Platelet rescue by macrophage depletion in obese ADAMTS-13-deficient mice at risk of thrombotic thrombocytopenic purpura.

Essentials Obesity is a potential risk factor for development of thrombotic thrombocytopenic purpura (TTP). Obese ADAMTS-13-deficient mice were triggered with von Willebrand factor (VWF). Depletion of hepatic and splenic macrophages protects against thrombocytopenia in this model. VWF enhances phagocytosis of platelets by macrophages, dose-dependently. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS-13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets. Objectives To evaluate the role of obesity and ADAMTS-13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia. Methods Lean or obese ADAMTS-13-deficient (Adamts-13-/- ) and wild-type (WT) mice were injected with 250 U kg-1 of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts-13-/- mice by injection of clodronate-liposomes, 48 h before injection of rVWF. Results Obese Adamts-13-/- mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts-13-/- mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts-13-/- mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts-13-/- mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose-dependent increase of platelet phagocytosis. Conclusions Obese Adamts-13-/- mice are more susceptible to the induction of TTP-related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

An open conformation of ADAMTS-13 is a hallmark of acute acquired thrombotic thrombocytopenic purpura.

Essentials Conformational changes in ADAMTS-13 are part of its mode-of-action. The murine anti-ADAMTS-13 antibody 1C4 discriminates between folded and open ADAMTS-13. ADAMTS-13 conformation is open in acute acquired thrombotic thrombocytopenic purpura (TTP). Our study forms an important basis to fully elucidate the pathophysiology of TTP. SUMMARY: Background Acquired thrombotic thrombocytopenic purpura (aTTP) is an autoimmune disorder characterized by absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies. Recently, it was shown that ADAMTS-13 adopts a folded or an open conformation. Objectives As conformational changes in self-antigens play a role in the pathophysiology of different autoimmune diseases, we hypothesized that the conformation of ADAMTS-13 changes during acute aTTP. Methods Antibodies recognizing cryptic epitopes in the spacer domain were generated. Next, the conformation of ADAMTS-13 in 40 healthy donors (HDs), 99 aTTP patients (63 in the acute phase versus 36 in remission), 12 hemolytic-uremic syndrome (HUS) patients and 63 sepsis patients was determined with ELISA. Results The antibody 1C4 recognizes a cryptic epitope in ADAMTS-13. Therefore, we were able to discriminate between a folded and an open ADAMTS-13 conformation. We showed that ADAMTS-13 in HDs does not bind to 1C4, indicating that ADAMTS-13 circulates in a folded conformation. Similar results were obtained for HUS and sepsis patients. In contrast, ADAMTS-13 of acute aTTP patients bound to 1C4 in 92% of the cases, whereas, in most cases, this binding was abolished during remission, showing that the conformation of ADAMTS-13 is open during an acute aTTP episode. Conclusions Our study shows that, besides absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies, an open ADAMTS-13 conformation is also a hallmark of acute aTTP. Demonstrating this altered ADAMTS-13 conformation in acute aTTP will help to further unravel the pathophysiology of aTTP and lead to improved therapy and diagnosis.

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13.

BACKGROUND: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF). OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

Assessment of dietary adequacy in a remote Inuvialuit population.

BACKGROUND: Traditional foods are rich sources of essential nutrients, but Inuvialuit in the Northwest Territories (NWT), Canada, have been undergoing a nutrition transition, characterised by an increased consumption of non-nutrient-dense foods. The present study aimed to characterise energy, nutrient and food intakes amongst adult Inuvialuit. METHODS: The study collected up to three 24-h dietary recalls on nonconsecutive days for each participant in spring/summer of 2008 in one remote community in the NWT. Recall data were analysed for energy and nutrient intake, dietary adequacy, most commonly reported foods, and food contributors to energy and nutrients. RESULTS: Participants included 14 men and 50 women (response rate 79%). Median daily energy intake was 9.4 (interquartile range=5.7) MJ for men and 8.3 (3.6) MJ for women. The majority of adult Inuvialuit did not meet the recommendation for vitamins A [median intake=344.7 (246.3) µg-RAE in men, 248.9 (213.8) µg-RAE in women], B(6) [0.9 (0.8) mg in men, 1.0 (0.5) mg in women] and E [2.4 (2.1) mg in men, 1.8 (1.0) mg in women], dietary fibre [7.7 (5.7) g in men, 8.7 (4.4) g in women], calcium [779.6 (842.0) mg in men, 610.4 (431.5) mg in women] and total folate [222.6 (57.7) µg in men, 264.6 (127.5) µg in women]. Vitamin D intake was below the recommendation for most women [median intake=100.0 (119.2) IU]. Traditional foods contributed substantially to protein and iron intake. Juices were the main contributors to energy, carbohydrate and calcium. CONCLUSIONS: The present study revealed an inadequate consumption of essential nutrients in an Inuvialuit population. If these nutrient deficiencies continue, this population will face an increased burden of chronic diseases and malnutrition.

Prevalence and risk factors for self-reported chronic disease amongst Inuvialuit populations.

BACKGROUND: Chronic disease prevalence amongst Canadian Arctic populations is increasing, but the literature amongst Inuvialuit is limited. The present study aimed to provide baseline data that could be used to monitor changes in chronic disease risk factors and long-term health in the Arctic by determining prevalence and risk factors of self-reported chronic disease amongst adult Inuvialuit in remote communities. METHODS: Self-reported demographics and history of chronic disease (hypertension, heart disease, diabetes and cancer) were collected in three communities between July 2007 and July 2008 in the Northwest Territories. Food frequency questionnaires recorded dietary intake, International Physical Activity Questionnaires recorded physical activity and anthropometric measures of height and weight were obtained. RESULTS: Response rates ranged from 65-85%. More than 20% of the 228 participants aged 19-84 years reported having a chronic disease. Age-adjusted prevalence was 28, 9, 9 and 6 per 100 for hypertension, heart disease, diabetes and cancer, respectively. Compared with non-cases, participants reporting hypertension were more likely to have a higher body mass index and a lower level of education. Hypertension was more common amongst participants reporting heart disease and diabetes than Inuvialuit not reporting these morbidities. CONCLUSIONS: Inuvialuit participants in this study were most affected by hypertension and diabetes compared with heart disease or any cancer. Female participants had a higher prevalence of heart disease compared with the Canadian average. Primary preventive strategies are necessary to mitigate the increasing rates of chronic disease risk factors in this population. Further studies with a larger sample size and measured chronic disease are necessary to confirm the findings obtained in the present study.

Food patterns and socioeconomic indicators of food consumption amongst Inuvialuit in the Canadian Arctic.

BACKGROUND: Inuvialuit in the Canadian Arctic have been experiencing a nutrition transition resulting in a decrease in nutrient-dense food consumption, which may, in part, explain this population's increasing chronic disease rates. Because the available literature is limited, the present study aimed to document the extent of this transition by examining current dietary patterns and socioeconomic factors affecting food group consumption. METHODS: This cross-sectional study was conducted in three Inuvialuit communities in the Northwest Territories between 2007 and 2008. A validated food frequency questionnaire determined intake frequency of fruit and vegetables (FV), traditional foods (TF) and non-nutrient-dense foods (NNDF). Socioeconomic status (SES) was assessed by questions on education, ownership of items in working condition used to create a Material Style of Life (MSL) scale and residents in household employed/on income support. Daily intake frequencies were compared by gender and age group using Wilcoxon rank sum test. SES association with food group intake was determined using logistic regression. RESULTS: The response rate was 65-85%. One hundred and seventy-five participants were female and 55 were male, aged 19-84 years [mean (SD) 44 (14)]. Mean frequencies of FV and TF consumption were 1.6 (1.5) and 1.6 (1.7) times per day, respectively. NNDF were reported 9.2 (3.0) times per day. The highest MSL score (>12) was significantly associated with higher fruit (≥0.7 times per day) and higher TF intake (≥1.1 times per day) compared with the lowest score (≤7). An intermediate MSL score (8-12) was related to higher vegetable consumption (≥0.4 times per day). CONCLUSIONS: NNDF were consumed approximately seven times more frequently than TF in the present study, indicating that the dietary transition is well underway amongst Inuvialuit. Participants with higher SES were more likely to consume nutrient-dense foods, suggesting possible cost barriers.

Important psychosocial factors to target in nutrition interventions to improve diet in Inuvialuit communities in the Canadian Arctic.

BACKGROUND: With increasing chronic disease amongst Inuvialuit in the Canadian Arctic, research on dietary behaviours and their determinants in this population is needed to develop nutritional behaviour change intervention strategies. The present study aimed to assess the knowledge, self-efficacy and intentions towards healthy eating and healthy eating behaviours of Inuvialuit adults in the Northwest Territories (NWT), Canada. METHODS: The Adult Impact Questionnaire was developed from behavioural theories and workshops held in the communities. It was conducted with adult Inuvialuit (≥19 years) from randomly selected households in three NWT communities to collect data on the psychosocial constructs of healthy food knowledge, self-efficacy and intentions, and the dietary behaviours of healthy and unhealthy food acquisition and preparation. Associations between demographic, socioeconomic, psychosocial constructs and behaviours were analysed using multivariate linear regression. RESULTS: The 228 participants [mean (SD) age 43.4 (13.6) years; response rates 65-85%] acquired non-nutrient-dense foods a mean (SD) of 2.7 (3.0) times more frequently than nutrient-dense, low sugar and low fat foods. Increased intention was associated with a greater frequency of acquiring healthy foods (ß=0.17, P=0.012) and a lower frequency of acquiring unhealthy foods (ß=-0.18, P=0.008). Overall, participants reported using food preparation methods that reduce fat content slightly more than methods that add fat [mean (SD) score 0.3 (1.9)]. Use of healthier food preparation methods was associated with higher levels of healthy food knowledge (ß=0.26, P<0.001), self-efficacy (ß=0.29, P<0.001) and intentions (ß=0.22, P=0.001). CONCLUSIONS: Healthy food intention was the construct most significantly associated with all three healthier dietary behaviours. Interventions that target intentions to change food choice and preparation may be effective strategies to improve dietary intake in Inuvialuit populations.

Inuvialuit adults in the Canadian Arctic have a high body mass index and self-reported physical activity.

BACKGROUND: Inuvialuit in Arctic Canada are undergoing nutrition and lifestyle transitions that encourage decreased physical activity (PA) and increased body mass index (BMI), although data specific to PA and BMI within this unique population are not currently available. The present study aimed to characterise current PA and BMI levels in a sample of Inuvialuit adults. METHODS: Inuvialuit adults randomly selected from a cross-sectional survey of three communities in the Northwest Territories, Canada, completed an adapted International Physical Activity Questionnaire (IPAQ) and an anthropometric questionnaire to obtain height and weight. Data were analysed using the standardised IPAQ protocol, and the results are presented by gender and age group (≤50 and >50 years). RESULTS: One hundred and ninety-six Inuvialuit adults [148 women, 48 men; mean (standard deviation (SD)) ages 45 (14) and 41 (14) years, respectively; age range 19-84 years] completed the IPAQ (response rates 65-85%). Seventy-one percent of men and 74% of women allowed height measurements, whilst 81% and 84% allowed weight measurements, respectively; the remainder provided self-reported anthropometrics. Mean (SD) BMIs were 28.6 (7.7) kg/m(2) for males and 30.5 (8.8) kg/m(2) for females. The greatest percentage of participants in both gender and age groups fell into the obese BMI category. Self-reported PA was high, with means for men and participants ≤50 years in the high category of PA and means for women and participants >50 years in the medium category. Almost two-thirds (65%) of participants were classified as being overweight or obese, although 89% reported medium or high levels of PA. CONCLUSIONS: The results obtained in the present study indicate widespread overweight/obesity accompanying high levels of self-reported PA.

Manual method for liquid-based cytology: a demonstration using 1,000 gynecological cytologies collected directly to vial and prepared by a smear-slide technique.

We report on the formulation and use of an alcoholic-agar additive solution generally useful for rapid, inexpensive liquid-based cytology slide preparation. Gynecological cytology specimens were collected from 1,000 women. Two hundred fifty aliquots each of CytoRich, CytoRich Red (Tripath Imaging, Inc., Burlington, NC), Preservcyt (Cytyc Corp., Boxborough, MA), and DINA*TRANS (DINA*CYT Corp., Portland, OR) fixatives were used for this study. Fixed cell suspensions from 1,000 women, seen consecutively by a general obstetrics and gynecology practice, were vortex-mixed and transferred into a premeasured amount of alcoholic-agar in test tubes. Test tubes were conventionally centrifuged, cells were trapped in a spontaneously formed agar-gel, and the supernatant solutions were decanted. Vortex-mixing cell buttons caused a rapid gel-to-sol transition, affording viscous cell suspensions that were applied to slides, smeared, and stained using Papanicolaou stains. Slides showed unclumped, monolayered, uniform, random cell-spreads. All of the fixatives afforded crisp presentation of normal and abnormal cells. There was an about 3-fold increase in HSIL+ (0.7-1.8%) and LSIL diagnoses (1.3-4.4%), and a 45% reduction in ASCUS diagnoses (3.3-1.8%), as compared to our cytology laboratory's previous year's patients' statistics with a concurrent 0.2% unsatisfactory rate, due solely to inadequate sampling. This manual method makes liquid-based cytology inexpensive and does not require specialized preparative devices.

Liquid-based cervical-cell collection with brushes and wooden spatulas: a comparison of 100 conventional smears from high-risk women to liquid-fixed cytocentrifuge slides, demonstrating a cost-effective, alternative monolayer slide preparation method.

Our purpose was to demonstrate that 17.5-mm liquid-based cytocentrifuge circle-slides made using a Hettich (Andreas Hettich Co., Tuttlingen, Germany) cytocentrifuge are at least as efficacious as conventional smears for detecting cervical abnormalities. One hundred conventional smears were collected with cytobrushes and wooden spatulas from high-risk women. Both devices were then placed into CytoRich Red (AutoCyte, Inc., Burlington, NC). Cells were concentrated from CytoRich Red by centrifugation and suspended in CytoRich Yellow. Two 17.5-mm circle-slides were produced, compared to each other in order to test reproducibility of diagnoses between slides, and compared to conventional slides. Sixty-five normals, three ASCUS, three LSIL, and two HSIL matched. Overall, cytocentrifugation yielded 27 additional findings among 25 cases. Also, it downgraded one conventional ASCUS to normal (immature metaplasia and chronic inflammation) and one conventional HSIL to normal (transitional cell metaplasia). Cytocentrifugation of 14 conventional normals afforded four ASCUS and 10 LSIL. Cytocentrifugation of 11 conventional ASCUS afforded nine LSIL and two HSIL (one with AIS). One HSIL + AIS was found with a conventional HSIL. Cytobrushes and wooden spatulas can be used to collect material for liquid-based cervico-vaginal cytology if they are placed into CytoRich Red. Hettich cytocentrifuge slides were more reliable than conventional smears in presenting well-dispersed, sharply imaged cells, affording greater diagnostic sensitivity and certainty. Compared to previous split-sample studies, three different outcomes were noted: 1) the endocervical component was consistently well-represented; 2) AIS was seen more often in liquid-based slides; and 3) the liquid-based slide did not underdiagnose its conventional companion. The liquid-based preparations downgraded one ASCUS to normal and one conventional HSIL to transitional-cell metaplasia. Histology correlation of these two cases agreed with the liquid-based cytology diagnoses, which validates their specificity. These improvements are ascribed to differences in fixation and processing. The low cost, ease of operation, and reusable chambers of the Hettich cytocentrifuge make it a cost-effective liquid-based cytology instrument, especially for small to intermediate-size laboratories. The claims that this paper makes must be proven by additional studies, and test implementation needs to be scrutinized by appropriate regulatory agencies whose standards may vary from country to country. Diagn. Cytopathol. 2000;22:86-91.

Rare epithelial sheets with "cancer-like" nuclei presenting against a background of cytologically normal-appearing endometrial epithelium in endometrial brush samplings: establishing a differential diagnosis.

Liquid-fixed Tao brush samples showing small quantities (less than 10%) of endometrial epithelial sheets with cancer-like nuclei that are admixed with benign endometrium raise a concern about false-positive cytology interpretations. This paper details 7 cases along with the outcomes of three prior papers that touch on the differential diagnosis of "small amounts of atypical epithelium with cancer-like nuclei." Liquid-fixed, cytocentrifuge-processed Tao brush endometrial samples from 7 women showing "small amounts of atypical epithelium with cancer-like nuclei" were followed up by hysterectomy. Clinical presentations, diagnostic tests, surgical procedures, and tissue outcomes are detailed. Four women had hyperplastic polyps (three with focal atypical complex hyperplasia, and one with focal atypical simple hyperplasia). One had endometrial microcarcinoma. One had p53-positive endometrial intraepithelial carcinoma (EIC). One had endometrial intraepithelial neoplasia (EIN). The differential diagnosis of the index cytological finding, "small quantities of endometrial epithelial sheets with cancer-like nuclei admixed with benign endometrium," includes hyperplastic polyp, EIC, microcarcinoma/EIN, and carcinoma metastatic to the endometrium. Combining endometrial brushing, endovaginal sonography/sonohysterography, and selective immunostaining may be useful for both detecting and sorting out these lesions. Diagn. Cytopathol. 1999;21:378-386.