Anneli Lauhio

Human Neutrophil Collagenase MMP-8 in Peri-implant Sulcus Fluid and its Inhibition by Clodronate

The exact molecular mechanisms of the loosening of a dental implant are not well-known. The characteristics of implant sulci are similar to those of periodontal sulci regarding gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF). Proteolytic enzymes, matrix metalloproteinases (MMPs), participate in peri-implant tissue remodeling. Clodronate is a well-tolerated bisphosphonate-group drug currently used in bone-resorption-related diseases in humans. The mechanisms of bisphosphonate action are not clarified. Collagenase activity in diseased PISF was significantly higher than in the clinically healthy group. Immunoblotting disclosed that diseased PISF contained increased immunoreactives MMP-8 compared with the healthy PISF. The residual latent collagenase activity in the diseased PISF was activated by gold thioglucose and inhibited completely by 100 pM of doxycycline closely resembling pure neutrophil collagenase (MMP-8). The presence of MMP-8 in diseased but not in clinically healthy PISF may prove to be a useful biochemical indicator to monitor peri-implant health and disease. Pure human neutrophil collagenase (MMP-8) and the MMP-8 present in PISF and in the GCF of both loosening implants and periodontitis-affected teeth were efficiently inhibited in vitro by clodronate (50% inhibition [IC50] was achieved by 150 uM of clodronate), an osteoactive, antiresorptive bisphosphonate. Furthermore, the new finding suggests an extended and hitherto-undescribed potential for clodronate in preventing the loosening of both implants and teeth, based on a dual beneficial effect: prevention of both bone resorption/osteolysis and of soft tissue/dental ligament destruction. Potential new therapeutic indications based on the collagenase-inhibiting effect of clodronate provide potential new therapeutic indications for a variety of diseases involving connective tissue breakdown, such as periodontal disease, arthritides, and tumor invasion.

Inhibition of Matrix Metalloproteinase-1 by Dichloromethylene Bisphosphonate (Clodronate)

Abstract. Interstitial collagenase present in human jaw cyst extract and purified human fibroblast-type collagenase (MMP-1) were both efficiently inhibited in vitro by clodronate, an osteoactive, antiresorptive bisphosphonate. The IC50 of clodronate to inhibit MMP-1 is 150 μM. These findings suggest an extended and hitherto undescribed properties for clodronate/biphosphonates in prevention and treatment of tissue degradation in both bone and soft tissue destructive diseases.

Serum MMP-8, -9 and TIMP-1 in sepsis: high serum levels of MMP-8 and TIMP-1 are associated with fatal outcome in a multicentre, prospective cohort study. Hypothetical impact of tetracyclines.

Recent evidence suggests that matrix metalloproteinases (MMPs) and their endogenous inhibitors are involved in the pathogenesis of sepsis. We studied serum levels of MMP-8, MMP-9 and TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) in a multicentre, prospective cohort study of patients with sepsis treated in Intensive Care Units (ICUs). We analyzed serum samples taken on ICU admission from 248 critically ill sepsis patients. MMP-8, -9 and TIMP-1 serum levels were analyzed by enzyme-linked immunosorbent assays. Serum MMP-8, MMP-9 and TIMP-1 levels were significantly higher in patients with severe sepsis than in healthy controls. Serum MMP-8 levels among non-survivors (n=33) were significantly (p=0.006) higher than among survivors (n=215). Serum TIMP-1 but not MMP-9 levels were significantly higher among non-survivors than survivors (p<0.0001, p=0.079, respectively). Systemic MMP-8 is upregulated in sepsis suggesting that MMP-8 may contribute to the host response during sepsis. High serum MMP-8 and TIMP-1 levels at ICU admission were seen among patients with fatal outcome. With this background, clinical studies examining the ability of MMP-inhibitors (such as the non-antimicrobial properties of tetracyclines) to diminish the MMP-mediated inflammatory response are needed to develop novel therapies in order to improve the outcome of sepsis.

A Nationwide Study of Campylobacter jejuni and Campylobacter coli Bacteremia in Finland Over a 10-Year Period, 1998–2007, With Special Reference to Clinical Characteristics and Antimicrobial Susceptibility

Campylobacter jejuni and Campylobacter coli bacteremia patients were mainly young and without severe underlying diseases. The bacterial isolates were typically susceptible to antimicrobial agents. The outcome was usually good, regardless of appropriate or inappropriate antimicrobial treatment given at the hospital.

Diagnosis of Lyme neuroborreliosis with antibodies to recombinant proteins DbpA, BBK32, and OspC, and VlsE IR6 peptide

Abstract.Three recombinant antigens, decorin binding protein A (DbpA), BBK32, and outer surface protein C (OspC), and IR6 peptide of borrelial VlsE protein, were evaluated for the diagnosis of neuroborreliosis (NB), using cerebrospinal fluid (CSF) and serum samples from 89 patients. Their performances in enzyme-linked immunosorbent assay (ELISA) were compared with that of commercial flagella antigen. IgG ELISAs were performed with three variants of each recombinant antigen originating from Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii, and with the IR6 peptide. IgM antibodies were analysed against OspC and flagella. Of the patients whose CSF contained elevated anti-flagella IgG antibodies, 93% were positive for at least three of the new antigens. Of those with negative or borderline CSF anti-flagella antibodies, 51% were positive for three new antigens. Antibodies to BBK32 were detectable mainly in early disease. Antibodies to DbpA and IR6 were observed in early and late NB. The use of the new antigens at presentation of the disease improved the laboratory diagnosis of NB. In IgG ELISAs, the diagnostic sensitivity of assays with the new antigens was between 75 and 88%, but was only 52% with the flagella antigen. The discriminatory power between patient and control samples appeared better in the CSF than in the serum. We suggest that assessment of CSF antibodies to at least two antigens, using either flagella and one of the new antigens or two of the new antigens, would improve the current diagnostic yield of NB.

Substrate Specificity and Activation Mechanisms of Collagenase from Human Rheumatoid Synovium

Substrate specificity studies of collagenase extracted from human rheumatoid synovium suggest that synovial pannus tissue overlying articular cartilage may not be particularly active in degradation of cartilage type II collagen, which, considering the poor inherent healing capacity of the articular hyaline cartilage, may exert a protective function against inadvertant tissue damage. Rheumatoid synovial tissue was also used to establish synovial fibroblast cell lines. Treatment of these cells in monolayer cultures with IL-1 leads to collagenase gene activation, increased collagenase production and an almost complete autoactivation of secreted collagenase. Interleukin-1 also activated stromelysin gene suggesting this as a possible mechanism effecting autoactivation. Latent human fibroblast and macrophage collagenase purified from culture medium were efficiently activated by phenylmercuric chloride but also by gold thioglucose, gold sodium thiomalate and HCIO. These new observations support the Cys73 switch activation mechanism. In contrast to neutrophil collagenase, the activation by gold(I) compounds and HCIO was associated with a change in the apparent molecular weight of the fibroblast procollagenase. In addition, gold(I) compounds rendered collagenase more susceptible to thermal denaturation. Thus the fibroblast-type interstitial collagenase, probably derived from fibroblast- and macrophage-like synoviocytes, seems to provide the predominant collagenolytic potential in human rheumatoid synovial tissue. Furthermore, the conditions in synovitis tissue may be such as to favor at least initial activation of collagenase synthesized and secreted in situ.

Urinary matrix metalloproteinase -8, -9, -14 and their regulators (TRY-1, TRY-2, TATI) in patients with diabetic nephropathy.

Matrix metalloproteinase-9 (MMP-9) has been shown to be involved in the development of diabetic nephropathy (DNP). We studied the levels, molecular forms, and degree of activation of urinary MMP-8, -9, -14, trypsin-1 and -2, as well as tumor-associated trypsin inhibitor (TATI) of DNP patients and healthy controls. Urinary samples were analyzed for MMPs by Western blotting and gelatin zymography and for trypsin-1, -2, and TATI by time-resolved immunofluorometric assays. Total MMP-8 immunoreactivity, the proportion of active MMP-9, and gelatinolytic activity in urine were significantly higher in DNP patients than in controls. In urine of DNP patients the proportion of active polymorphonuclear neutrophil (PMN)-type (but not fibroblast-type) MMP-8 was increased. MMP-8 and MMP-9 were found to form high molecular weight complexes in DNP urine. Total immunoreactivity of soluble urinary MMP-14 and the levels of trypsin (TRY)-1 and TRY-2, but not of TATI, were also significantly increased in DNP. Zymography, Western blotting, and immunofluorometric analysis of DNP urine showed a significant association especially between activation of MMP-9 as well as PMN-type MMP-8 and TRY-2. Our findings suggest that a trypsin-MMP cascade is involved in the pathogenesis of DNP, which may offer new possibilities for diagnosis and treatment of DNP with MMP inhibitors.

Serum matrix metalloproteinases in patients resuscitated from cardiac arrest. The association with therapeutic hypothermia

AIM To study the systemic levels of matrix metalloproteinases (MMP) -7, -8 and -9 and their inhibitor TIMP-1 in cardiac arrest patients and the association with mild therapeutic hypothermia treatment on the serum concentration of these enzymes. METHODS MMP-7, -8 and -9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) were analysed in blood samples obtained from 51 patients resuscitated from cardiac arrest. The samples were taken at 24 and 48 h from restoration of spontaneous circulation (ROSC). The biomarker levels were compared between patients (N=51) and healthy controls (N=10) and between patients who did (N=30) and patients who did not (N=21) receive mild therapeutic hypothermia. RESULTS MMP-7 (median 0.47 ng/ml), MMP-8 (median 31.16 ng/ml) and MMP-9 (median 253.00 ng/ml) levels were elevated and TIMP-1 levels suppressed (median 78.50 ng/ml) in cardiac arrest patients as compared with healthy controls at 24h from ROSC. Hypothermia treatment associated with attenuated elevation of MMP-9 (p=0.001) but not MMP-8 (p=0.02) or MMP-7 (p=0.69). Concentrations of MMPs -7, -8 and -9 correlated with the leukocyte count but not with C-reactive protein (CRP) or neurone-specific enolase (NSE) levels. CONCLUSION We demonstrated that the systemic levels of MMP-7, -8 and -9 but not TIMP-1 are elevated in cardiac arrest patients in the 48 h post-resuscitation period relative to the healthy controls. Patients who received therapeutic hypothermia had lower MMP-9 levels compared to non-hypothermia treated patients, which generates hypothesis about attenuation of inflammatory response by hypothermia treatment.

The role of unfinished root canal treatment in odontogenic maxillofacial infections requiring hospital care

ObjectivesThe aim of this study was to evaluate clinical and radiological findings and the role of periapical infection and antecedent dental treatment of infected focus teeth in odontogenic maxillofacial abscesses requiring hospital care.Materials and methodsIn this retrospective cohort study, we evaluated medical records and panoramic radiographs during the hospital stay of patients (n = 60) admitted due to odontogenic maxillofacial infection originating from periapical periodontitis.ResultsTwenty-three (38 %) patients had received endodontic treatment and ten (17 %) other acute dental treatment. Twenty-seven (45 %) had not visited the dentist in the near past. Median age of the patients was 45 (range 20–88) years and 60 % were males. Unfinished root canal treatment (RCT) was the major risk factor for hospitalisation in 16 (27 %) of the 60 cases (p = .0065). Completed RCT was the source only in 7 (12 %) of the 60 cases. Two of these RCTs were adequate and five inadequate.ConclusionsThe initiation of inadequate or incomplete primary RCT of acute periapical periodontitis appears to open a risk window for locally invasive spread of infection with local abscess formation and systemic symptoms. Thereafter, the quality of the completed RCT appears to have minor impact. However, a considerable proportion of the patients had not received any dental treatment confirming the importance of good dental health. Thus, thorough canal debridement during the first session is essential for minimising the risk for spread of infection in addition to incision and drainage of the abscess. If this cannot be achieved, tooth extraction should be considered.Clinical relevanceIncomplete or inadequate canal debridement and drainage of the abscess may increase the risk for spread of endodontic infection.

Discovery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profiling

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase–signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.