocclusion, whereas none of the patients in the PSI group needed reoperation. In conclusion, PSIs are reliable for use in orthognathic surgery, with no signs of infection associated complications.

Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer.

Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that resides in the gastrointestinal tracts of humans and animals. A. muciniphila has been linked with intestinal health and improved metabolic status in obese and type 2 diabetic subjects. Specifically, A. muciniphila has been shown to reduce high-fat-diet-induced endotoxemia, which develops as a result of an impaired gut barrier. Despite the accumulating evidence of the health-promoting effects of A. muciniphila, the mechanisms of interaction of the bacterium with the host have received little attention. In this study, we used several in vitro models to investigate the adhesion of A. muciniphila to the intestinal epithelium and its interaction with the host mucosa. We found that A. muciniphila adheres strongly to the Caco-2 and HT-29 human colonic cell lines but not to human colonic mucus. In addition, A. muciniphila showed binding to the extracellular matrix protein laminin but not to collagen I or IV, fibronectin, or fetuin. Importantly, A. muciniphila improved enterocyte monolayer integrity, as shown by a significant increase in the transepithelial electrical resistance (TER) of cocultures of Caco-2 cells with the bacterium. Further, A. muciniphila induced interleukin 8 (IL-8) production by enterocytes at cell concentrations 100-fold higher than those for Escherichia coli, suggesting a very low level of proinflammatory activity in the epithelium. In conclusion, our results demonstrate that A. muciniphila adheres to the intestinal epithelium and strengthens enterocyte monolayer integrity in vitro, suggesting an ability to fortify an impaired gut barrier. These results support earlier associative in vivo studies and provide insights into the interaction of A. muciniphila with the host.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

BACKGROUND: Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. METHODS: Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. RESULTS: Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. CONCLUSION: The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

Functional identification of conserved residues involved in Lactobacillus rhamnosus strain GG sortase specificity and pilus biogenesis.

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.

Peptides against Mac-1 do not sufficiently target leukemia or lymphoma in vivo.

BACKGROUND: ß2 Integrins (cluster of differentiation-18, CD18) are expressed only by leukocytes and serve as cell surface receptors, being involved both in inside-out and outside-in signalling, and in cell movement. Therefore, they are interesting targets for therapeutic intervention. Phage display-derived inhibitory peptides against αMß2 integrins (macrophage-1 antigen, Mac-1) have been found to be effective in preventing leukocyte movement in vitro and in vivo but little is known regarding their ability to target leukaemia and lymphoma in vivo. MATERIALS AND METHODS: Athymic nude mice were inoculated with human THP-1 acute monocytic leukemia (AML-M5 variant), U937 diffuse histiocytic lymphoma, and OCI-AML-3 acute-myeloidic leukemia cells, and then treated with Mac-1-inhibiting peptides ADGACILWMDDGWCGAAG (DDGW) or CPCLLGCC fused with green fluorescent protein (LLG-GFP). RESULTS: Mac-1-inhibiting DDGW peptide had no effect on leukemia and lymphoma burden in mice, and LLG-GFP fusion did not home to leukemia cells in vivo. CONCLUSION: Although peptides against Mac-1 are promising drugs and diagnostic tools based on earlier experiments in inflammation they exhibit compromised biological avidity as a therapeutic and diagnostic means for leukaemia and lymphoma.

In vivo targeting of activated leukocytes by a ß2-integrin binding peptide.

BACKGROUND: In immunopathological conditions, clinical diagnosis is commonly made on the basis of patient symptoms, measurement of blood leukocyte levels or proinflammatory biomarkers, non-specific radiological findings and, regarding infection, microbiological analysis. Nevertheless, frequently the exact spatial location of inflammation or even infection cannot be reliably identified, despite the use of up-to-date clinical, laboratory and imaging techniques. For this reason, new tools are warranted for use in advanced diagnosis and therapy targeting in patients. OBJECTIVE: The peptide CPCFLLGCC (LLG), known to bind activated ß2-integrins in vitro, was fused with green fluorescent protein (GFP) to test the ability of LLG fusions to target and bind activated leukocytes in vivo. METHODS: A murine skin scratch inflammation model was chosen for the convenient non-surgical detection of GFP. RESULTS: The murine skin lesion inflammation model demonstrated in vivo targeting of LLG-GFP to sites of inflammation. Targeting by LLG-GFP fusion construct depends on the ability of the LLG-moiety to bind activated leukocytes. Control construct unable to bind ß2-integrins appeared biologically inert. CONCLUSION: The data support the possibility of using this fluorescently labeled peptide as a tool for both the detection of and targeting to inflammatory sites characterized by robust leukocyte activation.

BopA does not have a major role in the adhesion of Bifidobacterium bifidum to intestinal epithelial cells, extracellular matrix proteins, and mucus.

The ability of bifidobacteria to adhere to the intestine of the human host is considered to be important for efficient colonization and achieving probiotic effects. Bifidobacterium bifidum strains DSM20456 and MIMBb75 adhere well to the human intestinal cell lines Caco-2 and HT-29. The surface lipoprotein BopA was previously described to be involved in mediating adherence of B. bifidum to epithelial cells, but thioacylated, purified BopA inhibited the adhesion of B. bifidum to epithelial cells in competitive adhesion assays only at very high concentrations, indicating an unspecific effect. In this study, the role of BopA in the adhesion of B. bifidum was readdressed. The gene encoding BopA was cloned and expressed without its lipobox and hydrophobic signal peptide in Escherichia coli, and an antiserum against the recombinant BopA was produced. The antiserum was used to demonstrate the abundant localization of BopA on the cell surface of B. bifidum. However, blocking of B. bifidum BopA with specific antiserum did not reduce adhesion of bacteria to epithelial cell lines, arguing that BopA is not an adhesin. Also, adhesion of B. bifidum to human colonic mucin and fibronectin was found to be BopA independent. The recombinant BopA bound only moderately to human epithelial cells and colonic mucus, and it failed to bind to fibronectin. Thus, our results contrast the earlier findings on the major role of BopA in adhesion, indicating that the strong adhesion of B. bifidum to epithelial cell lines is BopA independent.

Selective gelatinase inhibitor peptide is effective in targeting tongue carcinoma cell tumors in vivo.

BACKGROUND: Matrix metalloproteinases (MMP) are strongly associated with cancer progession. Broad-spectrum MMP inhibition is rarely beneficial clinically due to adverse effects. Of all MMPs, the gelatinases are associated with the spread of several types of cancer, including oral carcinoma. We have developed gelatinase-specific peptides, as well as their fusion with green fluorescent protein (GFP), capable of effectively targeting carcinomas. MATERIALS AND METHODS: Effects on tumor growth and lymphatic micrometastatic spread in vivo was studied by use of HSC-3-cell xenografted athymic nude mice. Antigelatinolytic mono- vs. polytherapies, as well as biological activity of peptide-GFP fusion, were also analyzed in vivo. RESULTS: Antigelatinolytic therapy effectively inhibited growth of xenografted tumors in mice but the proportion of enlarged lymph nodes remained the same; antigelatinolytic polytherapy seemed not to potentiate the antitumor effects. The peptide-GFP chimera sustained its activity in vivo and effectively homed to the primary tumors. CONCLUSION: Peptide gelatinase inhibitors are effective in inhibiting primary tumor growth but alone do not prevent the spread of carcinoma cells; however, their bioactive GFP fusion is a candidate for tumor characterization and imaging.

Liposomal Tumor Targeting in Drug Delivery Utilizing MMP-2- and MMP-9-Binding Ligands.

Nanotechnology offers an alternative to conventional treatment options by enabling different drug delivery and controlled-release delivery strategies. Liposomes being especially biodegradable and in most cases essentially nontoxic offer a versatile platform for several different delivery approaches that can potentially enhance the delivery and targeting of therapies to tumors. Liposomes penetrate tumors spontaneously as a result of fenestrated blood vessels within tumors, leading to known enhanced permeability and subsequent drug retention effects. In addition, liposomes can be used to carry radioactive moieties, such as radiotracers, which can be bound at multiple locations within liposomes, making them attractive carriers for molecular imaging applications. Phage display is a technique that can deliver various high-affinity and selectivity peptides to different targets. In this study, gelatinase-binding peptides, found by phage display, were attached to liposomes by covalent peptide-PEG-PE anchor creating a targeted drug delivery vehicle. Gelatinases as extracellular targets for tumor targeting offer a viable alternative for tumor targeting. Our findings show that targeted drug delivery is more efficient than non-targeted drug delivery.

A chimera of green fluorescent protein with gelatinase binding and tumor targeting peptide.

Matrix metalloproteinases (MMPs) are enzymes that can hydrolyze almost all constituents of extracellular matrix. An MMP subgroup, the gelatinases, has been focused during last years, since over-expression of gelatinase A (MMP-2) and gelatinase B (MMP-9) has been linked with severe homeostasis disorders such as tumor growth, metastasis formation, and chronic inflammation. In this study, a phage display library-derived novel antigelatinolytic decapeptide, the CTT-peptide, was expressed as a carboxyl terminal, histidine-tagged fusion with the green fluorescent protein (CTT-GFP) in Escherichia coli. In addition, a biologically intact chimera, in which residues in the CTT-peptide critical for gelatinase binding were replaced with alanine (Ala-CTT-GFP), was constructed. The GFP-fusion proteins were purified to homogeneity with a simple one-step procedure utilizing nickel affinity chromatography. The purified chimeras were tested for their binding properties to 4beta-phorbol-12,13-butyrate (PdBu) activated, MMP-9 expressing THP-1 cells, and it was demonstrated that the CTT-GFP strongly bound to the cells, whereas Ala-CTT-GFP lacked the binding ability. Furthermore, the adherence of the CTT-GFP to MMP-9 expressing cells was demonstrated to be mediated by the CTT-moiety, since the binding could be dose-relatedly inhibited with increasing concentrations of synthetic soluble CTT-peptide. In conclusion, this novel tool, combining the gelatinase binding ability of the CTT-peptide with the fluorescing property of the GFP, should clearly improve both experimental and clinical studies of the role and function of gelatinases.