Microchimeric cells promote production of rheumatoid arthritis-specific autoantibodies.

BACKGROUND: Women are more likely to develop autoimmune diseases than men. Contribution from microchimerism (Mc) has been proposed, as women naturally acquire Mc from more sources than men because of pregnancy. Women with Rheumatoid Arthritis (RA) who lack RA-associated HLA alleles have been found to harbor Mc with RA-associated HLA alleles in higher amounts than healthy women in prior work. However, an immunological impact of Mc remains to be elucidated. OBJECTIVES: To test the hypothesis that Mc with RA-risk associated HLA alleles can result in the production of RA-associated autoantibodies, when host genetic risk is absent. METHODS: DBA/2 mice are unable to produce RA-specific anti-citrullinated autoantibodies (ACPAs) after immunization with the enzyme peptidyl arginine deiminase (PAD) in a previously developed model. DBA/2 females were mated with C57BL/6 males humanized to express HLA-DR4, which is associated with RA-risk and production of ACPAs, to evaluate DR4+ fetal Mc contribution. Next, DBA/2 females born of heterozygous DR4+/- mothers were evaluated for DR4+ Mc of maternal or littermate origin. Finally, DBA/2 females from DR4+/- mothers were crossed with DR4+ males, to evaluate the contribution of any Mc source to ACPA production. RESULTS: After PAD immunization, between 20 % and 43 % of DBA/2 females (otherwise unable to produce ACPAs) had detectable ACPAs (CCP2 kit) after exposure to sources of Mc with RA-associated HLA alleles, compared to 0 % of unmated/unexposed DBA/2 females. Further the microchimeric origin of the autoantibodies was confirmed by detecting a C57BL/6-specific immunoglobulin isotype in the DBA/2 response. CONCLUSION: Our study demonstrates that Mc cells can produce "autoantibodies" and points to a role of Mc in the biology of autoimmune diseases, including RA.

Development and evaluation of a decision aid for women eligible for organized breast cancer screening according to international standards: A multi-method study.

BACKGROUND: and purpose: In France, women lack information to make a shared decision to start breast cancer screening. Decision aids are useful to facilitate this discussion, yet few meet international standards. The objective of this project was to build, validate and measure the quality of a decision aid for organized breast screening in France, in line with international standards, intended for both women and healthcare professionals. MATERIALS AND METHODS: This mixed-methods study was conducted between January 2017 and June 2022. The prototype was developed from a qualitative study, systematic review and targeted literature review and alpha tested during two Delphi rounds. Readability was evaluated with the Flesch score and content with International Patient Decision Aid Standards Instrument (IPSASi). RESULTS: An online decision aid, accessible at www.Discutons-mammo.fr, written in French was developed. The content included eligibility, information about breast screening the advantages and disadvantages of screening, patient preferences and a patient-based discussion guide using text, infographics, and videos. The Flesch readability test score was 65.4 and the IPDASi construct quality score was 176 out of 188. CONCLUSIONS: This decision aid complies with IPDASi standards and could help women eligible for breast screening in France make a shared decision with a specialized healthcare professional about whether or not to participate in organized breast screening.

In utero position matters for littermate cell transfer in mice: an additional and confounding source with maternal microchimerism.

Introduction: Feto-maternal cell transfer during pregnancy is called microchimerism (Mc). Its persistence in respective hosts is increasingly studied as to its potential role in immune tolerance, autoimmunity, cancer, and degenerative diseases. Murine models with transgenic reporter genes, heterozygously carried by the mother, allow maternal Mc tracking in wild-type (WT) offspring. However, as gestation in mice is multi-embryonic, an exchange of cells between fetuses carrying the same reporter gene as their mother and negative WT littermate, named littermate Mc (LMc), can occur and be confounded with the maternal source. We propose here to evaluate LMc contribution in mice. Methods: To avoid the maternal confounding source of Mc, transgenic males, heterozygous for a reporter gene, here, the human leukocyte antigen DRB1*04 (DR4+/-), were crossed with WT females (DR4-/-). DR4+/- LMc was specifically quantified by HLA-DR4 quantitative PCR, i) in utero in main organs from 15 DR4-/- fetuses from three litters of 11, nine, and five; and ii) after birth in two litters of eight pups: in two DR4-/- stillborns and four DR4-/- adult mice. Results: At embryonic stages, DR4-/- fetuses having one or two nearby DR4+/- littermates in the same uterine horn were almost seven times more frequently positive for DR4- microchimerism in their organs (p = 0.01) and had quantitatively more LMc (p = 0.009) than those without nearby DR4+/- littermates. Furthermore, LMc persists at birth and into adulthood with interindividual heterogeneity. Conclusions: This study identifies heterogeneity for LMc acquisition according to in utero position and different interpretation of previously published results on maternal Mc in mice.

Sustainable development in general practice.

BACKGROUND: As health care accounts for 4-5% of global carbon emissions, many health organisations have called for implementing sustainable development actions in health care. However, sustainable development measures in general practice are rarely implemented by physicians. The aim of this study was to explore the practices of general practitioners (GPs) in terms of sustainable development to identify which actions are appropriate and achievable. METHODS: A qualitative study was conducted in 12 French GPs using face-to-face or telephone interviews, transcribed verbatim and analysed through a global inductive analysis with constant comparison. Semi-structured interviews were focussed on waste management, relationships between health professionals, sustainable development, and GPs' activity. RESULTS: The mean age of the GPs was 42.8 years and they mainly worked in an urban environment. The interviews highlighted 4 themes. It appeared that a balance needs to be found between the environmental impact and the constraints related to medical care. To be able to think about integrating sustainable development into health care, GPs should make a personal commitment to change their routine. In practice, consumption should be reassessed, prescriptions and prevention reconsidered. These actions could be applied to the GPs' environment as role models for their patients, business leaders, and members of the healthcare system. CONCLUSION: GPs felt concerned by sustainable development and were already involved in its implementation in their practice. Tools are available to help GPs to continue to implement their actions described in this article, but their impact remains to be investigated.

In an era where global health is an increasing concern for the population, it appeared necessary to study the extent to which health professionals were willing to change their behaviours in their professional lives. This study was based on the interview of 12 French general practitioners and investigated their perspective on sustainable development and how they implemented it in their practice. Four main themes were highlighted. Some physicians did not spontaneously see the link between their practice and sustainable development. Our study showed that they were willing to adapt their practice despite organisational constraints and the difficulty in changing their behaviour.

Very Early Diagnosis and Management of Congenital Erythropoietic Porphyria.

Congenital erythropoietic porphyria (CEP), a rare form of porphyria, is caused by a defect in the heme biosynthesis pathway of the enzyme uroporphyrinogen III synthase (UROS). Uroporphyrinogen III synthase deficiency leads to an accumulation of nonphysiological porphyrins in bone marrow, red blood cells, skin, bones, teeth, and spleen. Consequently, the exposure to sunlight causes severe photosensitivity, long-term intravascular hemolysis, and eventually, irreversible mutilating deformities. Several supportive therapies such as strict sun avoidance, physical sunblocks, red blood cells transfusions, hydroxyurea, and splenectomy are commonly used in the management of CEP. Currently, the only available curative treatment of CEP is hematopoietic stem cell transplantation (HSCT). In this article, we present a young girl in which precocious genetic testing enabled early diagnosis and allowed curative treatment with HSCT for CEP at the age of 3 months of age, that is, the youngest reported case thus far.

Upscaling xylem phenology: sample size matters.

BACKGROUND AND AIMS: Upscaling carbon allocation requires knowledge of the variability at the scales at which data are collected and applied. Trees exhibit different growth rates and timings of wood formation. However, the factors explaining these differences remain undetermined, making samplings and estimations of the growth dynamics a complicated task, habitually based on technical rather than statistical reasons. This study explored the variability in xylem phenology among 159 balsam firs [Abies balsamea (L.) Mill.]. METHODS: Wood microcores were collected weekly from April to October 2018 in a natural stand in Quebec, Canada, to detect cambial activity and wood formation timings. We tested spatial autocorrelation, tree size and cell production rates as explanatory variables of xylem phenology. We assessed sample size and margin of error for wood phenology assessment at different confidence levels. KEY RESULTS: Xylem formation lasted between 40 and 110 d, producing between 12 and 93 cells. No effect of spatial proximity or size of individuals was detected on the timings of xylem phenology. Trees with larger cell production rates showed a longer growing season, starting xylem differentiation earlier and ending later. A sample size of 23 trees produced estimates of xylem phenology at a confidence level of 95 % with a margin of error of 1 week. CONCLUSIONS: This study highlighted the high variability in the timings of wood formation among trees within an area of 1 km2. The correlation between the number of new xylem cells and the growing season length suggests a close connection between the processes of wood formation and carbon sequestration. However, the causes of the observed differences in xylem phenology remain partially unresolved. We point out the need to carefully consider sample size when assessing xylem phenology to explore the reasons underlying this variability and to allow reliable upscaling of carbon allocation in forests.

How RA Associated HLA-DR Molecules Contribute to the Development of Antibodies to Citrullinated Proteins: The Hapten Carrier Model.

The risk to develop ACPA positive rheumatoid arthritis (RA), the most destructive type of autoimmune arthritis, is carried by HLA-DRB1 alleles containing a 5 amino acid motif: the shared epitope (SE). RA is preceded by the emergence of disease specific anti citrullinated protein antibodies (ACPA). SE positive HLA-DRB1 alleles are associated with ACPA and ACPA positive RA, not with ACPA negative RA, suggesting that ACPA contribute to the pathogenesis of RA. Understanding how HLA-DRB1 genotypes influence ACPA could lead to a curative or preventive treatment of RA. The "Shared epitope binds citrullinated peptides " hypothesis suggests that RA associated HLA-DR alleles present citrullinated peptides to T cells that help ACPA producing B cells. The "Hapten carrier model" suggests that PAD4 is the target of the T cells which help ACPA specific B cells through a hapten carrier mechanism in which PAD4 is the carrier and citrullinated peptides are the haptens. Direct binding assay of citrullinated peptides to purified HLA-DR molecules does not support the "shared epitope binds citrullinated peptides" hypothesis. The Odds Ratios to develop ACPA positive RA associated with each of 12 common HLA-DRB1 genotypes match the probability that the two HLA-DR molecules they encode can bind at least one peptide from PAD4, not from citrullinated fibrinogen. Thus, PAD4 tolerization might stop the carrier effect and switch off production of ACPA.

PAD2 immunization induces ACPA in wild-type and HLA-DR4 humanized mice.

Rheumatoid arthritis (RA) is associated with HLA-DRB1 alleles expressing the "shared epitope." RA is usually preceded by the emergence of anti-citrullinated protein autoantibodies (ACPAs). ACPAs recognize citrulline residues on numerous proteins. Conversion of arginine into citrulline is performed by enzymes called peptidyl arginine deiminases (PADs). We have previously demonstrated that C3H mice immunized with PADs can produce ACPAs by a hapten-carrier mechanism. Here, we address the influence of HLA-DR alleles in this model in mice expressing RA-associated HLA-DRB1*04:01 (KO/KI*04:01), HLA-DRB1*04:04 (KO/KI*04:04), or non-RA-associated HLA-DRB1*04:02 (KO/KI*04:02) after murine PAD2 immunization. Immunization with mPAD2 triggers production of ACPAs in wild-type (WT) and HLA-DR4 C57BL/6 mice. Both I-Ab and HLA-DR are involved in the activation of mPAD2-specific T lymphocytes. Among HLA-DR4 mice, mice expressing RA-associated HLA-DRB1*04:01 are the best responders to mPAD2 and the best anti-citrullinated peptide antibody producers.

PAD4 Immunization Triggers Anti-Citrullinated Peptide Antibodies in Normal Mice: Analysis With Peptide Arrays.

The critical immunological event in rheumatoid arthritis (RA) is the production of antibodies to citrullinated proteins (ACPAs), ie proteins on which arginines have been transformed into citrullines by peptidyl arginine deiminases (PAD). In C3H mice, immunization with PAD4 triggers the production of ACPAs. Here, we developed a peptide array to analyze the fine specificity of anti-citrullinated peptide antibodies and used it to characterize the ACPA response after hPAD4 immunization in mice expressing different H-2 haplotypes. Sera from C3H, DBA/2, BALB/c and C57BL/6 mice immunized with human PAD4 (hPAD4) or control-matched mice immunized with phosphate buffered saline (PBS) were used to screen peptide arrays containing 169 peptides from collagen, filaggrin, EBNA, proteoglycan, enolase, alpha and beta fibrinogen, histon and vimentin. Human PAD4 immunization induced antibodies directed against numerous citrullinated peptides from fibrinogen, histon 4 and vimentin. Most peptides were recognized under their arginine and citrullinated forms. DBA/2 and BALB/c mice (H-2d) had the lowest anti-citrullinated peptide IgG responses. C3H (H-2k) and BL6 mice (H-2b) had the highest anti-citrullinated peptide IgG responses. The newly developed peptide array allows us to characterize the ACPA production after hPAD4 immunization in mice on the H-2d, H-2k or H-2b backgrounds. This sensitive tool will be useful for further studies on mice for prevention of ACPA production by PAD tolerization.

What do women and healthcare professionals expect of decision aids for breast cancer screening? A qualitative study in France.

OBJECTIVE: Breast cancer screening decision aids (DAs) are designed to help women decide whether or not to participate in mammography-based programmes. We aimed to explore women's and healthcare professionals' expectations of a breast cancer screening DA, as part of the French DEDICACES study. METHODS: This French qualitative study was based on semistructured, individual interviews with women from the general population, general practitioners (GPs), midwives, gynaecologists, radiologists and screening centre managers. Sampling was purposive and used diversification criteria. The inductive analysis was based on grounded theory. RESULTS: Between April 2018 and May 2019, we interviewed 40 people: 13 women, 14 GPs, 4 gynaecologists, 3 midwives, 3 radiologists and 3 screening centre managers. The women and the healthcare professionals considered that a DA could help to improve levels of knowledge, harmonise medical practice and provide reliable, comprehensive information. Overall, the interviewees wanted an easy-to-use, intuitive, graphic-rich, interactive, computer-based, patient-centred DA. Use of the DA might be limited by a lack of familiarity with shared decision-making (SDM), the risk of misuse and a preference for asymmetric positive information. CONCLUSION: The present results are likely to facilitate the development of the first validated tool for SDM support in French breast cancer screening programmes.

In Rheumatoid Arthritis Patients, HLA-DRB1*04:01 and Rheumatoid Nodules Are Associated With ACPA to a Particular Fibrin Epitope.

Objectives: Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding the shared epitope (SE), a 5-amino acid motive. RA is usually preceded by the emergence of anti-citrullinated protein/peptide antibodies (ACPAs). Citrulline is a neutral amino acid resulting from post-translational modification of arginine involved in peptidic bounds (arginyl residue) by PeptidylArginine Deiminases (PADs). ACPAs recognize epitopes from citrullinated human fibrin(ogen) (hFib) and can be specifically detected by the AhFibA assay. Five citrullinated peptides derived from hFib together represent almost all of the epitopes recognized by patients with ACPA-positive RA, namely: α36-50cit, α171-185cit, α501-515cit, α621-635cit, and ß60-74cit. The use of antibody fine specificities as markers of clinical phenotypes has become a major challenge. Our objective was to study whether RA clinical characteristics and HLA-DRB1 genetic background were associated with a specific reactivity against the epitopes borne by the five peptides. Methods: 184 ACPA-positive RA patients fulfilling the 2010 ACR/EULAR criteria were studied. Patient characteristics including HLA-DRB1 genotype, were collected from their medical files. Anti-CCP2 antibodies, AhFibA, and antibodies against the five citrullinated hFib (hFib-cit) peptides were analyzed by ELISA. Results: Anti-α505-515cit antibodies were associated with HLA-DRB1*04:01 (OR = 5.52 [2.00 - 13.64]; p = 0.0003). High level anti-α505-515cit antibodies were associated with rheumatoid nodules (OR = 2.71 [1.00 - 7.16], p= 0.044). Conclusion: Immune complexes containing anti-α501-515cit antibodies and rheumatoid factors might be involved in the development of rheumatoid nodules on the HLA-DRB1*04:01 background. Apheresis of these epitope-specific antibodies might be a new therapeutic opportunity for patients with rheumatoid nodules.

Do RA associated HLA-DR molecules bind citrullinated peptides or peptides from PAD4 to help the development of RA specific antibodies to citrullinated proteins?

PURPOSE: Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding a five amino acid basic motive, the shared epitope SE). Each HLA-DRB1 genotype defines a genotype specific risk of developing RA. RA is preceded by the emergence of anti citrullinated protein antibodies (ACPAs). Citrullin is a neutral version of arginin, a basic amino acid, formed after post translational modification by Peptidyl Arginyl Deiminases (PADs). HLA-DRB1 genes associated with RA are also associated with ACPAs. Two models might explain this association. Here we tested both models for prediction of HLA-DRB1 genotypic risks of developing RA. METHODS: We calculated the likelihoods for the 2 HLA-DR molecules encoded by 12 common HLA-DRB1 genotypes to bind at least one randomly chosen peptide from PAD4 or fibrinogen(native or citrullinatd) and compared them with the 12 respective HLA-DRB1genotypic risks of developing RA. RESULTS: HLA-DRB1 Genotypic risks of developing RA correlate with likelihoods of binding PAD4 peptides, not citrullinated Fibrinogen peptides. Thus, the molecular basis for the association of HLA-DR and ACPA positive RA is most likely the capability for RA associated HLA-DR molecules to bind peptides(s) from PAD4.

Primary care practitioners' diagnostic action when the patient may have cancer: an exploratory vignette study in 20 European countries.

OBJECTIVES: Cancer survival rates vary widely between European countries, with differences in timeliness of diagnosis thought to be one key reason. There is little evidence on the way in which different healthcare systems influence primary care practitioners' (PCPs) referral decisions in patients who could have cancer.This study aimed to explore PCPs' diagnostic actions (whether or not they perform a key diagnostic test and/or refer to a specialist) in patients with symptoms that could be due to cancer and how they vary across European countries. DESIGN: A primary care survey. PCPs were given vignettes describing patients with symptoms that could indicate cancer and asked how they would manage these patients. The likelihood of taking immediate diagnostic action (a diagnostic test and/or referral) in the different participating countries was analysed. Comparisons between the likelihood of taking immediate diagnostic action and physician characteristics were calculated. SETTING: Centres in 20 European countries with widely varying cancer survival rates. PARTICIPANTS: A total of 2086 PCPs answered the survey question, with a median of 72 PCPs per country. RESULTS: PCPs' likelihood of immediate diagnostic action at the first consultation varied from 50% to 82% between countries. PCPs who were more experienced were more likely to take immediate diagnostic action than their peers. CONCLUSION: When given vignettes of patients with a low but significant possibility of cancer, more than half of PCPs across Europe would take diagnostic action, most often by ordering diagnostic tests. However, there are substantial between-country variations.

Granular Calcite Stimulates Natural Mycorrhization and Growth of White Spruce Seedlings in Peat-Based Substrates in Forest Nursery.

The acidity of peat-based substrates used in forest nurseries limits seedling mineral nutrition and growth as well as the activity of microorganisms. To our knowledge, no study has yet evaluated the use of granular calcite as a covering material to increase pH, calcium and CO2 concentrations in the rhizosphere and ectomycorrhizal development. The objective is to compare different covering treatments on early colonization of the roots by ectomycorrhizal fungi, as well as the growth and calcium nutrition of white spruce seedlings in the forest nursery. Three treatments were used to cover the plant cavities (Silica (29 g/cavity; control treatment), Calcite (24 g/cavity) and calcite+ (31 g/cavity)) and were distributed randomly inside each of the five complete blocks of the experimental design. The results show that calcite stimulates natural mycorrhization. Seedlings grown with calcite have significant gains for several growth and physiological variables, and that the periphery of their root plugs are more colonized by the extramatrical phase of ectomycorrhizal fungi, thus improving root-plug cohesion. The authors discuss the operational scope of the results in relation to the tolerance of seedlings to environmental stress and the improvement of their quality, both in the nursery and in reforestation sites.

Comparison of nine heart rate-based models to predict work metabolism of Forest workers.

Predicted work metabolism (WM) from 9 heart rate (HR)-based models were compared to measured WM obtained during work in 39 forest workers. Using measured (i.e. raw) HR in these models can overestimate actual WM since the HR increase associated with body heat accumulation is non-metabolic. Hence, accuracy of WM prediction was assessed on all possible combinations of models using raw HR and corrected HR (thermal component removed) and with five different estimates of maximum work capacity (MWC) for the models that require it as an input. The 50 model combinations produced a wide range of WM estimates. Three models using individual calibration produced the lowest RMSE and narrowest LoA with corrected HR (rRMSE ≤13%; LoA [rBias <5% ± 25%]). One of the models that requires neither determination of the thermal component nor individual calibration performed very well (rRMSE = 18%; LoA [rBias = 1% ± 36%]). Practitioner Summary: These results provide a better understanding of the accuracy of various HR-based work metabolism (WM) estimation models. This information should prove particularly useful to ergonomics professionals wishing to select a method that provides accurate estimation of WM from HR measurements during work in varied thermal environments. Abbreviations: BMI: body mass index; HR: heart rate (beats per min); HR99: HR value exceeded during 99% of the duration of the HR recording period; HRcorr: heart rate without thermal pulses; HRraw: measured heart rate; HRres: heart rate reserve; HRrest: heart rate at rest; LoA: limits of agreement; Mrest: resting metabolism; MWC: maximum work capacity; RMSE: root mean square error; VO2: oxygen consumption; VO2 max: maximum oxygen consumption; WM: work metabolism.

Peptidylarginine Deiminase Autoimmunity and the Development of Anti-Citrullinated Protein Antibody in Rheumatoid Arthritis: The Hapten-Carrier Model.

OBJECTIVE: The presence of autoantibodies to citrullinated proteins (ACPAs) often precedes the development of rheumatoid arthritis (RA). Citrullines are arginine residues that have been modified by peptidylarginine deiminases (PADs). PAD4 is the target of autoantibodies in RA. ACPAs could arise because PAD4 is recognized by T cells, which facilitate the production of autoantibodies to proteins bound by PAD4. We previously found evidence for this hapten-carrier model in mice. This study was undertaken to investigate whether there is evidence for this model in humans. METHODS: We analyzed antibody response to PAD4 and T cell proliferation in response to PAD4 in 41 RA patients and 36 controls. We tested binding of 65 PAD4 peptides to 5 HLA-DR alleles (DRB1*04:01, *04:02, *04:04, *01:01, and *07:01) and selected 11 PAD4 peptides for proliferation studies using samples from 22 RA patients and 27 controls. Peripheral blood lymphocytes from an additional 10 RA patients and 7 healthy controls were analyzed by flow cytometry for CD3, CD4, CD154, and tumor necrosis factor expression after PAD4 stimulation. RESULTS: Only patients with RA had both antibodies and T cell responses to PAD4. T cell response to peptide 8, a PAD4 peptide, was associated with RA (P = 0.02), anti-PAD4 antibodies (P = 0.057), and the shared epitope (P = 0.05). CONCLUSION: ACPA immunity is associated with antibodies to PAD4 and T cell responses to PAD4 and PAD4 peptides. These findings are consistent with a hapten-carrier model in which PAD4 is the carrier and citrullinated proteins are the haptens.

Mosaicism of XX and XXY cells accounts for high copy number of Toll like Receptor 7 and 8 genes in peripheral blood of men with Rheumatoid Arthritis.

The X chromosome, hemizygous in males, contains numerous genes important to immunological and hormonal function. Alterations in X-linked gene dosage are suspected to contribute to female predominance in autoimmunity. A powerful example of X-linked dosage involvement comes from the BXSB murine lupus model, where the duplication of the X-linked Toll-Like Receptor 7 (Tlr7) gene aggravates autoimmunity in male mice. Such alterations are possible in men with autoimmune diseases. Here we showed that a quarter to a third of men with rheumatoid arthritis (RA) had significantly increased copy numbers (CN) of TLR7 gene and its paralog TLR8. Patients with high CN had an upregulated pro-inflammatory JNK/p38 signaling pathway. By fluorescence in situ hybridization, we further demonstrated that the increase in X-linked genes CN was due to the presence of an extra X chromosome in some cells. Men with RA had a significant cellular mosaicism of female (46,XX) and/or Klinefelter (47,XXY) cells among male (46,XY) cells, reaching up to 1.4% in peripheral blood. Our results present a new potential trigger for RA in men and opens a new field of investigation particularly relevant for gender-biased autoimmune diseases.