Human C-reactive protein aggravates osteoarthritis development in mice on a high-fat diet.

OBJECTIVE: C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of 'metabolic' OA. DESIGN: Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n = 30) and wild-type littermates (n = 15) a 45 kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. RESULTS: Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. CONCLUSIONS: Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development.

Variable cartilage degradation in mice with diet-induced metabolic dysfunction: food for thought.

OBJECTIVE: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. DESIGN: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden.CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. RESULTS: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. CONCLUSIONS: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors.

Metabolic stress-induced inflammation plays a major role in the development of osteoarthritis in mice.

OBJECTIVE: Obesity is associated with systemic inflammation and is a risk factor for osteoarthritis (OA) development. We undertook this study to test the hypothesis that metabolic stress-induced inflammation, and not mechanical overload, is responsible for the development of high-fat diet-induced OA in mice. METHODS: Human C-reactive protein (CRP)-transgenic mice received a high-fat diet without or with 0.005% (weight/weight) rosuvastatin or 0.018% (w/w) rosiglitazone, 2 different drugs with antiinflammatory properties. Mice fed chow were included as controls. After 42 weeks, mice were killed and histologic OA grading of the knees was performed. To monitor the overall inflammation state, systemic human CRP levels were determined. RESULTS: Male mice on a high-fat diet had significantly higher OA grades than mice on chow and showed no correlation between OA severity and body weight. In male mice, high-fat diet-induced OA was significantly inhibited by rosuvastatin or rosiglitazone to OA grades observed in control mice. Both treatments resulted in reduced human CRP levels. Furthermore, a positive correlation was found between the relative individual induction of human CRP evoked by a high-fat diet on day 3 and OA grade at end point. CONCLUSION: High-fat diet-induced OA in mice is due to low-grade inflammation and not to mechanical overload, since no relationship between body weight and OA grade was observed. Moreover, the OA process was inhibited to a great extent by treatment with 2 drugs with antiinflammatory properties. The inflammatory response to a metabolic high-fat challenge may predict individual susceptibility to developing OA later in life. The use of statins or peroxisome proliferator-activated receptor γ agonists (e.g., rosiglitazone) could be a strategy for interfering with the progression of OA.

Adult hyperinsulinemic hypoglycemia not caused by an insulinoma: a report of two cases.

Nesidioblastosis is rare in adults and accounts for 0.5-5% of cases of organic hyperinsulinemia. The diagnosis of nesidioblastosis should be considered when peroperative imaging modalities fail to localize a lesion in patients with hyperinsulinism. Two female patients, aged 55 and 16 years, with hyperinsulinemic hypoglycemia are reported. Somatostatin receptor scintigraphy showed slight focal activity in both patients. The first patient underwent a Whipple procedure and became diabetic. The second patient underwent a distal hemi-pancreatectomy and suffered from recurrent hypoglycemic episodes 3 months after surgery, for which she is presently being treated with octreotide. Histological examination of the resected pancreata revealed focally increased islet tissue and a number of slightly hypertrophic beta cells. Such histological abnormalities have been related to functional changes of beta-cells. In infantile nesidioblastosis, a proportion of cases has been associated with mutations in one of several genes. Whether such mutations, leading to hyperinsulinism, also play a role in adult nesidioblastosis is presently unknown.

Proliferative index in phaeochromocytomas: does it predict the occurrence of metastases?

Evaluation of the malignant potential of phaeochromocytomas in the absence of metastases presents a formidable challenge to both clinicians and pathologists. Until now, no widely accepted clinical, histological, immunohistochemical or molecular method has become available to discriminate malignant from benign phaeochromocytomas. In other endocrine tumours, estimation of proliferative activity by MIB-1 immunostaining has emerged as a promising approach for the determination of metastatic potential. In this study, the utility of MIB-1 immunostaining as a predictive marker for the occurrence of metastases in phaeochromocytomas was evaluated. In addition, the density of S100-positive sustentacular cells was studied, since their depletion has been identified as a negative predictive marker in smaller series. Furthermore, several clinicopathological parameters were evaluated. One hundred and ten patients operated on for a total of 99 benign and 37 malignant phaeochromocytomas were studied. All malignant tumours had documented metastases. The histopathological diagnosis of primary tumours and metastases was reviewed and graded for angioinvasion, capsular extension, and intra-tumoural necrosis. The proliferative index (percentage of MIB-1-positive cells) and the density of S100-positive cells were assessed. In addition, age at resection, associated familial tumour syndromes, tumour size, and tumour location were recorded. Univariate analysis revealed statistically significant correlations between malignancy and proliferative index (p<0.0005) and depletion of S100-positive sustentacular cells (p<0.0005). Fifty per cent of the malignant, but none of the benign phaeochromocytomas had a proliferative index greater than 2.5%. Higher age at resection (p=0. 03), sporadic occurrence (p<0.0005), extra-adrenal location (p<0. 0005), tumour size (p<0.0005), and necrosis (p=0.03) were also significantly associated with malignancy. Logistic regression showed that proliferative index (p=0.0072), size (p=0.0022), and extra-adrenal location (p=0.0012) of the primary tumour were independently predictive for malignancy. In conclusion, this study indicates that assessing the proliferative activity of phaeochromocytomas by MIB-1 immunohistochemistry can predict the occurrence of metastases. The predictive value of S100 immunostaining, tumour size, and extra-adrenal location of the tumour was also confirmed.

Prognostic value of p53, bcl-2, and c-erbB-2 protein expression in phaeochromocytomas.

Many studies have tried to discriminate malignant from benign phaeochromocytomas, but until now no widely accepted histological, immunohistochemical, or molecular methods have been available. In this study of 29 malignant and 85 benign phaeochromocytomas from 102 patients, immunohistochemistry was performed with antibodies to the tumour suppressor gene product p53 and the proto-oncogene products bcl-2 and c-erbB-2, using the avidin-biotin complex method. Malignant phaeochromocytomas showed a statistically significant higher frequency of p53 (p=0.042) and bcl-2 (p=0.037) protein expression than their benign counterparts. The combination of both markers showed an even higher significance (p=0.004), to which both markers contributed equally. Overexpression of c-erbB-2 was associated with the occurrence of familial phaeochromocytomas (p=0. 001), but no difference was found between benign and malignant cases. In conclusion, p53, bcl-2, and c-erbB-2 all appear to be involved in the pathogenesis of a proportion of phaeochromocytomas. Immunoreactivity to p53 and bcl-2 proteins may help to predict the clinical behaviour of phaeochromocytomas.

Anti-CD25 therapy reveals the redundancy of the intragraft cytokine network after clinical heart transplantation.

BACKGROUND: Despite blockade of the interleukin-2/interleukin 2 receptor (IL-2/IL-2R) pathway by the murine anti-CD25 (i.e., IL-2R alpha chain) monoclonal antibody BT563, cardiac rejection can still occur. In these cases, growth factors other than IL-2 may contribute to allograft rejection. We studied the expression of IL-15, a macrophage-derived cytokine associated with T-cell activation, which interacts with the beta and gamma chains of the IL-2R during rejection episodes under anti-CD25 therapy. METHODS: We measured intragraft IL-15 mRNA expression and the number of IL-15- and CD68-positive cells in posttransplantation endomyocardial biopsies (EMBs; n=45) and in nontransplanted, donor-heart specimens (n=11) by competitive template reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: IL-15 mRNA expression was present in the majority of posttransplantation EMB specimens (91%, 41/45) and in nontransplanted donor-heart specimens (91%, 10/11). Relative IL-15 mRNA levels were neither associated with transplantation nor with rejection status. After transplantation, the number of IL-15- and CD68-positive cells significantly increased (P<0.001), but IL-15-positive cell counts did not reflect the histological rejection grade. Anti-CD25 treatment, in contrast to its effects on the IL-2/IL-2R complex, had no influence on intragraft IL-15 mRNA and protein production. In rejection EMB specimens, during (n=5) and after (n=8) anti-CD25 therapy, no differences in relative IL-15 mRNA levels, or in IL-15- and CD68-positive cell counts, were measured. CONCLUSIONS: After heart transplantation, high numbers of IL-15- and CD68-positive cells infiltrate the graft. This phenomenon is independent of the rejection status. IL-15 remains present during blockade of the IL-2/IL-2R pathway by anti-CD25 monoclonal antibodies, and it may participate in T cell-dependent donor-directed immune responses, thereby explaining the occurrence of rejection in the absence of IL-2.

Immunohistochemical localization of somatostatin receptor sst2A in human rheumatoid synovium.

OBJECTIVE: To identify the somatostatin receptor-expressing cells in rheumatoid synovium using a recently developed antiserum directed against the somatostatin receptor subtype 2A (sst2A). METHODS: We carried out immunohistochemical studies of synovial biopsies from 7 patients with rheumatoid arthritis (RA) and one non-RA patient, using a rabbit polyclonal antiserum directed against sst2A and monoclonal antibodies directed against phenotypic markers. RESULTS: SSt2A was expressed by the endothelial cells of the synovial venules but also by a subset of synovial macrophages. CONCLUSION: The identification of somatostatin receptors on macrophages, which are thought to be important effector cells in RA, may offer mechanistic insights into the potential therapeutic effect of somatostatin (analogs) in RA.

Immunohistochemical detection of somatostatin receptor subtypes sst1 and sst2A in human somatostatin receptor positive tumors.

Although in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the cellular localization of human sst1 and sst2A receptor proteins in both cryostat- and paraffin-embedded sections of 25 human tumor tissues using two recently developed polyclonal antibodies. Six somatostatin (SS) receptor (SSR) positive tumors (two gastrinomas, three carcinoids, one pheochromocytoma) and one SSR negative tumor (renal cell carcinoma), selected by positive and negative SSR autoradiography, respectively, were studied by both immunohistochemistry and Western blot analysis. The six SSR positive tumors expressed sst2A, while 4 of 5 expressed sst1 as well. The SSR negative tumor did not express either sst1 or sst2A. Western blot analysis of wheat germ agglutinin purified membrane proteins confirmed the presence of the sst1 and sst2A glycosylated receptors. The paraffin-embedded sections gave best information with respect to the subcellular localization. Sst1 immunoreactivity was observed both on the membrane and in the cytoplasm, while sst2A showed predominantly membrane-associated immunoreactivity. This subcellular distribution of sst1 or sst2A receptors was confirmed in paraffin-embedded sections of 8 additional intestinal carcinoids, 5 gastrinomas and 5 pheochromocytomas. Sst1 receptors were detected in 7 out of 8 carcinoids, in all gastrinomas, and in 4 out of 5 pheochromocytomas, while 6 out of 8 carcinoids, all gastrinomas, and 3 out of 5 pheochromocytomas expressed sst2A receptors. In conclusion, sst1 and sst2A receptors show a differential subcellular localization in human SSR positive tumors. The use of SSR subtype selective antibodies to detect the subcellular distribution of SSR subtypes in individual tumor cells is an important step forward to understand more about the pathophysiological role of the different SSR subtypes in human tumors.

Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions.

Cytological slides of serous fluids of 41 malignant mesotheliomas, 88 metastatic adenocarcinomas, and 25 reactive effusions were immunostained with the antibodies anti-CEA, MOC-31, Leu-M1, Ber-EP4, and B72.3. Most mesotheliomas and all reactive fluids failed to stain with these antibodies. The sensitivity of the five markers to detect carcinoma cells differed remarkably. Especially MOC-31, Ber-EP4, and B72.3 stained with a high number of carcinoma cases and the complemetary value of Ber-EP4 and B72.3 to immunostain carcinoma cells was impressive: 94% of the metastatic adenocarcinoma cases reacted with Ber-EP4 or B72.3 in contrast to 1 of 41 malignant mesotheliomas.

Overlapping stage models and reaction time additivity: effects of the activation equation.

Many experimental factors have been found to affect mean reaction time (RT) additively in factorial experiments. What sorts of RT models are compatible with this fact? Sternberg (1969) showed that serial, discrete-stage models are consistent with additivity, and as a result additivity has sometimes been regarded as evidence in favor of such models. However, McClelland (1979) showed that an alternative "cascade" model, which violates crucial assumptions of discrete-stage models, also predicts RT additivity in many cases. This article examines various modified versions of the cascade model, and shows that mean RT additivity arises from many overlapping stage models, including some with thresholds, information quantization, and nonlinear activation transformations. This suggests that other aspects of the data besides mean RT additivity should be examined to distinguish between serial and overlapping stage models.

Neural cell adhesion molecule distribution in primary and metastatic uveal melanoma.

Tumor cell adhesion, detachment, and aggregation play an important part in tumor invasion and metastasis, and a variety of cell adhesion molecules have been found on tumor cells. Cell adhesion molecules, including those of the immunoglobulin superfamily, are associated with the development of metastatic behavior in cutaneous melanomas. The neural cell adhesion molecule (NCAM) belongs to this family. To investigate its possible role in the development metastatic behavior of uveal melanomas, the authors studied immunohistochemically the expression of NCAM by using an antibody that recognizes all three major isoforms of NCAM and an antibody that recognizes the HNK-1 epitope present on some isoforms of NCAM. The authors studied 32 primary uveal melanomas from 32 patients (among these, 12 were rapidly metastasizing and 16 slowly metastasizing) and 29 metastases from 19 patients. From 13 patients the primary, as well as the metastatic, tumors were available. With one exception, all HNK-1 positive primary and metastatic tumors were also positive for NCAM. NCAM was significantly more expressed in aggressive, rapidly metastasizing primary tumors (P = .02 and .04, respectively) and in metastases. HNK-1 was significantly (P = .04) more expressed in larger tumors. In liver metastases HNK-1 immunoreactivity was significantly (P = .005) less frequently expressed than NCAM. Therefore, NCAM isoforms that lack the HNK-1 epitope might play a role in the organ specific metastatic behavior of uveal melanomas.

Fat-storing cells and myofibroblasts are involved in the initial phase of carbon tetrachloride-induced hepatic fibrosis in BN/BiRij rats.

This study on the appearance, distribution and kinetics of fibroblast-like cells (fat-storing cells, transitional cells, myofibroblasts and fibroblasts) after CCl4-treatment was undertaken to delineate further the respective roles of these cell types in liver fibrogenesis. The different cell types were distinguished on the basis of their immunophenotypic pattern with a combination of marker antibodies and on the basis of ultrastructural characteristics. Combined staining for alpha-smooth muscle actin (sma) and desmin (Des) revealed perisinusoidal fat-storing cells (FSC) as d+ sma- and myofibroblasts around the central veins of the normal rat liver as d+ sma+. During the initial phase of CCl4-induced hepatic fibrosis (week 1 and 2), the number of d+ sma+ cells increased in the degenerating area around the central veins and d+ sma+ cells appeared in the very thin fibrotic septa at week 2. Ultrastructural examination of the affected central areas showed the presence of myofibroblasts. These sma+ cells proliferated, as shown by double staining for bromodeoxyuridine (BrdU) and sma. In degenerating parenchymal areas, d+ sma- FSC were present. The FSC in the perisinusoidal space of areas which were not affected by CCl4 intoxification, remained d+ sma-. These immunostaining findings support the electron microscopical results, which show the presence of cells with the typical ultrastructural characteristics of FSC in both the degenerating areas and the perisinusoidal space of unaffected areas. After one week of CCl4-treatment, enhanced deposition of procollagen type III was observed around the central veins. Enhanced deposition of collagen type IV was seen subendothelially along the sinusoids, notably in degenerating parenchymal areas where the septa were later formed. FSC appear to be the principal source of collagen type IV during fibrogenesis. These observations further support and specify the role of FSC in early fibrogenesis. With the progression of the CCl4-induced fibrosis, d+ sma+ myofibroblasts remained localized in the fibrotic septa, but now along their outer edge. The majority of the cells in the septa were formed by d- sma- cells indicating a prominent role of fibroblasts in the septal formation. Septal fibroblasts are not only likely to produce matrix components, but also were shown to degrade collagen, as evidenced by the increased number of collagen-containing vacuoles during the course of fibrosis. In conclusion, myofibroblasts and FSC appear to be the main cell types involved in the initial phase of liver fibrogenesis induced by CCl4. Both myofibroblasts and FSC divide and transform.(ABSTRACT TRUNCATED AT 400 WORDS)

Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats.

Normal epithelial cell differentiation is characterized by the production of distinct cytokeratin proteins. It is well known that epithelia of several organs show squamous metaplasia in a vitamin A-deficient status. It is not yet known whether these histological changes are concomitant with a change in cytokeratin expression. Therefore, 3-week-old female rats (BN/BiRij) were fed a vitamin A-deficient diet for 8 weeks. The cytokeratin expression in epithelia of various organs was monitored immunohistochemically during the induction of vitamin A deficiency. Therefore, monoclonal antibodies specific for human cytokeratin 4, 5, 5 + 8, 7, 10, 14, 18 and 19 were used. In a normal vitamin A status, the distributional pattern for the different cytokeratins in rats was similar to that reported for human tissue. No change in cytokeratin expression was seen in trachea, skin, liver and colon at any time point studied. Squamous metaplasia in urinary bladder and salivary glands was observed after six weeks on the vitamin A-deficient diet. This was concomitant with a substitution of cytokeratins 4, 5 + 8, 7, 18 and 19 by cytokeratin 10. The latter cytokeratin is specific for keratinized squamous epithelium. A change in cytokeratin expression was observed in bladder, ureter, kidney, salivary glands, uterus and conjunctiva before histological alterations appeared. In conclusion, the changes in cytokeratin expression observed under vitamin A deficiency in epithelia in vivo are in agreement with those described in other studies for epithelial cells in vitro. The changes in cytokeratin expression and the subsequent differentiation into squamous cells occurs in basal cells of the bladder but not in transitional cells.(ABSTRACT TRUNCATED AT 250 WORDS)