Polyclonal and monoclonal B lymphocytes response in HCV-infected patients treated with direct-acting antiviral agents

Hepatitis C virus (HCV) chronic infection can be associated with extrahepatic manifestations such as mixed cryoglobulinaemia and lymphoproliferative disorders that are endowed with increased rates of morbidity and all-cause mortality. In this study, we used flow cytometry to evaluate the effect of interferon-free antiviral treatment on peripheral blood lymphocytes in HCV-infected patients with or without associated lymphoproliferative disorders. Flow cytometry analysis of peripheral blood lymphocytes was performed at baseline and at the end of treatment. In HCV-infected patients with lymphoproliferative disorders, we evaluated immunoglobulin (Ig) light chain \u3ba/\u3bb ratio variations as a measure of monoclonal B-cell response to antiviral therapy. Healthy volunteers were enrolled as controls. A total of 29 patients were included, nine with and 20 without lymphoproliferative disorders. Sustained virological response was achieved in 29 of 29 patients. We observed a significant reduction in the B-cell compartment (39% global reduction) in eight of nine HCV-infected patients with lymphoproliferative disorders after viral clearance. We recognized the same trend, even if less pronounced, in HCV-infected patients without lymphoproliferative disorders (9% global reduction). Among HCV-infected patients with lymphoproliferative disorders, three showed an improvement/normalization of the immunoglobulin light chain ratio, whereas in the remaining six patients monoclonal B cells persisted to be clonally restricted even 1\ua0year after the end of treatment. Our data show that DAAs treatment can be effective in reducing the frequency of pathological B cells in the peripheral blood of HCV-infected patients affected by HCV-associated lymphoproliferative disorders; however, monoclonal populations can persist after viral eradication

Care pathways models and clinical outcomes in disorders of consciousness

Objective: Patients with Disorders of consciousness, are persons with extremely low functioning levels and represent a challenge for health care systems due to their high needs of facilitating environmental factors. Despite a common Italian health care path-way for these patients, no studies have analyzed information on how each region have implemented it in its welfare system correlating data with patients’ clinical outcomes. Materials and Methods: A multicenter observational pilot study was realized. Clinicians collected data on the care pathways of patients with Disorder of consciousness by ask-ing 90 patients’ caregivers to complete an ad hoc questionnaire through a structured phone interview. Questionnaire consisted of three sections: sociodemographic data, description of the care pathway done by the patient, and caregiver evaluation of health services and information received.Results: Seventy- three patients were analyzed. Length of hospital stay was different across the health care models and it was associated with improvement in clinical diag-nosis. In long- term care units, the diagnosis at admission and the number of caregivers available for each patient (median value=3) showed an indirect relationship with worsening probability in clinical outcome. Caregivers reported that communication with professionals (42%) and the answer to the need of information were the most critical points in the acute phase, whereas presence of Non- Governmental Organizations (25%) and availability of psychologists for caregivers (21%) were often missing during long-term care. The 65% of caregivers reported they did not know the UN Convention on the Rights of Persons with Disabilities. Conclusion: This study highlights relevant differences in analyzed models, despite a recommended national pathway of care. Future public health considerations and ac-tions are needed to guarantee equity and standardization of the care process in all European countries

Hyperglycemia, Reduced Hematopoietic Stem Cells, and Outcome of COVID-19

: Admission hyperglycemia has emerged worldwide as a predictor of poor COVID-19 outcome. Hyperglycemia leads to a defect in circulating hematopoietic stem/progenitor cells (HSPCs) which, in turn, predicts diabetic complications. Here, we explored whether reduced HSPCs mediated at least part of the prognostic effect of hyperglycemia on COVID-19 outcome. We found that patients with COVID-19 (n=100) hospitalized in a nonintensive setting displayed dramatically (50-60%) reduced levels of HSPCs measured by flow cytometry as CD34+, CD34+CD45dim or CD34+CD133+ cells, compared with controls (n=595). This finding was highly significant (all p3-fold higher risk of adverse outcome. Upon formal analysis, reduction of HSPCs was a significant mediator of the admission hyperglycemia on COVID-19 outcome, being responsible for 28% of its prognostic effect

Analysis of Italian regulations on pathways of care for patients in a vegetative or minimally conscious state

Different rehabilitation models for persons diagnosed with disorders of consciousness have been proposed in Europe during the last decade. In Italy, the Ministry of Health has defined a national healthcare model, although, to date, there is a lack of information on how this has been implemented at regional level. The INCARICO project collected information on different regional regulations, analysing ethical aspects and mapping care facilities (numbers of beds and medical units) in eleven regional territories. The researchers found a total of 106 laws; differences emerged both between regions and versus the national model, showing that patients with the same diagnosis may follow different pathways of care. An ongoing cultural shift from a treatment-oriented medical approach towards a care-oriented integrated biopsychosocial approach was found in all the welfare and healthcare systems analysed. Future studies are needed to explore the relationship between healthcare systems and the quality of services provided

Baseline patients’ characteristics.

<p>The total number of cases (Cases), the male:female (M:F) ratio, the mean age (years) with minimum and maximum values (range) of patients subdivided according to the histologically confirmed diagnoses, are reported in the first three columns. Surgery indicates the number of cases subjected to pancreatoduodenectomy (PD), distal pancreatectomy (DP) palliative resection (PR). Blood and spleen columns report the number of cases for whom T cells or immature myeloid cells (M cells) subsets were available. PDAC = pancreatic ductal adenocarcinoma; NETs = pancreatic neuroendocrine tumors; BPNs = pancreatic borderline neoplasms; SCA = serous cystadenoma; ChrPa = chronic pancreatitis;</p>§<p>Other tumors included 3 papillary, 3 duodenal and 3 stromal tumors.</p>*<p>2/7 patients underwent middle pancreatectomies.</p

Ratio between splenic and circulating CD33⁺CD14⁻HLA-DR⁺ immature myeloid cells.

<p> Ref. = reference group made of patients with chronic pancreatitis and of patients with splenic non-neoplastic lesions; BPNs = Borderline pancreatic neoplasms; PDAC = Ductal adenocarcinoma; NETs = Neuroendocrine tumors. Boxes represent interquartile ranges with medians; bars represents minimum and maximum values.</p

Pancreatic cancer cell conditioned media effects on lymphocyte and immature myeloid cell subsets.

<p>A total of 17 healthy PBMC were analysed by flow cytometry after they have been cultured for 4 days in control medium or pancreatic cancer cell conditioned media. PBMC from 11 donors were cultured in Capan1 conditioned and in their respective control media, while PBMC from 6 donors were cultured in BxPC3 and MiaPaCa2 conditioned media and in their respective control media. The median and interquartile ranges (IQR) of the percentage of lymphocyte and CD33⁺ immature myeloid cell subsets are shown. The statistical analysis of data (Wilcoxon signed rank test) was made by pairing any conditioned media result with its own control. Asterisks highlight statistical significance.</p

Pancreatic cancer cell conditioned media effects on PDL1 and CTLA4 in immature myeloid cell subsets.

<p>Healthy PBMC were analysed by flow cytometry after they have been cultured for 4 days in control medium or in pancreatic cancer cell conditioned media. PBMC from 8 donors were cultured in Capan1 conditioned and in their respective control media, while PBMC from 6 donors were cultured in BxPC3 and MiaPaCa2 conditioned media and in their respective control media. In the series of 8 PBMC donors used for Capan1 experiments, CTLA4 data was available for a subset of 6 donors. Median and interquartile ranges (in brackets) of the percentage of CD33⁺ immature myeloid cells expressing PDL1 or CTLA4 are shown. Only few events among the CD33⁺CD14⁺HLA-DR⁻ cell population were obtained (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054824#pone-0054824-t002" target="_blank">table 2</a>) and this did not allow an accurate analysis of this subset, which was omitted from the table. The statistical analysis of data (Wilcoxon signed rank test) was made by pairing any conditioned media result with its own control. Asterisks highlight statistical significance.</p

CTLA4 negative dendritic cells suppress T cell proliferation.

<p>Panel A: CD33⁺CD14⁻HLA-DR⁺PDL1⁺ and CD33⁺CD14⁻HLA-DR⁺PDL1⁻ cells were FACS sorted and cocultured with allogenic T cells and proliferation was evaluated by (³H)-thymidine incorporation. Assay was performed in triplicate; data are mean ± SE of 4 independent experiments. Panel B: CTLA4⁺ and CTLA4⁻ dendritic cells were FACS sorted and cocultured with allogenic T cells and proliferation was evaluated by (³H)-thymidine incorporation. Assay was performed in triplicate; data are mean ± SE of 3–4 independent experiments.</p

Immature myeloid cells in peripheral blood.

<p>Panel A (upper left): a typical example of gating of low, intermediate (Int.) and high complexity sets among CD33⁺ cells in flow cytometry. Panel B (upper right): low, intermediate and high complexity CD33⁺ cells were analysed on the basis of CD14 and HLA-DR expression. A typical example is shown in this panel. Panel C (lower left): Blood low complexity CD33⁺CD14⁻HLA-DR⁺ cells. Panel D (lower right): blood low complexity CD33⁺CD14⁻HLA-DR⁻ cells. Ref. = reference group made of patients with chronic pancreatitis (open dots) and of patients with splenic non-neoplastic lesions; BPNs = Borderline pancreatic neoplasms; PDAC = Ductal adenocarcinoma; NETs = Neuroendocrine tumors. * = p<0.004 (adjusted p-value for significance) with respect to Reference.</p