Partial Replacement of Animal Proteins with Plant Proteins for 12 Weeks Accelerates Bone Turnover Among Healthy Adults: A Randomized Clinical Trial.

BACKGROUND: Plant-based diets may reduce the risk of chronic diseases, but can also lead to low calcium and vitamin D intakes, posing a risk for bone health. OBJECTIVES: We investigated whether partial replacement of animal proteins with plant-based proteins using a whole-diet approach affects bone and mineral metabolism in healthy adults in 3 groups fed diets differing in protein composition. METHODS: This 12-week clinical trial was comprised of 107 women and 29 men (20-69 years old; BMI mean ± SD, 24.8 ± 3.9) randomly assigned to consume 1 of 3 diets designed to provide 17 energy percent (E%) protein: "animal" (70% animal protein, 30% plant protein of total protein intake), "50/50" (50% animal, 50% plant), and "plant" (30% animal, 70% plant) diets. We examined differences in bone formation [serum intact procollagen type I amino-terminal propeptide (S-iPINP)], bone resorption [serum collagen type 1 cross-linked C-terminal telopeptide (S-CTX)], mineral metabolism markers (primary outcomes), and nutrient intakes (secondary outcomes) by ANOVA/ANCOVA. RESULTS: S-CTX was significantly higher in the plant group (mean ± SEM, 0.44 ± 0.02 ng/mL) than in the other groups (P values < 0.001 for both), and differed also between the animal (mean ± SEM, 0.29 ± 0.02 ng/mL) and 50/50 groups (mean ± SEM, 0.34 ± 0.02 ng/mL; P = 0.018). S-iPINP was significantly higher in the plant group (mean ± SEM, 63.9 ± 1.91 ng/mL) than in the animal group (mean ± SEM, 55.0 ± 1.82 ng/mL; P = 0.006). In a subgroup without a history of vitamin D supplement use, plasma parathyroid hormone was significantly higher in the plant than in the animal group (P = 0.018). Vitamin D and calcium intakes were below recommended levels in the plant group (mean ± SEM, 6.2 ± 3.7 µg/d and 733 ± 164 mg/d, respectively). CONCLUSIONS: Partial replacement of animal proteins with plant-based proteins for 12 weeks increased the markers of bone resorption and formation among healthy adults, indicating a possible risk for bone health. This is probably caused by lower vitamin D and calcium intakes from diets containing more plant-based proteins, but it is unclear whether differences in protein intake or quality play a major role. This trial was registered at clinicaltrials.gov as NCT03206827.

Serum biomarkers for Modic changes in patients with chronic low back pain.

PURPOSE: Lumbar Modic change (MC) can serve as a diagnostic marker as well as an independent source of chronic low back pain (CLBP). This study aimed to test for the existence of serum biomarkers in CLBP patients with MC. METHODS: Age- and sex-matched CLBP patients with confirmed MC on lumbar MRI (n = 40) and pain-free controls (n = 40) were assessed. MC was classified into M1, predominating M1, predominating M2 and M2. MC volumes were calculated. Fasting blood samples were assessed for inflammatory mediators, signalling molecules, growth factors and bone turnover markers. Serum concentrations of 46 biomarkers were measured. RESULTS: Median concentrations of interleukin (IL)-15 (p < 0.001), IL-8 (p < 0.001), tumour necrosis factor (TNF)-alpha (p < 0.001), Eotaxin-1 (p < 0.05), Eotaxin-3 (p < 0.001), monocyte chemotactic protein (MCP)-1 (p < 0.05), macrophage inflammatory protein (MIP)-1alpha (p < 0.01), TEK receptor tyrosine kinase (Tie)-2 (p < 0.001), vascular cell adhesion molecule (VCAM)-1 (p < 0.001), RANTES (p < 0.001), C telopeptide of type I collagen (CTX)-1 (p < 0.001), vascular endothelial growth factor (VEGF)-C (p < 0.001), VEGF-D (p < 0.05), fms-related tyrosine kinase (Flt)-1 (p < 0.01) and intercellular adhesion molecule (ICAM)-1 (p < 0.01) were significantly higher among controls. IL-1sRII (23.2 vs. 15.5 ng/ml, p < 0.001) and hepatocyte growth factor (HGF)-1 (169 vs. 105 pg/ml, p < 0.01) concentrations were significantly higher among patients. Type or volume of MC was not associated with biomarker concentrations. CONCLUSIONS: This is the first study to assess the blood serum biomarker profile in individuals with CLBP with MC. Several biomarkers were suppressed, while two markers (IL-1sRII and HGF) were elevated among MC patients, irrespective of MC type or size, with CLBP compared with asymptomatic controls.

Elevated preoperative serum levels of collagen I carboxyterminal telopeptide predict better outcome in early-stage luminal-B-like (HER2-negative) and triple-negative subtypes of breast cancer.

Type 1 collagen is an important part of the extracellular matrix and changes in its metabolism and distribution are essential in breast cancer induction and progression. Serum concentrations of type 1 collagen synthesis (aminoterminal propeptide (PINP)) and degradation markers (carboxyterminal telopeptide (ICTP)) have previously been studied in early and metastatic breast cancer, but no data are available on specific breast cancer subtypes. We assayed 662 preoperative serum samples for PINP and ICTP and 109 postoperative serum samples for ICTP. The results were linked to prospectively collected clinical data and the cases were divided into breast cancer subtypes for survival analyses. The concentrations of both pre- and postoperative ICTP serum levels increased linearly from ductal in situ carcinoma to stage I-II tumors, stage III tumors, and finally to those with concomitant primary metastases (preoperative ICTP, p = 0.009; postoperative ICTP, p = 0.016). High-preoperative ICTP levels were associated with better breast cancer-specific survival in connection with luminal-B-like (HER2-negative) tumors (p = 0.017), which was confirmed in Cox regression analysis (relative risk = 3.127; 95% confidence interval = 1.081-9.049, p = 0.035), when T-class (relative risk = 4.049; 95% confidence interval = 1.263-12.981; p = 0.019) and nodal status (relative risk = 3.896; 95% confidence interval = 1.088-13.959; p = 0.037) were included in the analysis. In patients with triple-negative breast cancer, a high-preoperative ICTP level was a significant predictor of local relapse-free survival in univariate (p = 0.0020) and multivariate analyses (relative risk = 13.04; 95% confidence interval = 1.354-125.5; p = 0.026; for T-class, relative risk = 2.128 and 95% confidence interval = 0.297-15.23; p = 0.452; for N-class, relative risk = 0.332 and 95% confidence interval = 0.033-3.307; p = 0.347). A preoperatively elevated serum ICTP level appears to be an important marker of better prognosis in triple-negative breast cancer and luminal-B-like (HER2-negative) subtypes.

Autoimmune antibodies to collagen XIII in myasthenia gravis patients.

INTRODUCTION: Evaluation of the nerve fascicular structure can be useful in diagnosing nerve damage, but it is a very challenging task with 3T MRI because of limited resolution. In this pilot study, we present the feasibility of high-resolution 7T MRI for examining the nerve fascicular structure. METHODS: A 3-dimensional (3D) gradient-spoiled sequence was used for imaging peripheral nerves in extremities. Images acquired with different in-plane resolutions (0.42 × 0.42 mm vs. 0.12 × 0.12 mm), and different main field strengths (7T vs. 3T) were compared. RESULTS: The individual nerve fascicles were identified at 0.12 × 0.12 mm resolution in both field strengths but not at 0.42 × 0.42 mm resolution. The fascicular structure was more sharply depicted in 7T images than in 3T images. DISCUSSION: High-resolution 3D imaging with 7T MRI demonstrated feasibility for imaging nerve fascicular structures. Muscle Nerve 57: 506-510, 2018.

The interplay of matrix metalloproteinase-8, transforming growth factor-ß1 and vascular endothelial growth factor-C cooperatively contributes to the aggressiveness of oral tongue squamous cell carcinoma.

BACKGROUND: Matrix metalloproteinase-8 (MMP-8) has oncosuppressive properties in various cancers. We attempted to assess MMP-8 function in oral tongue squamous cell carcinoma (OTSCC). METHODS: MMP-8 overexpressing OTSCC cells were used to study the effect of MMP-8 on proliferation, apoptosis, migration, invasion and gene and protein expression. Moreover, MMP-8 functions were assessed in the orthotopic mouse tongue cancer model and by immunohistochemistry in patient samples. RESULTS: MMP-8 reduced the invasion and migration of OTSCC cells and decreased the expression of MMP-1, cathepsin-K and vascular endothelial growth factor-C (VEGF-C). VEGF-C was induced by transforming growth factor-ß1 (TGF-ß1) in control cells, but not in MMP-8 overexpressing cells. In human OTSCC samples, low MMP-8 in combination with high VEGF-C was an independent predictor of poor cancer-specific survival. TGF-ß1 treatment also restored the migration of MMP-8 overexpressing cells to the level of control cells. In mouse tongue cancer, MMP-8 did not inhibit metastasis, possibly because it was eliminated in the peripheral carcinoma cells. CONCLUSIONS: The suppressive effects of MMP-8 in OTSCC may be mediated through interference of TGF-ß1 and VEGF-C function and altered proteinase expression. Together, low MMP-8 and high VEGF-C expression have strong independent prognostic value in OTSCC.

Tenascin-C and fibronectin expression divide early stage tongue cancer into low- and high-risk groups.

BACKGROUND: Oral tongue squamous cell carcinoma (OTSCC) metastasises early, especially to regional lymph nodes. There is an ongoing debate on which early stage (T1-T2N0) patients should be treated with elective neck dissection. We need prognosticators for early stage tongue cancer. METHODS: Mice immunisation with human mesenchymal stromal cells resulted in production of antibodies against tenascin-C (TNC) and fibronectin (FN), which were used to stain 178 (98 early stage), oral tongue squamous cell carcinoma samples. Tenascin-C and FN expression in the stroma (negative, moderate or abundant) and tumour cells (negative or positive) were assessed. Similar staining was obtained using corresponding commercial antibodies. RESULTS: Expression of TNC and FN in the stroma, but not in the tumour cells, proved to be excellent prognosticators both in all stages and in early stage cases. Among early stages, when stromal TNC was negative, the 5-year survival rate was 88%. Correspondingly, when FN was negative, no cancer deaths were observed. Five-year survival rates for abundant expression of TNC and FN were 43% and 25%, respectively. CONCLUSIONS: Stromal TNC and, especially, FN expressions differentiate patients into low- and high-risk groups. Surgery alone of early stage primary tumours might be adequate when stromal FN is negative. Aggressive treatments should be considered when both TNC and FN are abundant.

Bone markers in polycystic ovary syndrome: A multicentre study.

OBJECTIVE: Hyperandrogenism, hyperinsulinaemia and obesity, known characteristics of polycystic ovary syndrome (PCOS), may influence bone mineral density and biochemical markers of bone turnover (BTMs) can provide a noninvasive assessment of bone turnover. To this end, the serum concentrations of BTMs and 25-hydroxyvitamin D (25OHD) were analysed in women with PCOS, and their possible associations with metabolic parameters of PCOS were determined. SUBJECTS AND METHODS: Bone formation markers procollagen type I amino-terminal propeptide (PINP) and osteocalcin (OC), and bone resorption marker carboxy-terminal cross-linking telopeptide of type I collagen (CTX), along with 25OHD, were measured in 298 women with PCOS and 194 healthy controls. RESULTS: Serum levels of PINP (47.0 ± 20.2 vs 58.1 ± 28.6 µg/L, P < .001) and OC (18.2 ± 7.5 vs 20.6 ± 9.8 µg/L, P < .001) were decreased in women with PCOS compared with controls, whereas no significant differences were found in CTX and 25OHD levels. Age-stratified analyses suggested that PINP (50.5 ± 21.7 vs 68.2 ± 26.6 µg/L, P < .001) and OC levels (20.4 ± 7.6 vs 25.5 ± 9.6 µg/L, P < .001) were decreased only in the younger age group (≤30 years) women with PCOS compared with controls. The formation markers and resorption marker decreased with age in both study groups. CONCLUSIONS: Bone formation markers were decreased in younger women with PCOS when compared with healthy women, which may affect bone mass in these women.

Neoplastic extracellular matrix environment promotes cancer invasion in vitro.

The invasion of carcinoma cells is a crucial feature in carcinogenesis. The penetration efficiency not only depends on the cancer cells, but also on the composition of the tumor microenvironment. Our group has developed a 3D invasion assay based on human uterine leiomyoma tissue. Here we tested whether human, porcine, mouse or rat hearts as well as porcine tongue tissues could be similarly used to study carcinoma cell invasion in vitro. Three invasive human oral tongue squamous cell carcinoma (HSC-3, SCC-25 and SCC-15), melanoma (G-361) and ductal breast adenocarcinoma (MDA-MB-231) cell lines, and co-cultures of HSC-3 and carcinoma-associated or normal oral fibroblasts were assayed. Myoma tissue, both native and lyophilized, promoted invasion and growth of the cancer cells. However, the healthy heart or tongue matrices were unable to induce the invasion of any type of cancer cells tested. Moreover, when studied in more detail, small molecular weight fragments derived from heart tissue rinsing media inhibited HSC-3 horizontal migration. Proteome analysis of myoma rinsing media, on the other hand, revealed migration enhancing factors. These results highlight the important role of matrix composition for cancer invasion studies in vitro and further demonstrate the unique properties of human myoma organotypic model.

Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures.

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-ß1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

Efficacy and Toxicity of Intrathecal Liposomal Cytarabine in First-line Therapy of Childhood Acute Lymphoblastic Leukemia.

We investigated efficacy and toxicity of replacing conventional triple (cytarabine, methotrexate, and hydrocortisone) intrathecal therapy (TIT) with liposomal cytarabine during maintenance therapy among 40 acute lymphoblastic leukemia patients. Twenty-eight of 29 patients in the TIT arm received TIT and 9/11 in the liposomal cytarabine arm received liposomal cytarabine. Arachnoiditis occurred in all initial 5 patients given liposomal cytarabine and intrathecal prednisolone succinate. Subsequently liposomal cytarabine was given with systemic dexamethasone. Neurotoxicity occurred at 6/27 liposomal cytarabine administrations with concomitant dexamethasone (22%). More liposomal cytarabine-treated patients experienced neurotoxicity in relation to intrathecal therapy during at least 1 cycle compared with TIT-treated patients (6/9 [67%] vs. 3/28 [11%], P=0.002). Apart from intermittent lower extremity sensory pain in 1 liposomal cytarabine-treated patient, no permanent adverse neurological sequelae were observed. In intention-to-treat analysis, projected 5-year event-free survival (pEFS-5y) was borderline higher for patients in the liposomal cytarabine arm compared with the TIT arm (1.0 vs. 0.69, P=0.046). However, pEFS-5y and projected 5-year relapse-free survival did not differ signficantly between patients treated with liposomal cytarabine or TIT (1.0 vs. 0.73, P=0.10; 1.0 vs. 0.76, P=0.12). Larger prospective trials are needed to explore whether liposomal cytarabine should be used as first-line prevention of relapse.

Automated immunoassays for the autoantibodies to carbamylated or citrullinated telopeptides of type I and II collagens.

BACKGROUND: The aim of the study was to describe automated immunoassays for autoantibodies to homocitrulline or citrulline containing telopeptides of type I and II collagen in various disease categories in an early arthritis series. METHODS: Serum samples were collected from 142 patients over 16 years of age with newly diagnosed inflammatory joint disease. All samples were analyzed with an automated inhibition chemiluminescence immunoassay (CLIA) using four different peptide pairs, each consisting of a biotinylated antigen and an inhibiting peptide. Assays were performed with an IDS-iSYS analyzer. Autoantibodies binding to homocitrulline and citrulline containing C-telopeptides of type I (HTELO-I, TELO-I) and type II collagens (HTELO-II, TELO-II) were analyzed. RESULTS: The mean ratio of HTELO-I inhibition in seropositive and seronegative rheumatoid arthritis (RA) was 3.07 (95% CI 1.41-11.60), p=0.003, and in seropositive and seronegative undifferentiated arthritis (UA) 4.90 (1.85-14.49), p<0.001. The respective mean ratios in seropositive and seronegative RA and UA were in TELO-I 8.72 (3.68-58.01), p<0.001 and 3.13 (1.49-6.16), p=0.008, in HTELO-II 7.57 (3.18-56.60), p<0.001 and 2.97 (1.23-6.69), p=0.037, and in TELO-II 3.01 (1.30-9.51), p=0.002 and 3.64 (1.86-7.65), p=0.008. In reactive arthritis, ankylosing spondylitis, psoriatic arthritis and unspecified spondyloarthritis the inhibition levels were similar to those observed in seronegative RA or UA. CONCLUSIONS: Autoantibodies binding to homocitrulline or citrulline containing telopeptides of type I and II collagen did not differ significantly. They were highest among patients with seropositive disease and they differentiated seropositive and seronegative arthritis.

The hypoxic tumor microenvironment regulates invasion of aggressive oral carcinoma cells.

Invasion is an important hallmark of cancer involving interactions between the tumor microenvironment and the cancer cells. Hypoxia, low oxygen level, is related to increased invasion and metastasis in many cancers. The aim was to elucidate the effect of hypoxia on invasion of oral squamous cell carcinoma cells (OSCCs), and the applicability of a novel 3-dimentional myoma organotypic invasion model in hypoxia experiments. OSCC cell lines (primary oral carcinoma derived cells UT-SCC-43A, recurrent oral carcinoma cells UT-SCC-43B and aggressive tongue carcinoma cells HSC-3) were studied for their migration and invasion capabilities under normoxia, hypoxia, and in the presence a hypoxia-mimicker cobalt chloride. As expected, the recurrent UT-SCC-43B cells were significantly more aggressive than the primary tumor derived cells. In contrast to tongue carcinoma HSC-3 cells, they only mildly responded to hypoxia in the migration or invasion assays, indicating a cell line specific response of hypoxia on the invasive potential. The modification of the organotypic human tissue-derived matrix via the removal of various yet unidentified soluble factors by rinsing the tissue resulting in stripped matrix substantially changed the invasion pattern of HSC-3 cells and the outcomes of hypoxic treatments. Only in the stripped tissue hypoxia significantly increased invasion, whereas in native intact tissue the induced invasion was not observed. This demonstrates the importance of the soluble factors to the invasion pattern and to the hypoxia response. A metastasis and poor prognosis marker, hypoxia-regulated lysyl oxidase (LOX), was present in the myoma tissue, but could be removed by rinsing. The inhibition of LOX resulted in a decrease in invasion area, but only very mildly in invasion depth. Thus, it may have a role in the modulation of the invasion pattern. Another hypoxia-related poor prognosis marker carbonic anhydrase 9 (CAIX) was induced in HSC-3 cells both by the hypoxic exposure and interestingly in invading HSC-3 cells inside the tissue even in normoxic conditions. In conclusion, this suggests that the intact myoma organotypic model offers optimally hypoxic surroundings, thus being an excellent human tumor microenvironment mimicker.

Procollagen I-expressing renin cell precursors.

Renin-expressing cells in the kidney normally appear as mural cells of developing preglomerular vessels and finally impose as granulated juxtaglomerular cells in adult kidneys. The differentiation of renin-expressing cells from the metanephric mesenchyme in general and the potential role of special precursor stages in particular is not well understood. Therefore, it was the aim of this study to search for renin cell precursors in the kidney. As an experimental model, we used kidneys of aldosterone synthase-deficient mice, which display a prominent compensatory overproduction of renin cells that are arranged in multilayered perivascular cell clusters. We found that the perivascular cell clusters contained two apparently distinct cell types, one staining positive for renin and another one staining positive for type I procollagen (PC1). It appeared as if PC1 and renin expression were inversely related at the cellular level. The proportion of renin-positive to PC1-positive cells in the clusters was inversely linked to the rate of salt intake, as was overall renin expression. Our findings suggest that the cells in the perivascular cell clusters can reversibly switch between PC1 and renin expression and that PC1-expressing cells might be precursors of renin cells. A few of those PC1-positive cells were found also in adult wild-type kidneys in the juxtaglomerular lacis cell area, in which renin expression can be induced on demand.

Different amounts of protein-bound citrulline and homocitrulline in foot joint tissues of a patient with anti-citrullinated protein antibody positive erosive rheumatoid arthritis.

BACKGROUND: Antibodies binding to citrullinated proteins are a frequent finding in rheumatoid arthritis patients and may precede the onset of clinical symptoms several years. The antibodies are a predisposing factor for bone erosions but their origin is unknown. In this study we analyze in detail the levels of protein bound citrulline and homocitrulline in several tissue samples of a single erosive arthritic surgery patient. METHODS: Serum antibodies binding to CCP, MCV and citrulline- or homocitrulline-containing type I and II collagen carboxytelopeptides were measured. Tissue samples of a single RA patient, taken in two separate operations performed with two-year time span were hydrolyzed and analyzed for citrulline and homocitrulline content by HPLC. RESULTS: Protein-bound citrulline and homocitrulline were found in several joint tissues of a RA patient with ACPA-positive erosive disease. The amount of homocitrulline stayed relatively constant between the different tissues. The amount of citrulline in erosive tissue was 3-times higher than in non-erosive tissue in the first operation. In the samples of the second operation 3-4-times higher mean amounts of citrulline were found in two out of the six tissues investigated. CONCLUSIONS: Homocitrulline is present in rheumatoid nodule together with citrulline. There is more variation in the amount of citrulline than in the amount of homocitrulline between the tissues. The tissue sample containing the most citrulline was the most erosive.

New insights to the role of aryl hydrocarbon receptor in bone phenotype and in dioxin-induced modulation of bone microarchitecture and material properties.

Bone is a target for high affinity aryl hydrocarbon receptor (AHR) ligands, such as dioxins. Although bone morphology, mineral density and strength are sensitive endpoints of dioxin toxicity, less is known about effects on bone microarchitecture and material properties. This study characterizes TCDD-induced modulations of bone tissue, and the role of AHR in dioxin-induced bone toxicity and for normal bone phenotype. Six AHR-knockout (Ahr(-/-)) and wild-type (Ahr(+/+)) mice of both genders were exposed to TCDD weekly for 10 weeks, at a total dose of 200µg/kgbw. Bones were examined with micro-computed tomography, nanoindentation and biomechanical testing. Serum levels of bone remodeling markers were analyzed, and the expression of genes related to osteogenic differentiation was profiled using PCR array. In Ahr(+/+) mice, TCDD-exposure resulted in harder bone matrix, thinner and more porous cortical bone, and a more compact trabecular bone compartment. Bone remodeling markers and altered expression of a number of osteogenesis related genes indicated imbalanced bone remodeling. Untreated Ahr(-/-) mice displayed a slightly modified bone phenotype as compared with untreated Ahr(+/+) mice, while TCDD exposure caused only a few changes in bones of Ahr(-/-) mice. Part of the effects of both TCDD-exposure and AHR-deficiency were gender dependent. In conclusion, exposure of adult mice to TCDD resulted in harder bone matrix, thinner cortical bone, mechanically weaker bones and most notably, increased trabecular bone volume fraction in Ahr(+/+) mice. AHR is involved in bone development of a normal bone phenotype, and is crucial for manifestation of TCDD-induced bone alterations.

Four automated 25-OH total vitamin D immunoassays and commercial liquid chromatography tandem-mass spectrometry in Finnish population.

BACKGROUND: Comparing four fully automated 25-OH-D immunoassays to a commercially available liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with human serum samples from Finnish population. METHODS: 400 samples were analyzed with the Liaison Total Vitamin D, the IDS-iSYS 25-Hydroxy Vitamin D, the ARCHITECT 25-OH Vitamin D, the ADVIA Centaur Vitamin D Total, and a commercially available LC-MS/MS 25-OH D (PerkinElmer) method. RESULTS: The Liaison method mean value (95% confidence intervals) was 65.6 nmol/L (62.6 - 68.6); the IDS-iSYS mean was 70.3 nmol/L (67.4 - 73.1); the ARCHITECT mean was 69.0 nmol/L (65.5 - 72.5); ADVIA Centaur mean was 71.6 nmol/L (68.9 - 74.3), and the LC-MS/MS mean was 82.8 nmol/L (79.4 - 86.2). The regression coefficients (r) between the LC-MS/MS and immunoassays were 0.650 for Liaison, 0.757 for IDS-iSYS, 0.721 for ARCHITECT and 0.684 for ADVIA Centaur. With the Passing-Bablok analysis, none of the immunoassays gave results equivalent to LC-MS/MS. Two of the four automated 25-OH-vitamin D assays (IDS-iSYS, ADVIA Centaur) were overall in good clinical agreement with LC-MS/MS, even though the results obtained with all compared methods were not equivalent. CONCLUSIONS: In conclusion, in routine clinical laboratory both immunoassays and LC-MS/MS are useful for measuring 25-OH-vitamin D provided that these methods are correctly standardized and especially sample pretreatment is carefully performed.

Extracellular matrix alterations and acute inflammation; developing in parallel during early induction of pulmonary fibrosis.

Pulmonary fibrosis is a hallmark of several systemic diseases such as systemic sclerosis. Initiation and early development is not well characterized, as initiation usually is unnoticed in patients, yet fibrosis has been considered a late event, occurring after an inflammatory phase. By utilizing an animal model, the starting point can be defined and the initiation process and early development thoroughly investigated. To investigate these processes from a systemic perspective, we choose a systemic administration route, instead of the more commonly used local administration. The aim of this work was to study the initiation of pulmonary fibrosis in an animal model and to investigate early alterations in connective tissue, cell turnover and acute immune response in lung parenchyma. Animals were injected subcutaneously with bleomycin, three times a week (w) for 1-4w (controls received saline). Total collagen was histologically assessed by Picro Sirius Red and Masson's Trichrome, collagen production by antibodies directed against N-terminal of procollagens I and III, proliferation by labeling with proliferating cell nuclear antigen, apoptosis by TUNEL and innate immunity by detecting neutrophils and macrophages. Total collagen was significantly increased at 1, 2 and 4w compared with controls. Procollagen I, was increased at 1w and remained increased, whereas procollagen III-staining was increased at 2w, compared with controls. Myofibroblasts were increased at all times as were proliferation, whereas apoptosis was increased from 2w. Neutrophils peaked at 1w (2779±820 cells/mm²) and gradually decreased, whereas macrophages peaked at 2w (135±29 cells/mm²). Subcutaneously administered bleomycin induces rapid alterations in connective tissue and cell turnover, suggesting a plasticity of the connective tissue. A transient neutrophilia is detected and increased number of macrophages likely represents a clearance process of said neutrophils. The study suggests fibrosis initiation and acute inflammation to occur in parallel in this model.

Antibodies binding to citrullinated telopeptides of type I and type II collagens and to mutated citrullinated vimentin synergistically predict the development of seropositive rheumatoid arthritis.

OBJECTIVE: To assess whether antibodies binding to citrullinated carboxy-terminal telopeptides of type I and type II collagens (TELO-I and TELO-II, respectively), mutated citrullinated vimentin (MCV) and cyclic citrullinated peptides (CCP) predict the development of rheumatoid arthritis (RA). METHODS: A case-control study was nested within a Finnish cohort of 36 000 adults who had neither arthritis nor a history of arthritis at baseline examination in 1966-1972. Among them, 151 subjects developed seropositive (ie, positive for rheumatoid factor) RA, and 67 subjects developed seronegative RA by late 1989. One or two control subjects were chosen for every case. Preillness serum specimens were analysed for antibodies against synthetic TELO-I, TELO-II, MCV and CCP. RESULTS: The mean levels of anti-TELO-I, anti-TELO-II, anti-CCP and anti-MCV antibodies were higher in subjects who later developed seropositive or seronegative RA than in controls. In subjects who later developed seronegative RA, anti-TELO-I and anti-TELO-II antibodies were statistically significantly higher compared with controls (p=0.005 and p=0.013). In the highest tertiles of anti-MCVs and anti-TELO-I levels, the OR for rheumatoid-factor-positive RA were 2.66 (95% CI 1.48 to 4.77) or 2.51 (CI 1.48 - 4.28), respectively. The subjects ranked into the highest tertiles of both anti-TELO-I and anti-MCV antibodies had a 4.56 OR (95% CI 1.82 to 11.46) of developing seropositive RA compared with those in the lowest tertiles of these antibodies. For the co-occurrence for high anti-TELO-II and anti-MCV antibodies, the corresponding OR was 3.62 (95% CI 1.37 to 9.54). CONCLUSION: Antibodies to TELO-I or TELO-II and MCV exert a synergistic effect on the risk of developing seropositive RA.

Trypsin-2 enhances carcinoma invasion by processing tight junctions and activating ProMT1-MMP.

Enhanced proteolysis and altered tight junction (TJ) proteins associate with carcinoma invasion. We hypothesized that trypsin-2, a tumor-associated serine proteinase, induces tongue carcinoma invasion by activating pro-membrane type-1 matrix metalloproteinase (MT1-MMP) and disturbing the TJs. The effects of invasion were analyzed using trypsin-2 over-expressing human tongue squamous cell carcinoma cells (Try2-HSC-3) in vitro and in vivo. The invasion of Try2-HSC-3 cells was increased in mouse xenografts and human organotypic model. Trypsin-2 activated proMT1-MMP, as well as altered the expression of TJ protein claudin-7. In conclusion, trypsin-2 over-expression enhanced tongue carcinoma cell invasion by various genetic and proteolytic mechanisms.

Comparison of automated 25-OH vitamin D immunoassays with liquid chromatography isotope dilution tandem mass spectrometry.

BACKGROUND: Comparing two fully automated 25-OH-D immunoassays with a validated liquid chromatography isotope dilution tandem mass spectrometry (LC-IDMS/MS) method in human serum samples. METHODS: 180 samples were analyzed with the Liaison TOTAL Vitamin D, the IDS-iSYS 25-Hydroxy Vitamin D, and a validated LC-IDMS/MS 25-OH D method. RESULTS: The Liaison method mean +/- standard deviation (SD) was 64.3 +/- 23.5 nmol/L; the IDS-iSYS 70.1 +/- 27.0 nmol/L, and the LC-IDMS/MS 68.9 +/- 24.6 nmol/L. The correlation coefficient (r) between the Liaison and LC-IDMS/MS was 0.723, and the r between the IDS-iSYS and LC-IDMS/MS was 0.799. When the samples were grouped by females and males, the r between the Liaison and LC-IDMS/MS was 0.680 and 0.846, being 0.781 and 0.863 between the IDS-iSYS and LC-IDMS/MS, respectively. With the Passing-Bablok analysis, only the IDS-iSYS gave equivalent results to LC-IDMS/MS. CONCLUSIONS: The automated 25-OH-D immunoassays were in clinically good overall agreement with the LC-IDMS/MS method.