data_sheet_1_SHIP1 Deficiency in Inflammatory Bowel Disease Is Associated With Severe Crohn’s Disease and Peripheral T Cell Reduction.DOCX

<p>In our previous study, we observed a severe reduction in the Src homology 2-containing-inositol-phosphatase-1 (SHIP1) protein in a subpopulation of subjects from a small adult Crohn’s Disease (CD) cohort. This pilot study had been undertaken since we had previously demonstrated that engineered deficiency of SHIP1 in mice results in a spontaneous and severe CD-like ileitis. Here, we extend our analysis of SHIP1 expression in peripheral blood mononuclear cells in a second much larger adult Inflammatory Bowel Disease (IBD) cohort, comprised of both CD and Ulcerative Colitis patients, to assess contribution of SHIP1 to the pathogenesis of human IBD. SHIP1 protein and mRNA levels were evaluated from blood samples obtained from IBD subjects seen at UCSF/SFVA, and compared to healthy control samples. Western blot analyses revealed that ~15% of the IBD subjects are severely SHIP1-deficient, with less than 10% of normal SHIP1 protein present in PBMC. Further analyses by flow cytometry and sequencing were performed on secondary samples obtained from the same subjects. Pan-hematolymphoid SHIP1 deficiency was a stable phenotype and was not due to coding changes in the INPP5D gene. A very strong association between SHIP1 deficiency and the presence of a novel SHIP1:ATG16L1 fusion transcript was seen. Similar to SHIP1-deficient mice, SHIP1-deficient subjects had reduced numbers of circulating CD4⁺ T cell numbers. Finally, SHIP1-deficient subjects with CD had a history of severe disease requiring multiple surgeries. These studies reveal that the SHIP1 protein is crucial for normal T cell homeostasis in both humans and mice, and that it is also a potential therapeutic and/or diagnostic target in human IBD.</p

Specific SHIP1 activity measurement.

<p>(A) Specificity of the assay was determined by immunoprecipitation (IP) of SHIP1 from multiple myeloma cell lines (U266 and OPM2) that express SHIP1 (see Western blot insert of whole cell lysates) and breast cancer cell line (MCF7) that does not. Phosphatase activity of immunoprecipitates was determined by adding the SHIP1 substrate PtdIns(3,4,5)P₃, followed by Malachite Green reaction. Human recombinant SHIP2 was used as positive control for the phosphatase assay. Only immunoprecipitates from SHIP1-competent cells show phosphatase activity. (B) OCRL1, SHIP2 or SHIP1 were immunoprecipitated from SHIP1-competent U266 cells and SHIP1-negative MDA-MB-231 (MDA) breast cancer cells. IPs were run on Western blot and probed for SHIP1, SHIP2 and OCRL1 (upper panel). Whole cell lysates (WCL) were also subjected to Western blot analysis of these phosphatases (lower panels). OCRL1 and SHIP2 were precipitated from both MDA-MB-231 and OPM2 cells, whereas SHIP1 was only observed in OPM2, emphasizing specificity of the IP. (C) Malachite Green phosphatase assay (wells shown in lower panels) shows no activity in SHIP1 precipitates from MDA-MB-231 cells, whereas SHIP1 activity was observed in U266 cells. In contrast, OCRL1 phosphatase activity was detected in both cell lines.</p

Patient characteristics (patients included in the SHIP activity assay).

<p>Patient characteristics (patients included in the SHIP activity assay).</p

Aberrant SHIP1 activity and expression in adult CD patients.

<p>(A) PBMCs from CD patients and healthy controls (HC) were lysed, phosphatase assay performed. Examples of 3 of 5 CD patients with abrogated SHIP1 expression are shown. Lower panels show SHIP1, PTEN and actin protein in the same PBMC lysates of CD patients and healthy controls. Phosphatase activity in these patients was tested in duplicate, with identical results. (B) Correction of SHIP1 phosphatase activity for the amount of SHIP1 protein in the lysates shows that intrinsic SHIP1 enzymatic activity is significantly lower in CD patients compared to HC. (C) Increased mRNA and protein (examples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182308#pone.0182308.s003" target="_blank">S2A Fig</a>) expression compensates for the reduced intrinsic enzymatic activity (CD patients [n = 47] vs healthy controls [n = 42], 1.68 fold increase, p = 0.0042). RNA was available of three of the five SHIP1-deficient patients (open circles). (C) PTEN protein expression in PBMC lysates from CD patients and healthy controls (HC) were determined by Western blot analysis, and corrected for Actin levels in the same samples. Quantification of individual experiments, including mean ±SD are shown. (D,E) PTEN (D) and SHIP2 (E) protein expression in PBMC lysates from CD patients and healthy controls (HC) were determined by Western blot analysis, and corrected for Actin levels in the same samples. Quantification of individual experiments, including mean ±SD are shown (examples in Fig 2A lower panel and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182308#pone.0182308.s003" target="_blank">S2B Fig</a>).</p

Specific SHIP1 activity measurement.

<p>(A) Specificity of the assay was determined by immunoprecipitation (IP) of SHIP1 from multiple myeloma cell lines (U266 and OPM2) that express SHIP1 (see Western blot insert of whole cell lysates) and breast cancer cell line (MCF7) that does not. Phosphatase activity of immunoprecipitates was determined by adding the SHIP1 substrate PtdIns(3,4,5)P₃, followed by Malachite Green reaction. Human recombinant SHIP2 was used as positive control for the phosphatase assay. Only immunoprecipitates from SHIP1-competent cells show phosphatase activity. (B) OCRL1, SHIP2 or SHIP1 were immunoprecipitated from SHIP1-competent U266 cells and SHIP1-negative MDA-MB-231 (MDA) breast cancer cells. IPs were run on Western blot and probed for SHIP1, SHIP2 and OCRL1 (upper panel). Whole cell lysates (WCL) were also subjected to Western blot analysis of these phosphatases (lower panels). OCRL1 and SHIP2 were precipitated from both MDA-MB-231 and OPM2 cells, whereas SHIP1 was only observed in OPM2, emphasizing specificity of the IP. (C) Malachite Green phosphatase assay (wells shown in lower panels) shows no activity in SHIP1 precipitates from MDA-MB-231 cells, whereas SHIP1 activity was observed in U266 cells. In contrast, OCRL1 phosphatase activity was detected in both cell lines.</p

SHIP1 expression and activity are independent of <i>ATG16L1</i> SNP status.

<p>ATG16L1 rs2241880 SNP status was determined for those subjects for whom genomic DNA was available. Groups were stratified according to protective (AA), heterozygous (AG) or risk (GG) alleles. (A) SHIP1 activity corrected for total amount of SHIP1 present in the lysates is shown. The number of risk alleles does not affect SHIP1 intrinsic activity levels. (B) SHIP1 mRNA levels corrected for RPLP1 mRNA levels are shown for patients and controls, stratified according to <i>ATG16L1</i> SNP status. The number of <i>ATG16L1</i> risk alleles does not affect SHIP1 mRNA expression.</p

Data_Sheet_1_Determinants of HIV late presentation among men who have sex with men in Portugal (2014–2019): who’s being left behind?.docx

IntroductionHIV late presentation (LP) remains excessive in Europe. We aimed to analyze the factors associated with late presentation in the MSM population newly diagnosed with HIV in Portugal between 2014 and 2019.MethodsWe included 391 newly HIV-1 diagnosed Men who have Sex with Men (MSM), from the BESTHOPE project, in 17 countrywide Portuguese hospitals. The data included clinical and socio-behavioral questionnaires and the viral genomic sequence obtained in the drug resistance test before starting antiretrovirals (ARVs). HIV-1 subtypes and epidemiological surveillance mutations were determined using different bioinformatics tools. Logistic regression was used to estimate the association between predictor variables and late presentation (LP).ResultsThe median age was 31 years, 51% had a current income between 501–1,000 euros, 28% were migrants. 21% had never been tested for HIV before diagnosis, with 42.3% of MSM presenting LP. 60% were infected with subtype B strains. In the multivariate regression, increased age at diagnosis, higher income, lower frequency of screening, STI ever diagnosed and higher viral load were associated with LP.ConclusionOur study suggests that specific subgroups of the MSM population, such older MSM, with higher income and lower HIV testing frequency, are not being targeted by community and clinical screening services. Overall, targeted public health measures should be strengthened toward these subgroups, through strengthened primary care testing, expanded access to PrEP, information and promotion of HIV self-testing and more inclusive and accessible health services.</p

Data_Sheet_1_Determinants of HIV late presentation among men who have sex with men in Portugal (2014–2019): who’s being left behind?.docx

IntroductionHIV late presentation (LP) remains excessive in Europe. We aimed to analyze the factors associated with late presentation in the MSM population newly diagnosed with HIV in Portugal between 2014 and 2019.MethodsWe included 391 newly HIV-1 diagnosed Men who have Sex with Men (MSM), from the BESTHOPE project, in 17 countrywide Portuguese hospitals. The data included clinical and socio-behavioral questionnaires and the viral genomic sequence obtained in the drug resistance test before starting antiretrovirals (ARVs). HIV-1 subtypes and epidemiological surveillance mutations were determined using different bioinformatics tools. Logistic regression was used to estimate the association between predictor variables and late presentation (LP).ResultsThe median age was 31 years, 51% had a current income between 501–1,000 euros, 28% were migrants. 21% had never been tested for HIV before diagnosis, with 42.3% of MSM presenting LP. 60% were infected with subtype B strains. In the multivariate regression, increased age at diagnosis, higher income, lower frequency of screening, STI ever diagnosed and higher viral load were associated with LP.ConclusionOur study suggests that specific subgroups of the MSM population, such older MSM, with higher income and lower HIV testing frequency, are not being targeted by community and clinical screening services. Overall, targeted public health measures should be strengthened toward these subgroups, through strengthened primary care testing, expanded access to PrEP, information and promotion of HIV self-testing and more inclusive and accessible health services.</p

Data_Sheet_1_Determinants of HIV late presentation among men who have sex with men in Portugal (2014–2019): who’s being left behind?.docx

IntroductionHIV late presentation (LP) remains excessive in Europe. We aimed to analyze the factors associated with late presentation in the MSM population newly diagnosed with HIV in Portugal between 2014 and 2019.MethodsWe included 391 newly HIV-1 diagnosed Men who have Sex with Men (MSM), from the BESTHOPE project, in 17 countrywide Portuguese hospitals. The data included clinical and socio-behavioral questionnaires and the viral genomic sequence obtained in the drug resistance test before starting antiretrovirals (ARVs). HIV-1 subtypes and epidemiological surveillance mutations were determined using different bioinformatics tools. Logistic regression was used to estimate the association between predictor variables and late presentation (LP).ResultsThe median age was 31 years, 51% had a current income between 501–1,000 euros, 28% were migrants. 21% had never been tested for HIV before diagnosis, with 42.3% of MSM presenting LP. 60% were infected with subtype B strains. In the multivariate regression, increased age at diagnosis, higher income, lower frequency of screening, STI ever diagnosed and higher viral load were associated with LP.ConclusionOur study suggests that specific subgroups of the MSM population, such older MSM, with higher income and lower HIV testing frequency, are not being targeted by community and clinical screening services. Overall, targeted public health measures should be strengthened toward these subgroups, through strengthened primary care testing, expanded access to PrEP, information and promotion of HIV self-testing and more inclusive and accessible health services.</p