Angiotensin‐converting enzyme 2: The old door for new severe acute respiratory syndrome coronavirus 2 infection

Coronavirus (CoV) disease 2019 (COVID‐19) is an ongoing pandemic caused by severe acute respiratory syndrome CoV 2 (SARS‐CoV‐2). The highly contagious SARS‐CoV‐2 belongs to the genus Betacoronavirus, and it is phylogenetically closely related to SARS‐CoV, a human CoV that caused an outbreak back in 2002 to 2003. Both SARS‐CoV‐2 and SARS‐CoV enter human cells via the interactions between viral crown‐like spike protein and human angiotensin‐converting enzyme 2 (ACE2) receptor. Here, we aim to review the involvement of ACE2 in human CoV infections by discussing the roles of ACE2 in CoV evolution, cross‐species transmissibility, and COVID‐19 susceptibility. We also provide our perspectives on COVID‐19 treatment and prevention.     

Evidence for genetic heterogeneity in D‐2‐hydroxyglutaric aciduria

We performed molecular, enzyme, and metabolic studies in 50 patients with D ‐2‐hydroxyglutaric aciduria (D ‐2‐HGA) who accumulated D ‐2‐hydroxyglutarate (D ‐2‐HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D ‐2‐hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D ‐2‐hydroxyglutarate dehydrogenase (D ‐2‐HGDH). Enzyme assay of D ‐2‐HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D ‐2‐HGA whose enzyme activity was normal did not have mutations. Significantly lower D ‐2‐HG concentrations in body fluids were observed in mutation‐positive D ‐2‐HGA patients than in mutation‐negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D ‐2‐HG. Accordingly, we suggest a new classification: D ‐2‐HGA Type I associates with D ‐2‐HGDH deficiency, whereas idiopathic D ‐2‐HGA manifests with normal D ‐2‐HGDH activity and higher D ‐2‐HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D ‐2‐HGA patients with diverse genetic loci will be revealed in future studies. Hum Mutat 31:1–5, 2010. © 2009 Wiley‐Liss, Inc.     

Novel L2HGDH mutations in 21 patients with L-2-hydroxyglutaric aciduria of Portuguese origin

We studied 21 patients, from 18 families, with L-2-hydroxyglutaric aciduria (L-2-HGA), a rare neurometabolic disorder with a homogeneous presentation: progressive neurodegeneration with extrapyramidal and cerebellar signs, seizures, and subcortical leukoencephalopathy. Increased levels of L-2-hydroxyglutaric acid in body fluids proved the diagnosis of L-2-HGA in all 21 patients. We analyzed the L-2-HGA gene (L2HGDH), recently found to be mutated in consanguineous families with L-2-HGA, and identified seven novel mutations in 15 families. Three mutations appeared to be particularly prevalent in this Portuguese panel: a frameshift mutation (c.529delC) was detected in 12 out of 30 mutant alleles (40%), a nonsense mutation (c.208C>T; p.Arg70X) in 7/30 alleles (23%), and a missense mutation (c.293A>G; p.His98Arg) in four out of 30 mutant alleles (13%), suggesting that common origin may exist. Furthermore, two novel missense (c.169G>A; p.Gly57Arg, c.1301A>C; p.His434Pro) and two splice error (c.257-2A>G, c.907-2A>G) mutations were found. All the mutations presumably lead to loss-of-function with no relationship between clinical signs, progression of the disease, levels of L-2-HGA and site of the mutation. In the three remaining families, no pathogenic mutations in the L-2-HGA were found, which suggests either alterations in regulatory regions of the gene or of its intervening sequences, compound heterozygosity for large genomic deletion and, or further genetic heterogeneity.     

Cyclooxygenase‐2 immunoreactivity in collagenous colitis

Collagenous colitis (CC) is an inflammatory bowel disease of unknown aetiology and pathogenesis. In ulcerative colitis and Crohn's disease, prostaglandins may be involved in the pathogenesis of inflammation, and increased expression of cyclo‐oxygenase‐2 (COX‐2) has been detected. The purpose of this study was to examine the presence and cellular localization of COX‐2 in colonic mucosa of patients with CC. Using immunohistochemistry, immunoflouresence and Western blot analysis, COX‐2 expression was evaluated in colonic mucosal biopsies from 10 patients with active untreated CC, and compared with samples from eight normal controls, and samples from eight patients with ulcerative colitis or Crohn's disease. Specimens from patients with CC expressed COX‐2 protein in increased amounts compared with controls, but similar to patients with ulcerative colitis and Crohn's disease. COX‐2 expression was localized to the mononuclear cells of the lamina propria. COX‐2 expression was most evident in macrophages. Co‐localization of COX‐2 and macrophages was increased in number in comparison with controls. In conclusion COX‐2 is expressed in increased amounts primarily in the macrophage subpopulation of the inflammatory infiltrate of lamina propria in CC. Increased recruitment of macrophages, increased expression of COX‐2 and increased prostaglandin synthesis may be involved in the pathogenesis of CC.     

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.     

The distribution of fibrillin-2 and LTBP-2, and their co-localisation with fibrillin-1 in adult bovine tail disc

We investigated the distribution of fibrillin-2 and LTBP-2 (latent TGF-β binding protein-2) in the intervertebral disc of the adult bovine tail. The association of fibrillin-2 and of LTBP-2 with fibrillin-1 was examined by dual immunofluorescence staining. Both fibrillin-2 and LTBP-2 were found extensively distributed in all regions of the disc with the organisation of the network varying significantly region to region. In the outer annulus fibrosus (OAF) both fibrillin-2 and LTBP-2 co-localised with fibrillin-1 forming fibres running parallel to the collagen fibres of the lamellae with the microfibrillar network staining densely in between the adjacent lamellae and also at the boundaries of the collagen bundle compartments. In the inner annulus fibrosus (IAF) and nucleus pulposus (NP), co-localised fibrillin-1,2 and LTBP-2 formed a chondron-like structure around the cell. By contrast, the inter-territorial matrix of the IAF and NP contained a dense network of fibrillin-2 but only sparse/filamentous fibres of fibrillin-1 and LTBP-2. Dual immunostaining revealed that in this region, fibrillin-2 was highly colocalised with elastin. The LTBP-2 network co-localised well with that of fibrillin-1 in all regions and indeed is reported to bind strongly to fibrillin-1. However, interestingly LTBP-2 but not fibrillin-1 or fibrillin-2 was removed by hyaluronidase but not collagenase pre-digestion. Our results suggest that fibrillin-2 and LTBP-2 could play an important role in disc function.     

Polymorphisms in the matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 and the risk of human adenomyosis

The matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) may contribute to the development of adenomyosis. The aim of the present study was to investigate whether three single nucleotide polymorphisms (SNPs) in the promoter regions of MMP-2 (-1306C/T and -735C/T) and TIMP-2 (-418G/C) genes were related to the risk of adenomyosis development. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in 180 adenomyosis patients and 324 frequency-matched control women in a Chinese population. There were significant differences in allele frequencies and genotype distributions of the MMP-2 -1306C/T polymorphism between patients and control women (P = 0.01 and 0.04, respectively). The frequency of C allele in patients (92.2%) was significantly higher than in the controls (87.0%) (P = 0.01). Compared with the C/T+T/T genotypes, the C/C genotype could significantly increase the risk of adenomyosis development, with an odds ratio of 1.83 (95% CI = 1.13-2.96). However, no statistically significant difference was found in allele frequencies and genotype distributions of MMP-2 -735C/T and TIMP-2 -418G/C SNPs between the two groups (all P values > 0.05). Two polymorphisms of MMP-2 displayed linkage disequilibrium (D' = 0.74). The haplotype analysis suggested no significant association of four haplotypes with the risk of adenomyosis development. Our results indicated an association of MMP-2 -1306C/T polymorphism with the risk of adenomyosis, suggesting a potential role in adenomyosis development in North Chinese women.     

Cisplatin decreases renal cyclooxygenase-2 expression and activity in rats

Aim: Cisplatin (CP) induced acute renal failure (ARF) has previously been associated with decreased urinary prostaglandin E2 (PGE2) excretion and reduced aquaporin 2 (AQP2) expression in kidney collecting duct. In this study we examined the expression of cyclooxygenase (COX)‐1 and ‐2 as well as AQP2 and the Na‐K‐2Cl cotransporter in kidneys from rats with CP induced ARF. Methods: Rats were treated with either CP or saline and followed for 5 days. Kidneys were dissected into three zones and prepared for immunoblotting, quantitative polymerase chain reaction (QPCR) and immunohistochemistry. Renal content and urinary PGE2 excretion was measured. Results: Cisplatin treatment was associated with polyuria and a significant decreased creatinine clearance. Inner medullary PGE2 content and urinary PGE2 excretion was decreased in CP‐treated rats. QPCR and semiquatitative immunoblotting demonstrated that CP treatment reduced COX‐2, AQP2 and Na‐K‐2Cl cotransporter abundance in the different kidney zones, whereas no change in COX‐1 was observed. Results were confirmed by immunohistochemistry. Conclusion: Cyclooxygenase‐2 expression is decreased in inner medulla and cortex. Consistent with this urinary PGE2 levels were reduced. These data suggest that downregulation of COX‐2 is responsible for impaired de novo generation of vasodilatory prostaglandins which may play an important role for the CP induced renal vasoconstriction and development of nephropathy.     

Direct Insertion Mass Spectrometric Analysis of Thermal Degradation of Poly(2‐alkyl‐2‐oxazoline)

Direct pyrolysis mass spectrometry is applied to investigate the thermal behavior of poly(2‐isopropyl‐2‐oxazoline) (PIPOX, a thermoresponsive polymer) and poly[2‐(3‐butenyl)‐2‐oxazoline] (PBOX, a “clickable” polymer). It is found that the thermal degradation of PIPOX is started by a loss of side chains. At slightly higher temperatures the degradation of the polymer backbone occurs by random chain scission processes. In the case of PBOX, vinyl polymerization of the side chains produces chains with variable thermal stabilities. The number of repeating units of the polymer has almost no effect on the thermal behavior of PIPOX, but significantly affects both thermal stability and degradation product distribution of PBOX.     

D‐2‐hydroxyglutaric aciduria Type I: Functional analysis of D2HGDH missense variants

D‐2‐hydroxyglutaric aciduria Type I (D‐2‐HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D‐2‐hydroxyglutarate dehydrogenase (D‐2‐HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D‐2‐HGDH catalytic activity. Site‐directed mutagenesis was used to introduce 31 missense variants in the pCMV5‐D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D‐2‐HGDH enzyme activity was evaluated based on the conversion of [²H₄]D‐2‐HG to [²H₄]2‐ketoglutarate, which was subsequently converted into [²H₄]L‐glutamate and the latter quantified by LC‐MS/MS. Eighteen variants resulted in almost complete ablation of D‐2‐HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.     

Prostaglandin E2 and its receptor EP2 trigger signaling that contributes to YAP‐mediated cell competition

Cell competition is a biological process by which unfit cells are eliminated from “cell society.” We previously showed that cultured mammalian epithelial Madin‐Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by “normal” MDCK cells. However, the molecular mechanism underlying the elimination of active YAP‐expressing cells was unknown. Here, we used high‐throughput chemical compound screening to identify cyclooxygenase‐2 (COX‐2) as a key molecule triggering cell competition. Our work shows that COX‐2‐mediated PGE₂ secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase‐cyclic AMP‐PKA pathway that, in the presence of active YAP, induces E‐cadherin internalization leading to apical extrusion. Thus, COX‐2‐induced PGE₂ appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.     

Neuropilin-2 expression in cancer

Jubb A M, Sa S M, Ratti N, Strickland L A, Schmidt M, Callahan C A & Koeppen H
(2012) Histopathology  61, 340–349 Neuropilin‐2 expression in cancer Aims: Neuropilin‐2 is a coreceptor for vascular endothelial growth factor family members. Blockade of neuropilin‐2 is able to suppress lymphogenous metastasis in preclinical models. The aim of this study was to validate a protocol for the evaluation of neuropilin‐2 protein expression in situ, by comparison with in‐situ hybridization, western blotting, and mRNA expression levels. Methods and results: Immunohistochemistry was performed on normal human tissues, and whole sections for 79 primary non‐small‐cell lung carcinomas, 65 primary breast carcinomas, 79 primary colorectal cancers, and 52 metastases. Neuropilin‐2 expression was observed in lymphatic and blood vessels from all normal and malignant tissues examined. In addition, 32% of primary non‐small‐cell lung carcinomas, 15% of primary breast carcinomas and 22% of primary colorectal cancers showed tumour cell expression. Fifty‐five primary and nine secondary malignant melanomas were also examined for neuropilin‐2 expression by in‐situ hybridization. All showed vascular expression, and 85% of primary malignant melanomas showed tumour cell expression. Conclusions: In the majority of lung, breast and colorectal cancers, the effects of anti‐neuropilin‐2 are likely to be restricted to the vasculature. These results will assist in pharmacokinetic evaluations, tolerability assessments and the choice of setting to evaluate the activity of anti‐neuropilin‐2 therapies.     

TEG-1 CD2BP2 regulates stem cell proliferation and sex determination in the C. elegans germ line and physically interacts with the UAF-1 U2AF65 splicing factor

Background : For a stem cell population to exist over an extended period, a balance must be maintained between self‐renewing (proliferating) and differentiating daughter cells. Within the Caenorhabditis elegans germ line, this balance is controlled by a genetic regulatory pathway, which includes the canonical Notch signaling pathway. Results : Genetic screens identified the gene teg‐1 as being involved in regulating the proliferation versus differentiation decision in the C. elegans germ line. Cloning of TEG‐1 revealed that it is a homolog of mammalian CD2BP2, which has been implicated in a number of cellular processes, including in U4/U6.U5 tri‐snRNP formation in the pre‐mRNA splicing reaction. The position of teg‐1 in the genetic pathway regulating the proliferation versus differentiation decision, its single mutant phenotype, and its enrichment in nuclei, all suggest TEG‐1 also functions as a splicing factor. TEG‐1, as well as its human homolog, CD2BP2, directly bind to UAF‐1 U2AF65, a component of the U2 auxiliary factor. Conclusions : TEG‐1 functions as a splicing factor and acts to regulate the proliferation versus meiosis decision. The interaction of TEG‐1 CD2BP2 with UAF‐1 U2AF65, combined with its previously described function in U4/U6.U5 tri‐snRNP, suggests that TEG‐1 CD2BP2 functions in two distinct locations in the splicing cascade. Developmental Dynamics 241:505–521, 2012.© 2012 Wiley Periodicals, Inc.     

GATA-binding protein-3 regulates T helper type 2 cytokine and ifng loci through interaction with metastasis-associated protein 2

GATA‐binding protein‐3 (GATA‐3) regulates the T helper type 2 (Th2) cytokine locus through induction of chromatin remodelling. However, the molecular mechanism for this is poorly understood. To understand this mechanism better, we screened GATA‐3 interacting proteins using affinity purification and mass spectrometry. We found that GATA‐3 bound to metastasis‐associated protein 2 (MTA‐2), a component of the NuRD chromatin remodelling complex. GATA‐3 and MTA‐2 in turn bound to several regulatory regions of the Th2 cytokine locus and the ifng promoter. Cell transfection assay showed that MTA‐2 acted as an antagonist with GATA‐3 in the expression of Th2 cytokines, but co‐operated with GATA‐3 in the repression of the ifng gene expression. These results suggest that GATA‐3 interacts with MTA‐2 to co‐ordinately regulate Th2 cytokine and ifng loci during T helper cell differentiation.     

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β2m‐free heavy chains

Since HLA‐E heavy chains accumulate free of their light β₂‐microglobulin (β₂m) subunit, raising mAbs to folded HLA‐E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA‐E‐restricted mAbs (MEM‐E/02, MEM‐E/07, MEM‐E/08, DT9, and 3D12) were tested on denatured, acid‐treated, and natively folded (both β₂m‐associated and β₂m‐free) HLA‐E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β₂m‐free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM‐E/02, MEM‐E/07, and MEM‐E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4–6 residues and are “hidden” in folded HLA‐E heterodimers. They contain at least one HLA‐E‐specific residue that cannot be replaced by single substitutions with polymorphic HLA‐A, HLA‐B, HLA‐C, HLA‐F, and HLA‐G residues. Finally, also the MEM‐E/02 and 3D12 epitopes are spatially distinct. In summary, HLA‐E‐specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA‐E heterodimers, and HLA‐E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.     

Expansion of regulatory T cells via IL‐2/anti‐IL‐2 mAb complexes suppresses experimental myasthenia

Human autoimmune diseases are often characterized by a relative deficiency in CD4⁺CD25⁺ regulatory T cells (Treg). We therefore hypothesized that expansion of Treg can ameliorate autoimmune pathology. We tested this hypothesis in an experimental model for autoimmune myasthenia gravis (MG), a B‐cell‐mediated disease characterized by auto‐Ab directed against the acetylcholine receptor within neuromuscular junctions. We showed that injection of immune complexes composed of the cytokine IL‐2 and anti‐IL‐2 mAb (JES6‐1A12) induced an effective and sustained expansion of Treg, via peripheral proliferation of CD4⁺CD25⁺Foxp3⁺ cells and peripheral conversion of CD4⁺CD25^?Foxp3^? cells. The expanded Treg potently suppressed autoreactive T‐ and B‐cell responses to acetylcholine receptor and attenuated the muscular weakness that is characteristic of MG. Thus, IL‐2/anti‐IL‐2 mAb complexes can expand functional Treg in vivo, providing a potential clinical application of this modality for treatment of MG and other autoimmune disorders.     

Functionalization of WS2 Nanotubes with Fluorescent C‐dots and Conductive Polythiophenes

Multifunctional nanoparticles have attracted significant interest for their multifunctional applications. Described herein is the building of a new hybrid nanocomposite particle, conjugating tungsten disulfide (WS₂) nanotubes (NTs) to fluorescent carbon dots (C‐dots). Hybrid WS₂‐C‐dots hyb‐NTs nanomaterials are prepared by a hydrothermal reaction of PEDOT_Ac‐WS₂ NTs, where the PEDOT_Ac (poly 2,3‐dihydrothieno[3,4‐b][1,4]dioxine‐2‐carboxylic acid) polymer serves as the C‐dots precursor. The physical and chemical properties of the as‐synthesized WS₂‐C‐dots hyb‐NTs are determined by X‐ray powder diffraction (XRD), transmission electron microscopy (TEM), CHNSO analysis, and fluorescence, Raman, and UV–vis absorption spectroscopy. The WS₂‐C‐dots hyb‐NTs are shown to be very stable at normal room temperature and pressure and could be useful for multicolor cell imaging and targeted cell killing.     

Markers aiding the diagnosis of chondroid tumors: an immunohistochemical study including osteonectin,bcl‐2, cox‐2, actin,calponin, D2‐40 (podoplanin), mdm‐2, CD117 (c‐kit), and YKL‐40

Chondroid tumors comprise a heterogenous group of benign to overt malignant neoplasms, which may be difficult to differentiate from one another by histological examination. A group of 43 such tumors was stained with nine relevant antibodies in an attempt to find consistent marker profile(s) for the different subgroups. Archival material from three extraskeletal myxoid chondrosarcomas, five chordomas, five chondromyxoid fibromas, five chondroblastomas and 25 chondrosarcomas was stained with antibodies against osteonectin, bcl‐2, cox‐2, actin, calponin, D2‐40 (podoplanin), mdm‐2, CD117 (c‐kit) and YKL‐40. All 25 chondrosarcomas showed a positive staining reaction for D2‐40, none for actin and CD117, and a partial reactivity for bcl‐2 (36%). Chondroblastomas (5/5) and chondromyxoid fibromas (2/5) were the only tumors with a positive reaction for actin, and all chondroblastomas (n=5) and extraskeletal myxoid chondrosarcomas (n=3) were positive for bcl‐2. In contrast to all other tumors, two of three extraskeletal myxoid chondrosarcomas were also positive for CD17 and negative for osteonectin, cox‐2, mdm‐2 and actin. All five chordomas were negative for D2‐40 and positive for mdm‐2 and YKL‐40. The diagnosis of chondrosarcoma may be aided by its positivity for D2‐40 and YKL‐40 and its lack of reactivity for actin and CD117. This should be seen in the light of no reaction for D2‐40 in chordomas and a corresponding lack of reaction for osteonectin, cox‐2, mdm‐2 and actin in extraskeletal myxoid chondrosarcomas. A convincing immunoreactivity for calponin and/or actin in chondromyxoid fibromas and chondroblastomas may also be helpful in differentiating these tumors from chondrosarcomas.     

Activation of pro-MMP-2 mediated by MT1-MMP in human salivary gland carcinomas: possible regulation of pro-MMP-2 activation by TIMP-2

Matrix metalloproteinases (MMPs), a family of extracellular matrix-degrading enzymes, are considered to play important roles in cancer invasion and metastasis. The present study examined the production levels of eight different MMPs (MMP-1, 2, 3, 7, 8, 9 and 13, and MT1-MMP) and two tissue inhibitors of metalloproteinases (TIMP-1 and 2) in homogenates of human salivary gland carcinomas [mucoepidermoid carcinomas (MECs), adenoid cystic carcinomas (ACCs), and adenocarcinomas (ADEs)] and non-neoplastic control salivary glands using sandwich enzyme immunoassay systems. The levels of MMP-1, MMP-2, MMP-13, MT1-MMP, and TIMP-1 were significantly higher in the carcinoma samples than in the controls (p < 0.05). Gelatin zymography demonstrated that the activation ratio of the MMP-2 zymogen (pro-MMP-2) was significantly higher in the carcinomas than in the controls (p < 0.05). In addition, the activation ratio in MECs was significantly higher than that in ACCs or ADEs (p < 0.01) and also correlated with histological grade and lymph node metastasis in MECs (p < 0.05), whereas the ratio showed no such correlation in ACCs or ADEs. Although the production levels of pro-MMP-2 and MT1-MMP were similar among these carcinoma groups, TIMP-2 levels were significantly higher in ACCs and ADEs than in MECs (p < 0.01). In carcinoma samples, the pro-MMP-2 activation ratio correlated directly with the MT1-MMP/TIMP-2 ratio (r = 0.736, n = 23; p < 0.01). Immunohistochemistry and in situ zymography demonstrated localization of MMP-2, MT1-MMP, and TIMP-2 to carcinoma cells, but only in MECs did carcinoma cell nests exhibit gelatinolytic activity, which was inhibited by 1,10-phenanthroline. These results suggest that enhanced activation of pro-MMP-2 mediated by MT1-MMP is implicated in the invasion and metastasis of MECs and that TIMP-2 may regulate pro-MMP-2 activation in salivary gland carcinomas.