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Collection of peripheral blood stem cells (PBSCs) for autologous stem cell transplant (ASCT) requires mobilization from the bone marrow. There is variation in mobilization choice; during the COVID-19 pandemic BSBMT&CT guidelines recommended using granulocyte-colony stimulating factor (G-CSF) alone to minimize the use of chemotherapy. We report on the impact of mobilization regimen on stem cell collection, and whether IMiD-containing induction therapy impacts on mobilization and consequently transplant engraftment times for 83 patients undergoing ASCT at Leeds Teaching Hospitals. Cyclophosphamide plus G-CSF (cyclo-G) mobilization yielded more CD34 + cells (8.94 vs. 4.88 ×10 6 /kg, p  = < 0.0001) over fewer days (1.6 vs. 2.4 days, p  = 0.007), and required fewer doses of salvage Plerixafor than G-CSF only (13.6% vs. 35%, p  = 0.0407). IMiD-containing induction impaired all of these factors. CD34 + doses > 8×10 6 /kg were more frequent with Cyclo-G (62% vs. 11%, p  = 0.0001), including for those receiving IMiD 1st line induction (50% vs. 13.3%, p = 0.0381). Note that 92.6% of those receiving IMiD-free inductions were mobilized with Cyclo-G. The novel agents used in modern induction regimens (e.g Daratumumab) have been shown to impair yields, increasing the importance of optimizing mobilization regimens in the first instance. Furthermore, as cellular therapies become established in the management of multiple myeloma emerging data highlights the potential benefits of stem cell top up in the management of the haematological toxicities of these therapies. Our findings support re-adoption of Cyclo-G as the gold standard for mobilization to optimize PBSC harvesting and ensure sufficient cells for subsequent ASCTs., Competing Interests: The authors have no competing interests to declare, (© 2023 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Transfusion-associated microchimerism implies the presence of allogeneic hematopoietic cells in an individual, following the transfusion of a blood product. It is a transfusion-related adverse effect/long-term consequence, which has not been well-investigated among regularly transfused patients with thalassemia., Patients and Methods: We investigated 64 regularly transfused, homozygous β-thalassemic patients and 21 never-transfused healthy volunteer blood donors (controls) for the presence of microchimerism in their sera, using real-time PCR targeting circulating allogeneic, both, Human Leukocyte Antigen-DR isotype (HLA-DR) and non-HLA alleles. The investigation was longitudinally repeated in patient subsets for more than 2 years. Results were correlated with clinical and laboratory parameters, peripheral blood lymphocyte immunophenotype, blood storage time, and donor's gender to identify potential contributing factors for microchimerism generation., Results: Overall, microchimerism was detected in 52 of the 64 patients (81.2%) and in 6 of the 21 controls (28.5%, p = 0.0001). Forty-four patients (68.7%) exhibited long-term microchimerism (persisted for more than 6 months), confirmed at all time-points investigated. Microchimerism was more frequent among elderly, women, splenectomized and more heavily transfused patients, and among those who exhibit higher serum ferritin levels. In these patients, a distinct descending pattern of CD16 dim +CD56 dim + natural killer (NK)-cells ( p < 0.001) and an ascending pattern of CD4+CD25 bright CD127- regulatory T-cells ( p = 0.022) for increasing allelic burden were noticed, suggesting the establishment of recipient immune tolerance against the donor-derived chimeric alleles. Both splenectomized and non-splenectomized thalassemic patients exhibited the same trend. The storage time of transfused blood products and donor/gender mismatch had no impact on the development of microchimerism., Discussion-Conclusive Remarks: Transfusion-associated microchimerism appears to be a very common complication among multi-transfused thalassemic patients. The potential clinical consequences of this phenomenon remain as yet unclear. Immune tolerance attributed to disease itself and to repeated transfusions might at least in part explain its appearance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Matsagos, Verigou, Kourakli, Alexis, Vrakas, Argyropoulou, Lazaris, Spyropoulou, Labropoulou, Georgara, Lykouresi, Karakantza, Alepi and Symeonidis.)

Background: Patients' experience of symptoms often goes undetected during consultation in an outpatient clinic, and the use of a patient-reported outcome measure (PRO) in such a setting could be useful to aid treatment decision-making. A new PRO measure, the HM-PRO (Hematological Malignancy Specific Patient-Reported Outcome Measure) has been recently developed to evaluate hematological malignancy (HM) patients' health-related quality of life (HRQoL) and their symptom experience in daily clinical practice as well as in research. The objectives of the study were to assess: the internal consistency of the scores for Part A (impact) and its four domains (physical behavior; social well-being; emotional behavior; and eating and drinking habits) and Part B (signs and symptoms); and the test-retest reliability of the individual items of the newly developed hematological malignancy specific composite measure, the HM-PRO., Methods: This was a prospective longitudinal observational study where 150 patients with different HMs and different stage of disease (male n = 98 (65.3%); mean age 64.9 ± 14.4 years, range 17.9-89.2 years; mean time since diagnosis 3.7 ± 4.9 years, range 0.04-25.8 years) completed the HM-PRO at baseline (assessment 1 at t1) and after 7 days (assessment 2 at t2). Data analysis was performed using IBMSPSS 23 statistical software., Results: The Cronbach's alpha estimates of the HM-PRO for both assessment points (t1 and t2) were above 0.9 for Part A, and above 0.8 for Part B, showing strong stability of the measurement. The level of agreement for the reproducibility between the two assessments, using intra-class correlation coefficients (ICC), was very strong with Part A: ICC = 0.93 (95% CI = 0.90-0.95), and Part B: ICC = 0.91 (0.88-0.93). The ICC for the four domains of Part A ranged from 0.85-0.91. The ICC was greater than 0.8 for overall score of Part A and Part B for all the 10 diagnoses, confirming strong reliability., Conclusion: This study clearly indicates that the HM-PRO possesses strong test-retest reliability for both Part A and Part B. The Cronbach's alpha confirmed acceptable internal consistency. The extensive reliability testing described in this study supports the generic nature of the HM-PRO for use in hematological malignancies in both routine clinical practice, to aid treatment decisions, as well as in research., (Copyright © 2020 Goswami, Oliva, Ionova, Else, Kell, Fielding, Jennings, Karakantza, Al-Ismail, Collins, McConnell, Langton, Al-Obaidi, Oblak and Salek.)

Background: Our aim was to identify health-related quality-of-life (HRQoL) issues and symptoms in patients with haematological malignancies (HMs) and develop a conceptual framework to reflect the inter-relation between them., Methods: A total of 129 patients with HMs were interviewed in a UK multicentre qualitative study. All interviews were audio recorded, transcribed and analysed using NVivo-11., Results: Overall, 34 issues were reported by patients and were grouped into two parts: quality of life (QoL) and symptoms. The most prevalent HRQoL issues were: eating and drinking habits; social life; physical activity; sleep; and psychological well-being. Furthermore, most prevalent disease-related symptoms were: tiredness; feeling unwell; breathlessness; lack of energy; and back pain. The most prevalent treatment side effects were: tiredness; feeling sick; disturbance in sense of taste; and breathlessness., Conclusions: Both HMs and their treatments have a significant impact on patients' HRQoL, in particular on issues such as job-role change, body image and impact on finances., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest., (© The Author(s), 2020.)

Background: Validity is the ability of an instrument to measure what it claims to measure. It means the degree to which the empirical evidence supports the trustworthiness of interpretations based on the calculated scores. The hematological malignancy (HM) specific patient reported outcome measure (HM-PRO), is a newly developed instrument for use in daily clinical practice as well as in research. This study, provides the evidence for construct validity of the HM-PRO, specifically focusing on the convergent and divergent validity compared to the other established instruments used in hematology., Methods: This validation study adopted a prospective cross-sectional design where a heterogeneous group of patients diagnosed with different HMs and different disease state were recruited. A total of 905 patients were recruited from seven secondary care hospitals in the UK and online through five patient organizations. Patients were asked to complete the HM-PRO and other cancer specific PRO's, FACT-G and EORTC QLQ C-30. Data analysis was performed using IBM SPSS 23 statistical software., Results: A total of 486 males (53.7%) and 419 females (46.3%), with a mean age of 64.3 (± 12.4) years and mean time since diagnosis of 4.6 ( ± 5.2) were recruited. The total score of Part A of the HM-PRO highly correlated with the five functional scales of the EORTC QLQ-C30 (Physical = -0.71, Role = -0.72, Emotional = -0.64, Cognitive = -0.58, Social = -0.74-p < 0.001). With respect to correlation with FACT-G, the total score of Part A of the HM-PRO highly correlated with Physical (-0.74), Emotional (-0.57), Functional (-0.66) domains and overall score of FACT-G (-0.74). Similarly, the total score of Part B of the HM-PRO highly correlated with three symptoms scales of EORTC QLQ-C30 (Fatigue scale = -0.74, Nausea and Vomiting = -0.52, Pain = -0.59-p < 0.001) and individual symptom items (Dyspnea = 0.51, Insomnia= 0.43, Appetite loss = 0.54-p < 0.001)., Conclusion: The construct validity evidence presented in this research is a testimony to the HM-PRO's ability to measure HRQoL issues which it intends to measure. This is of utmost importance when a PRO is used in routine clinical practice so that the interpretation of the scores or response to an individual item is understood by the clinicians/nurses as intended by the patients., (Copyright © 2020 Goswami, Oliva, Ionova, Else, Kell, Fielding, Jennings, Karakantza, Al-Ismail, Collins, McConnell, Langton, Al-Obaidi, Oblak and Salek.)

Background: The quality of life of patients at all stages of hematological malignancy is greatly affected by the disease and its treatment. There is a wide range of health-related quality of life (HRQoL) issues important to these patients. Any new instrument developed to measure HRQoL of such patients should be content valid, i.e., the items should be comprehensively relevant to the patients and their health condition. The aim of the present study was to examine content validity of a hematological malignancy specific patient reported outcome measure (HM-PRO) developed for use in routine clinical practice., Methods: Following literature review and semi-structured interviews, the generated themes and sub-themes were discussed to develop the prototype version of the HM-PRO. A 4-step approach was used for content validation: initial testing and cognitive interviewing; item rating; content validity panel meeting; final field testing and cognitive interviewing. Additional questions related to patients' perception of recall period and preferred sentence structure (i.e., question or statement) of the items were also asked during cognitive interviews., Results: The content analysis of 129 transcribed semi-structured interviews resulted in the prototype version of the instrument consisting of 58 items grouped into two parts: Part A (impact/HRQoL - 34 items) and Part B (signs and symptoms - 24 items). The initial testing showed intra-class correlation coefficient (ICC) of >0.8 for both Part A and Part B. Item rating for language clarity, completeness, relevance, and response scale by experts and patients showed content validity index for scales average >0.8 for both Part A and Part B, except 0.64 for relevance for Part A by the patient panel. The final testing of the revised version of the instrument showed the Cronbach's alpha value of 0.91 for Part A and 0.76 for Part B, suggesting high internal consistency, and ICC of 0.91 for Part A and 0.76 for Part B. The recall period of "today" for Part-A and "last 3 days" for Part-B were the patients' preferred "recall period." Furthermore, the patients expressed preference to the HM-PRO items as statements., Conclusion: The findings of this study confirm that the HM-PRO possesses a strong content validity, includes all the issues important to patients and is easy to read, understand and respond to spontaneously., (Copyright © 2020 Goswami, Oliva, Ionova, Else, Kell, Fielding, Jennings, Karakantza, Al-Ismail, Collins, McConnell, Langton and Salek.)

Background: Patients with diabetes mellitus (DM) exhibit alterations in their immune response when infected by several types of micro-organisms. The increased susceptibility of diabetics to infections is particularly related to abnormalities in the function of neutrophils such as chemotaxis, adhesion and intracellular killing, leading to increased mortality rates. Aims of the study were to assess the phagocytic activity and the expression of antigens HLA-DR and CD64 of monocytes and neutrophils in diabetics with sepsis and evaluate their significance as prognostic factors., Methods: This is an observational prospective study conducted in a tertiary medical center, referring to a population of 51 diabetic patients who were treated for sepsis. Samples of whole blood were received from the selected patients and were evaluated for the expression of surface antigens HLA-DR and CD64 on monocytes and neutrophils, and for their phagocytic activity as well., Results: Alterations in the phagocytic activity were found in the diabetic patients who developed sepsis, and these were addressed as an elevation in the expression of CD64 on monocytes (CD64M), and a reduction in the expression of HLA-DR on monocytes (HLA-DRM) at least in the initial phase of the acute infection. A significant elevation was also noticed in the phagocytosis rate of both neutrophils and monocytes on day of admission. Survivors had higher rates of both CD64 and HLA-DR on monocytes when compared to non-survivors. No correlation was found between glycemic control, values of inflammatory markers on admission, phagocytosis rate and the survival of diabetics with sepsis. A reduced expression of CD64O, HLA-DRM and the co-expression of CD64/HLA-DR on monocytes in the initial phase of sepsis and poor glycemic control (hemoglobin A1c (HbA1c) > 8.5) was found., Conclusions: In the present study of diabetic patients with sepsis the phagocytic activity of neutrophils and monocytes is elevated at the initial phase of an acute infection and only the values of CD64 and HLA-DR on monocytes were significantly related to outcome. Further evaluation of these results with large prospective studies is warranted., Competing Interests: The authors declare that they do not have any conflict of interest to report., (Copyright 2020, Lekka et al.)

Aim: To determine measurement equivalence of paper and electronic application of the hematological malignancy-patient-reported outcome (HM-PRO), a specific measure for the evaluation of patient-reported outcomes in HMs. Patients & methods: Following International Society of Pharmacoeconomics and Outcomes Research ePRO Good Research Practice Task Force guidelines, a total of 193 adult patients with different HMs were recruited into a multicenter prospective study. The paper and the electronic version of the instrument were completed in the outpatient clinics in a randomized crossover design with a 30 min time interval to minimize the learning effect. Those who completed the paper version first, completed the electronic version after 30 min and vice versa. Instrument version and order effects were tested on total score of the two parts of the HM-PRO (Part A: quality of life and Part B: signs & symptoms) in a two-way ANOVA with patients as random effects. Intraclass correlation coefficients (95% CI) and Spearman's rank correlation coefficients were used to evaluate test-retest reliability and reproducibility. The effects of instrument version and order were tested on total score of the two parts of HM-PRO. Results: The questionnaire version and administration order effects were not significant at the 5% level. There were no interactions found between these two factors for HM-PRO (Part A [quality of life]; p = 0.95); and (part B [signs and symptoms]; p = 0.72]. Spearman's rank correlation coefficients were greater than 0.9, and intraclass correlation coefficients ranged from 0.94 to 0.98; furthermore, the scores were not statistically different between the two versions, showing acceptable reliability indexes. Noteworthy, the difference between the completion time for both paper (mean = 6:38 min) and electronic version (mean = 7:29 min) was not statistically significant (n = 100; p = 0.11). Patients did not report any difficulty in completing the electronic version during cognitive interviews and were able to understand and respond spontaneously. Conclusion: Measurement equivalence has been demonstrated for the paper and electronic application of the HM-PRO.

We investigated peripheral blood T-lymphocyte subpopulations and intracellular expression of IFN-γ, IL-4, IL-10, and IL-13, by whole blood flow cytometry, in 22 type I Gaucher disease (GD) patients. Results were compared with those of 19 sex- and age-matched controls. Patients with GD exhibited decreased frequencies and absolute numbers of CD3+/CD4+ helper T lymphocytes (40.8 ± 9.8% vs. 49.4 ± 5.7%, p = 0.002, and 0.77 ± 0.33 vs. 1.04 ± 0.28 × 10(9)/μL, p = 0.011), as well as increased frequencies of CD3+CD8+ suppressor T lymphocytes (23.8 ± 8.0% vs. 18.4 ± 3.8%, p = 0.010), resulting in a significantly decreased CD4/CD8 cell ratio (p < 0.001). Moreover, they had significantly increased percentages of IFNγ-producing both CD4+ and CD8+ T cells (p = 0.0003 and p = 0.023, respectively), implying a TH-1 polarization pattern. Finally, patients with GD had decreased percentages and absolute numbers of CD4+CD25(dim) T lymphocytes (p = 0.033 and p = 0.007, respectively), of CD4+CD25(high) T lymphocytes (p = 0.039 and p = 0.016, respectively), and of CD4+CD25(high)FOXP3+ regulatory T cells (p = 0.036 and p = 0.019, respectively). Our results demonstrate that patients with GD have a significant numerical impairment of T-helper lymphocytes and a constitutive TH1 direction pattern of activation of both CD4+ and CD8+ cells, associated with a significant decrease of T-regs. Ineffective T-cell control may explain the chronic inflammatory reaction and the increased incidence of lymphoid malignancies, which have been repeatedly reported among patients with GD.

Descriptive epidemiology of the myelodysplastic syndromes (MDS) is always interesting and may reveal time-dependent and geographical variations, as well as occupational exposure. Epidemiological data in Greece are not available by now. We have collected and analyzed medical records of all patients with a documented diagnosis of MDS, performed by an expert hematologist and/or hematopathologist, in the geographical area of Western Greece, during the 20-year period, defined between 1990 and 2009. We have then calculated and described demographic and clinical features of the diagnosed MDS patient population, and assessed the incidence and prevalence rates of MDS in Western Greece, during the above-mentioned period. A total of 855 patients with newly diagnosed MDS have been identified. Refractory anemia was the most common subtype in both FAB and WHO classification systems and in both genders. Del-5q and RARS were more commonly encountered among females, and the dysplastic subtype of chronic myelomonocytic leukemia among males. Trisomy 8 was the most common single cytogenetic abnormality. The crude mean annual incidence rate of MDS was 6.0 per 100,000 inhabitants aged ≥15 years old (all subtypes according to FAB), and it was 4.8 per 100,000 when CMML and RAEB-T were excluded. Crude incidence rate was higher in rural than in urban areas, but this finding was not confirmed after age standardization. Age-standardized mean annual incidence rate in men was 7.9/100,000 and in women 3.4/100,000. A continuously increasing incidence rate of MDS has been observed throughout the study period.

Chronic hepatitis C (CHC) and iron overload are the main causes of liver disease in β-thalassemia major (βTM). There is limited data regarding the course of CHC in this population. All patients (n=144) from the thalassemia centre of the University Hospital of Patras were evaluated (January 1981 to June 2012). Patients were classified into group A (n=57), which consisted of patients with CHC, who either had received antiviral treatment (n=49) or not (n=8), and group B which included 87 patients without CHC. Nineteen patients died during follow-up (median: 257.5 months (1-355)). Survival rates were 84.2 % and 88.5 % for group A and B, respectively. The causes of death were heart failure (63.2 %), accident (10.5 %), sepsis (5.3 %), liver failure (5.3 %), hepatocellular carcinoma (HCC) (5.3 %), non-Hodgkin lymphoma (5.3 %) and multiorgan failure (5.3 %). There were no differences in total survival between the two groups (p=0.524). In the multivariate analysis, survival was neither correlated with CHC (p=ns), nor with anti-HCV treatment (p=ns), whereas independent negative predictors were presence of heart failure (p<0.001), presence of malignancy other than HCC (p=0.001) and non-adherence to chelation treatment (p=0.013). Predictive factors for the development of cirrhosis were: CHC (p<0.001), age>35 years (p=0.007), siderosis grade 3/4 (p=0.029) and splenectomy (p=0.001); however, multivariately, only siderosis grade 3/4 was found to be significant (p=0.049). In this study, survival of patients with βTM was mainly associated with heart failure, presence of malignancy other than HCC and non-adherence to chelation treatment, rather than with liver disease. Multicentre studies need to be designed to define more accurately the indications of antiviral treatment in this population.

An insufficient cellular immune response seems to be critical for the immunopathogenesis of chronic hepatitis B virus infection. We have previously demonstrated no differences of T-lymphocyte subsets in blood between inactive hepatitis B s antigen (HBsAg) carriers and patients with HBeAg-negative chronic active hepatitis B. This study investigated the peripheral blood cytokine profile in patients with HBeAg-negative chronic active hepatitis B infection (Group A, n = 21) and inactive HBsAg carriers (Group B, n = 13). Serum cytokines [interferon (IFN)-γ, tumor necrosis factor-α, interleukin (IL)-1b, IL-4, IL-12, IL-10, IL-2, IL-5, IL-8] were analyzed by using flow cytometry. Patients with chronic active disease presented with significantly decreased levels of IFN-γ and IL-10 compared to inactive carriers (P = 0.048 and P = 0.008, respectively). In HBeAg-negative chronic active hepatitis B patients, a significant negative correlation of IFN-γ levels with serum hepatitis B viral load was noted (P = 0.021). In conclusion, patients with HBeAg-negative chronic active hepatitis B and HBsAg inactive carriers display a different cytokine profile. Decreased Th1 response observed in patients with chronic active hepatitis B could be implicated in the persistence of virus replication and ongoing progression of liver disease.

The value of prophylactic donor lymphocyte infusion (pDLI) is unclear and differs among diseases and transplantation protocols. Experience with this approach in patients with acute leukemia undergoing hematopoietic cell transplantation (HCT) with an alemtuzumab-incorporating conditioning protocol is lacking. We conducted a single-center prospective study to investigate the applicability and efficacy of prophylactic donor lymphocyte infusion (pDLI) in patients with leukemia undergoing HCT with a low-dose alemtuzumab-containing conditioning regimen. Inclusion criteria were high-risk acute myelogenous leukemia, acute lymphoblastic leukemia, or increasing mixed chimerism. All patients included were tapered off of immunotherapy. Exclusion criteria were a history of ≥ grade II or active graft-versus-host disease (GVHD). Of the 56 consecutive patients who underwent HCT with an alemtuzumab-containing regimen, 15 patients (8 with acute myelogenous leukemia and 7 with acute lymphoblastic leukemia) met the study inclusion criteria and received prophylactic DLI (total of 45 infusions) from 7 sibling donors and 8 unrelated donors. The first infusion was given at a median of 162 days posttransplantation. The median number of DLIs was 3, and the median cumulative CD3(+) cell dose was 2 × 10(6)cells/kg. Six of the 8 patients (75%) who received pDLI while in mixed chimerism converted to stable, complete donor chimerism. Some 47% of DLI recipients developed GVHD (4 acute GVHD and 3 with chronic GVHD) after a median cumulative dose of 2 × 10(6) CD3(+) cells/kg. After a median follow-up of 575 days, 11 (73%) pDLI recipients were alive. All 4 deaths were due to GVHD-related causes. None of the patients who received pDLIs relapsed. Patients with leukemia who received low-dose pDLI after conditioning with alemtuzumab are at low risk for relapse; however, this approach is associated with a relatively high incidence of severe GVHD., (Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34(+) irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34(+) from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.

Aim: To evaluate the immune response in peripheral blood and liver tissue, through the measurement of T-cell subsets, in patients with chronic hepatitis B (CHB) and C (CHC)., Patients and Methods: Thirty-four patients with CHB (21 with active HBV infection and 13 inactive HBV carriers) and 20 patients with CHC were included in the study. We also evaluated 21 biopsies from patients with active CHB infection and 20 patients with CHC. We measured CD3, CD4, CD8 and CD4/CD8 ratio in peripheral blood and liver tissue., Results: We found no differences in the numbers of all T-lymphocyte subpopulations between patients with active HBV infection and inactive carriers. We found a significant increase in the absolute numbers of CD3(+), CD4(+) and CD8(+) T-lymphocytes in CHC compared to CHB patients (p=0.005, p=0.034 and p<0.0001 respectively). There was a significant increase in the number of CD3(+) and CD8(+) T-lymphocytes in the area of portal tracts (p=0.012 and p=0.009 respectively) and lobules (p=0.011 and p=0.01 respectively) in patients with CHC compared to those with CHB. In both groups there was a direct correlation between CD3(+) cells in portal tracts and HAI score (r=0.783, p=0.008), while we noted a correlation between CD8(+) cells in portal tracts and HAI score only in patients with CHC. Interface hepatitis correlated to CD3(+) cells in lobules of patients with CHC and CHB but a direct relationship between CD8(+) cells and HAI score was found only in those with CHC., Conclusion: Insufficient cellular immune response is critical for the ineffective virus clearance and liver damage in chronic hepatitis B, while in chronic hepatitis C, immune response, as represented by CD8(+) T-cells, is present in the peripheral blood and the liver. However, there is an immunological escape of HCV, which seems to survive in the presence of an adequate immune response. The significant correlation between portal and periportal CD8(+) T-lymphocyte expression and interface hepatitis may be considered evidence of the occurrence of cytotoxic immune-mediated toxicity.

Background: Infections by a variety of pathogens are a significant cause of morbidity and mortality during perinatal period. The susceptibility of neonates to bacterial infections has been attributed to immaturity of innate immunity. It is considered that one of the impaired mechanisms is the phagocytic function of neutrophils and monocytes. The purpose of the present study was to investigate the phagocytic ability of neonates at birth., Methods: The phagocytic ability of neutrophils and monocytes of 42 neonates was determined using the Phagotest flow cytometry method, that assesses the intake of E. Coli by phagocytes, in cord blood and in peripheral blood 3 days after birth. Fifteen healthy adults were included in the study as controls., Results: The phagocytic ability of neutrophils in the cord blood of neonates was significantly reduced compared to adults. The 3rd postnatal day the reduction of phagocytic ability of neutrophils was no longer significant compared to adults. The phagocytic ability of monocytes did not show any difference from that of adults either at birth or the 3rd postnatal day., Conclusions: Our findings indicate that the intake of E. Coli by phagocytes is impaired at birth in both preterm and full term neonates compared to adults. This defect is transient, with the phagocytic ability in neonates reaching that of the adults 3 days after birth.

Functional single-nucleotide polymorphisms (SNPs) of inflammatory cytokines have been previously related to the occurrence of an ischemic stroke (IS). We investigated whether five functional SNPs (i.e., TNF-α-308G>A, IL6-174G>C, IL12B 1188A>C, IL4-589C>T, and IL10-1082G>A) might be associated with the age of onset and 6-month outcome of an acute IS. A probe-free real-time PCR methodology was used to genotype 145 consecutively admitted cases with a first-ever IS. Simple Kaplan-Mayer and adjusted Cox regression analyses showed no association between inflammatory genotypes and the age of IS onset. IL6-174G>C, IL12B 1188A>C, IL4-589C>T, and IL10-1082G>A were not found to significantly contribute to the long-term outcome of the disease. However, carriage of the TNF-α-308 GG genotype was significantly associated with reduced odds for an adverse outcome. Larger studies are needed to confirm our results.

We have recently reported that the activation of focal adhesion kinase (FAK) and its downstream targets upon pathogen challenge regulate phagocytosis in medfly haemocytes. The goal of this study was to further explore the signalling pathway underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate the haemocyte surface receptor, through which the extracellular signals in response to bacteria are transmitted intracellularly. The presented data demonstrate the expression of a beta integrin subunit in the surface of medfly haemocytes that transmits signals upon pathogen triggering to FAK and its downstream targets, Src, MAP kinases and Elk-1-like protein, for the engulfment of pathogen. Interestingly LPS is not internalized through integrins.

In the rapidly advancing field of flow cytometry, methodologies facilitating automated clinical decision support are increasingly needed. In the case of B-chronic lymphocytic leukemia (B-CLL), discrimination of the various subpopulations of blood cells is an important task. In this work, our objective is to provide a useful paradigm of computer-based assistance in the domain of flow-cytometric data analysis by proposing a Bayesian methodology for flow cytometry clustering. Using Bayesian clustering, we replicate a series of (unsupervised) data clustering tasks, usually performed manually by the expert. The proposed methodology is able to incorporate the expert's knowledge, as prior information to data-driven statistical learning methods, in a simple and efficient way. We observe almost optimal clustering results, with respect to the expert's gold standard. The model is flexible enough to identify correctly non canonical clustering structures, despite the presence of various abnormalities and heterogeneities in data; it offers an advantage over other types of approaches that apply hierarchical or distance-based concepts.

Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug resistance in AML. c-Jun N-terminal kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

The role of the phagocytic function of monocytes and neutrophils in sepsis has been poorly investigated. The present study evaluated the impact of the phagocytic activity of neutrophils and monocytes on the outcome of patients with severe sepsis. Thirty-one patients and 30 healthy individuals were enrolled in the study. The phagocytic activity of monocytes and neutrophils was evaluated during 24 h after admission and the results were correlated to the expression of CD64 on neutrophils and monocytes, CD14 antigen on monocytes, the Simplified Acute Physiology Score II and the patients' survival. A reduced phagocytic activity of neutrophils during the first 24 h after admission was a negative predictor for survival. Increased expression of CD64 antigen on polymorphonuclear cells (PMNs) and monocytes was favourably correlated to the patients' survival. In multivariate analysis the phagocytic activity of PMNs was the only independent predictor factor for survival. Patients with PMN phagocytic activity <37% had lower expression of CD64 on monocytes and PMNs and worse outcome, while those with phagocytic activity >37% had higher expression of CD64 on monocytes and PMNs and better outcome. Reduced phagocytic activity of neutrophils may represent a state of neutrophil inactivation similar to that previously described for monocytes during the compensatory anti-inflammatory response.

Local humoral and cellular immune responses modulate the inflammatory processes involved in the development of atherosclerotic lesions, as well as in the evolution of brain infarcts in stroke patients. The role of systemic adaptive immunity on the progression of such disease manifestations is less clear. In the current study, we evaluated the percentages of T helper 1 (Th1) [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T cells in 23 patients with a history of ischaemic stroke (IS) at the chronic stable phase of the disease (median post-stroke time 34.5 months). Seven stroke-free individuals matched for age and vascular risk factors (matched controls, MC) were collected for comparison. To measure cytokine values at baseline and after stimulation, we used a flow cytometry method of intracellular cytokine staining. Intrinsic Th1 and Th2 cytokine production in unstimulated T cells was negligible in all study participants. Following mitogenic stimulation with phorbol 12-myristate13-acetate/ionomycin, both the IS and the MC groups exhibited a similarly strong Th1 response; IL-2 production predominated in the CD4+ T cells and IFN-gamma in the CD8+ T cells. However, when measuring the Th2 cytokine-production capacity post-stimulation, a significant increase in the percentage of IL-4-producing T cells was observed in the IS groups, compared with the MC group, resulting in a significantly lower ratio of IFN-gamma-/IL-4-producing T cells. No such Th2 enhancement could be confirmed for the case of IL-10. We propose that in IS patients there is a systemic shift of the immune system towards Th2 responses at the late post-acute phase of stroke.

Background: The integration of biomedical information is essential for tackling medical problems. We describe a data model in the domain of flow cytometry (FC) allowing for massive management, analysis and integration with other laboratory and clinical information. The paper is concerned with the proper translation of the Flow Cytometry Standard (FCS) into a relational database schema, in a way that facilitates end users at either doing research on FC or studying specific cases of patients undergone FC analysis, Results: The proposed database schema provides integration of data originating from diverse acquisition settings, organized in a way that allows syntactically simple queries that provide results significantly faster than the conventional implementations of the FCS standard. The proposed schema can potentially achieve up to 8 orders of magnitude reduction in query complexity and up to 2 orders of magnitude reduction in response time for data originating from flow cytometers that record 256 colours. This is mainly achieved by managing to maintain an almost constant number of data-mining procedures regardless of the size and complexity of the stored information., Conclusion: It is evident that using single-file data storage standards for the design of databases without any structural transformations significantly limits the flexibility of databases. Analysis of the requirements of a specific domain for integration and massive data processing can provide the necessary schema modifications that will unlock the additional functionality of a relational database.

Post-traumatic splenectomy is associated with increased postoperative morbidity and mortality and long-term impairment of humoral and cellular immunity. Alternatives to surgery have been developed to minimize or avoid the immediate and/or long-term complications of splenectomy. Herein we investigated the long-term effect of non-operative management (NOM) of the traumatic rupture of the spleen on the distribution of peripheral blood (PB) lymphocyte populations and cytokine production by T cells. PB samples were drawn from six NOM patients, 13 age-matched adults who had undergone splenectomy after trauma (SP patients) and 31 age-matched controls. Cellular phenotypes and the intracellular production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-10 cytokines in T cells were determined in whole blood +/- mitogens by flow cytometry. NOM patients did not show any changes in the absolute numbers of lymphocytes or the distribution of their subsets, compared to the controls. In contrast, SP patients showed a sustained increase in the percentage and/or absolute numbers of lymphocytes, CD8 T cells, activated CD8 T cells, natural killer (NK) T cells, NK cells and gammadelta T cells, and a reduction in naive CD4 T cells. The constitutive or induced cytokine production by T cells of the NOM group was similar to the control group, whereas SP patients had increased percentages of constitutive IL-2- and IFN-gamma-producing CD8 T cells and IFN-gamma-producing CD4 T cells. Our findings indicate collectively that the healing process in NOM does not affect the architecture of the spleen to such an extent that it would lead to long-term alterations of the proportions of PB lymphocytes or the T cell cytokine profiles.

Antiphospholipid syndrome (APS) is an autoimmune condition characterized by thrombosis and/or recurrent fetal loss as well as the presence of autoantibodies against epitopes present on phospholipid-binding proteins. The role of cellular immunity in the pathogenesis of the syndrome remains unclear. We studied the cellular phenotype and the production of type 1 [interferon (IFN)-gamma, interleukin (IL)-2] and type 2 (IL-4, IL-10) cytokines by CD4+ and CD8+ T-lymphocyte subsets in 13 patients with untreated primary APS (PAPS) and in 32 healthy controls. The production of cytokines was determined in T cells after a 5-h culture with or without mitogenic stimulation using a flow cytometric method of intracellular cytokine staining. In six of the patients these studies were repeated 6 months later. In PAPS patients we found a reduced percentage of circulating CD4+CD45RA+ and an increased percentage and absolute number of CD8+HLA-DR+ cells. A type 1 response was observed in the patients' unstimulated cells, indicated by an increase in IFN-gamma-producing CD8+, IL-2-producing CD4+ T cells, and a decrease in IL-4-producing CD4+ and CD8+ T cells. Similar results were obtained in the patients at follow-up. Taken together, these results suggest a chronic in vivo stimulation of CD4+ and CD8+ T cells in PAPS patients exhibiting a type 1 polarization. Changes of cellular immunity may contribute to the pathogenesis of the clinical manifestations of the syndrome and might be proven to be useful targets for therapeutic interventions in the future.

Derangement of cellular immunity is central in the pathophysiology of adult autoimmune/idiopathic thrombocytopenic purpura (ITP). Herein we investigated cytokine gene expression in peripheral blood mononuclear cells (PBMCs) of adult chronic ITP patients and attempted to correlate cytokine polarization with the degree of thrombocytopenia. We used semiquantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) to measure the expression of type-1 (interleukin-2 [IL-2], interferon gamma [IFN-gamma]) and type-2 (IL-4, IL-5, IL-10, IL-3, IL-13) cytokines by PBMCs from 21 patients and 11 controls. Plasma transforming growth factor beta1 (TGF-beta1) levels were measured by enzyme-linked immunoassay (ELISA). T helper 1 (Th1)/Th2 ([IL-2 + IFN-gamma]/[IL-4 + IL-5]) cytokine mRNA ratios, thought to reflect the Th deviation of the pathogenic disease-specific T cells, and type-1/type-2 mRNA ratios, thought to reflect the overall immune response polarization, were significantly increased in ITP patients. The Th1/Th2 ratio was inversely correlated with platelet counts. TGF-beta1 levels appeared suppressed in patients with active disease, though not significantly. Our findings show a clear type-1 cytokine polarization of the autoimmune response in adult ITP that persists irrespective of disease status.

This study was performed to evaluate the impact of pro- and anti-inflammatory molecules and human leukocyte antigen DR (HLA-DR) expression as markers of immune status for the final outcome of septic patients. The study included 30 patients with severe sepsis due to community-acquired infections. Concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-8, IL-10, and transforming growth factor beta1 (TGF-beta1) in serum, as well as monocyte HLA-DR expression, were determined on admission and on days 3, 10, 13, and 17 during hospitalization. Of the 30 patients enrolled, 13 survived, while 17 died during their hospital stay. All patients had significantly lower HLA-DR expression and higher pro- and anti-inflammatory cytokine levels than healthy individuals. HLA-DR expression was significantly decreased in nonsurvivors at almost all time points. In nonsurvivors, higher levels in serum of TNF-alpha on days 13 and 17; IL-6 levels on day 3; and IL-10 on days 3, 10, and 13 were found. Baseline levels of TGF-beta1 were significantly higher in survivors. Independent risk factors of mortality were IL-10 levels on days 3 and 10, while monocyte HLA-DR expression on admission was a good predictor for survival. Several pro- and anti-inflammatory cytokines are oversynthesized during severe infections, especially in patients with a poor outcome. Monocyte HLA-DR expression is an early and constant predictive marker for survival in severe sepsis, while serum IL-10 levels on days 3 and 10 have negative prognostic value for the final outcome.