Peripheral Cell Counts Research Articles

Peripheral Endothelial Cell Count Is a Predictor of Disease Severity in Advanced Fuchs Endothelial Corneal Dystrophy.

In advanced Fuchs endothelial corneal dystrophy (FECD), central endothelial changes do not correlate with disease severity. The peripheral endothelial cell count (ECC) has not been studied as a marker of FECD severity. The goal of this study was to determine the relationship between the peripheral ECC and known clinical markers of FECD in advanced cases. Patients with FECD examined between January 1, 2013, and September 1, 2016, by 1 cornea specialist were identified. Medical records from all previous visits were reviewed to include eyes with high-quality central and peripheral in vivo confocal microscopy images performed on the same day as a clinical evaluation. Endothelial photographs were used to perform manual cell counts centrally and peripherally. Clinical grading of FECD from 1 to 4 was performed at the slit-lamp. We identified 154 eyes of 126 patients that met criteria for inclusion. With higher disease grades, central ECC and peripheral ECC decreased, visual acuity worsened, and central corneal thickness (CCT) increased (all P < 0.05). In patients with advanced disease (defined as either grade 3 or 4, CCT >700, or central ECC <350), the peripheral ECC was the best predictor of disease severity and had the highest number of statistically significant correlations with other clinical markers compared with competing variables. In advanced FECD, severity is best determined by the peripheral ECC compared with the central ECC, visual acuity, clinical disease grade, and CCT. The peripheral ECC should be added to the clinical parameters used to evaluate FECD severity.

Analysis of long term CD4+CD25highCD127- T-reg cells kinetics in peripheral blood of lung transplant recipients

BackgroundThe role of CD4+CD25highCD127− T-reg cells in solid-organ Transplant (Tx) acceptance has been extensively studied. In previous studies on kidney and liver recipients, peripheral T-reg cell counts were associated to graft survival, while in lung Tx, there is limited evidence for similar findings. This study aims to analyze long term peripheral kinetics of T-reg-cells in a cohort of lung recipients and tests its association to several clinical variables.MethodsFrom jan 2009 to dec 2014, 137 lung Tx recipients were submitted to an immunological follow up (median: 105.9 months (6.7–310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD4+CD25highCD127− T and FOXP3+ cells. We tested the association between T-reg and relevant variables by linear OR regression models for repeated measures, adjusting for time from Tx. Also, by ordered logistic models for panel data, the association between Chronic Lung Allograft Dysfuncton (CLAD) onset/progression and T-reg counts in the previous 3 months was tested.ResultsAmong all variables analyzed at multivariate analysis: Bronchiolitis Obliterans Syndrome (OR −6.51, p < 0.001), Restrictive Allograft Syndrome (OR −5.19, p = 0.04) and Extracorporeal photopheresis (OR −5.65, p < 0.001) were significantly associated to T-reg cell. T-reg cell counts progressively decreased according to the severity of CLAD. Furthermore, patients with higher mean T-reg counts in a trimester had a significantly lower risk (OR 0.97, p = 0.012) of presenting CLAD or progressing in the graft dysfunction in the following trimester.ConclusionsOur present data confirm animal observations on the possible role of T-reg in the evolution of CLAD.

Clinical analysis of 55 infants with group B streptococcus blood stream infection

Objective To summarize the clinical characteristics and outcome of infants with group B streptococcus (GBS) blood stream infection. Methods The medical records of 55 cases with GBS blood stream infection who were hospitalized in Shenzhen Children's Hospital from 1st January 2010 to 31st December 2015 were retrospectively analyzed. Results There were 30 boys and 25 girls in this study. The age ranged from 1 hour to 78 days. Six cases (10.9%) were early-onset and 49 cases (89.1%) were late-onset. Forty cases (72.7%) were neonates and 15 cases were infants. The meningitis was diagnosed in 20 patients (36.4%). Peripheral white blood cell (WBC) counts declined in 10 cases (18.2%), and elevated in 32 (58.2%) cases. Increased levels C-reactive protein were found in 41 cases (74.5%). All of isolates were susceptible to penicillin, ampicillin, linezolid and vancomycin, while the resistance rates to erythromycin, clindamycin, tetracycline and ciprofloxacin were 56.6% (30/53), 77.4% (41/53), 98.1% (52/53), and 1.9% (1/53), respectively. Meropenem was used in 18 cases, and penicillins or cephalosporins were used in 37 cases. Combined therapy with linezolid was used in 13 cases, combined therapy with vancomycin was used in 3 cases, and combined therapy with two kinds of antibiotics was used in 37 cases (67.3%). In clinical outcome analysis, 54 children (98.2%) were improved and 1 child (1.8%) died of complicated multiple organ dysfunction syndrome and intracranial hemorrhage. Conclusions GBS blood stream infection occurs commonly in the infants aged younger than 3 months, more than one third cases complicated with purulent meningitis. All of isolates are susceptible to penicillin, while the resistant rates are high to erythromycin and clindamycin. The percentage of combination therapy is high. The outcomes are not good. Key words: Streptococcus agalactiae; Infant; Blood stream infection

Descemetorhexis Without Grafting for Fuchs Endothelial Dystrophy-Supplementation With Topical Ripasudil.

To report the safety and efficacy of descemetorhexis without grafting as a primary intervention in Fuchs dystrophy, and the use of a ROCK inhibitor, ripasudil as a salvage agent in failing cases. Twelve eyes of 11 patients underwent central descemetorhexis not exceeding 4 mm. All had Fuchs dystrophy-producing visual symptoms, requesting intervention. Exclusion criteria were a peripheral endothelial cell count <1000 and central edema. Corneal clearance and visual parameters were recorded monthly until corneal clearance was observed, then at intervals of 6 months. Cases failing to clear by month 2 were considered for salvage treatment. This consisted of treatment with 1 of 2 formulations of Rho-associated kinase inhibitor eye drops. Endothelial keratoplasty was planned as the final salvage procedure in unsuccessful cases. Nine of 12 eyes cleared spontaneously between 2 and 6 months. One eye failed to clear by month 5 and topical Y-27632 was administered, without success. Endothelial keratoplasty was performed. In 2 eyes, healing stalled at 3 and 2 months. In both cases, topical ripasudil administered 6 times a day for 2 weeks resulted in complete corneal clearance. In cases achieving corneal clearance, best spectacle corrected visual acuity improved from a mean of 0.26 to 0.125 (logMAR) with subjective improvement in quality of vision. In Fuchs dystrophy with visual degradation due to central guttae, descemetorhexis without grafting is a viable procedure for visual rehabilitation. Careful patient selection is required, but the advent of topical ripasudil as a salvage agent suggests that a broader application of the surgery may be possible. Further study into the use of this agent is now needed.

Association of peripheral NK cell counts with Helios+ IFN-γ- Tregs in patients with good long-term renal allograft function.

Little is known about a possible interaction of natural killer (NK) cells with regulatory T cells (Treg ) in long-term stable kidney transplant recipients. Absolute counts of lymphocyte and Treg subsets were studied in whole blood samples of 136 long-term stable renal transplant recipients and 52 healthy controls using eight-colour fluorescence flow cytometry. Patients were 1946 ± 2201 days (153-10268 days) post-transplant and showed a serum creatinine of 1·7 ± 0·7 mg/dl. Renal transplant recipients investigated >1·5 years post-transplant showed higher total NK cell counts than recipients studied <1·5 years after transplantation (P = 0·006). High NK cells were associated with high glomerular filtration rate (P = 0·002) and low serum creatinine (P = 0·005). Interestingly, high NK cells were associated with high CD4+ CD25+ CD127- forkhead box protein 3 (FoxP3+ ) Treg that co-express the phenotype Helios+ interferon (IFN)-γ- and appear to have stable FoxP3 expression and originate from the thymus. Furthermore, high total NK cells were associated with Treg that co-express the phenotypes interleukin (IL)-10- transforming growth factor (TGF)-β+ (P = 0·013), CD183+ CD62L- (P = 0·003), CD183+ CD62+ (P = 0·001), CD183- CD62L+ (P = 0·002), CD252- CD152+ (P < 0·001), CD28+ human leucocyte antigen D-related (HLA-DR- ) (P = 0·002), CD28+ HLA-DR+ (P < 0·001), CD95+ CD178- (P < 0·001) and CD279- CD152+ (P < 0·001), suggesting that these activated Treg home in peripheral tissues and suppress effector cells via TGF-β and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). The higher numbers of NK and Treg cell counts in patients with long-term good allograft function and the statistical association of these two lymphocyte subsets with each other suggest a direct or indirect (via DC) interaction of these cell subpopulations that contributes to good long-term allograft acceptance. Moreover, we speculate that regulatory NK cells are formed late post-transplant that are able to inhibit graft-reactive effector cells.

Anti-CD20 Cell Therapies in Multiple Sclerosis-A Fixed Dosing Schedule for Ocrelizumab is Overkill.

Anti-CD 20 therapies have found significant uses in multiple sclerosis (MS). Based singularly on the accumulated evidence with the use of rituximab (RTX; Rituxan, Genentech, and Biogen) in neuroimmunological diseases, ocrelizumab (OCR; Ocrevus, Genentech) was developed as a treatment option for MS and selectively targets CD20 B cells, a cell surface antigen found on pre-B cells, mature, and memory B cells, but not on lymphoid stem cells and plasma cells. On the basis of indirect evidence, elimination of the antigen-presenting capabilities and antigen nonspecific immune functions of B cells appear to be central to the therapeutic efficacy of anti-CD20 B-cell therapies. An important question is this—Why does the drug need to be dosed at fixed intervals and not based on a measurable endpoint, such as tracking peripheral CD20 cell counts? There is minimal scientific validity in infusing the drug every 6 months particularly if CD20 cell counts are negligible in the peripheral blood. In this analysis, a case is made for following CD19 cell populations as a surrogate for CD20 cells on a monthly basis to guide OCR redosing parameters and does not follow a scheduled dosing parameter.

Confocal biomicroscopic changes of the corneal layers following femtosecond laser-assisted MyoRing implantation in keratoconus

PurposeTo evaluate the effect of the femtosecond laser-assisted MyoRing implantation on the confocal biomicroscopic findings in different corneal layers of the patients with keratoconus. MethodsTwelve eyes of 12 patients with mild to moderate keratoconus (keratometry between 48 and 52 diopters) and intolerance to hard contact lens entered the study. All the included patients underwent femtosecond laser-assisted MyoRing (Dioptex GmBH, Linz, Austria) implantation. The confocal biomicroscopy of the cornea was performed for all corneal layers in the center and periphery preoperatively and 3 and 6 months postoperatively. The cell counts and the qualitative findings in each layer of the cornea were compared between preoperative and 3 and 6 months postoperative images. ResultsCompared with preoperative values, the central epithelial and the central and peripheral midstromal cell counts were significantly decreased 6 months after MyoRing implantation (P = 0.015, P = 0.010 and 0.005, respectively). Furthermore, compared with preoperative values, the peripheral posterior stromal cell count was significantly decreased 3 months after MyoRing implantation (P = 0.033). In the qualitative analysis, highly reflective nuclei in the basal epithelium, transient disruption in the subepithelial nerve plexus, increase in the reflectivity of the stromal keratocyte, and normal endothelial cell morphology were seen. ConclusionsOur study demonstrated some findings similar to that reported in intrastromal corneal ring segments (ICRS): decreased central epithelial cell counts, highly reflective nuclei in the basal epithelium, transient disruption in the subepithelial nerve plexus, and normal endothelial cell count and morphology. In addition, a decrease in the central and peripheral midstromal, transient decrease in posterior stromal cell counts, and absence of amorphous depositions were in contrast with the findings reported in ICRS.

Successful Treatment of a Diamond-Blackfan Anemia (DBA) Patient with Eltrombopag

▪DBA is a rare bone marrow failure syndrome clinically defined by onset of hypoproliferative anemia in early childhood accompanied by a grossly normocellular bone marrow. With age, a significant fraction of patients progress to pancytopenia and a hypocellular bone marrow, with some increased risk of progression to leukemia. Autosomal dominant or sporadic heterozygous mutations in genes encoding for ribosomal subunit proteins have been etiologically linked to DBA. How loss of function mutations in ribosomal proteins results in selective loss of erythroid progenitors with variable severity is incompletely understood. Chronic transfusion therapy and corticosteroid treatment can alleviate anemia, but both cause severe long term toxicities. Hematopoietic stem cell transplantation, when available, is the only definitive treatment modality for the hematological manifestations, and the only known treatment for DBA patients once pancytopenia develops.Here we report on a 28 year old female patient diagnosed with DBA at age of one month based on clinical criteria. Recent mutational analysis identified a novel mutation in intron 4(c.356+3>C) of the RPS19 gene affecting a known splice donor site that has previously reported in DBA patients. She did not respond to corticosteroids and was maintained on transfusions. She failed various experimental therapies including danazol, hematopoietic growth factors including IL-3 and GM-CSF, and anti-thymocyte globulin/cyclosporine without sustained clinical response. After treatment with daclizumab in 2006 her transfusion requirements decreased for about 4 years. In 2012 she presented with progressive pancytopenia and hypocellular bone marrow without overt dysplasia, and normal cytogenetic analysis. She required red cell transfusions. The patient was enrolled in a clinical research protocol investigating the efficiency and safety of the thrombopoietin receptor agonist eltrombopag (EPAG) in patients with moderate aplastic anemia or those with bone marrow failure with unilineage cytopenia (ClinicalTrials.gov: NCT01328587). The study is designed as a non-randomized, phase II, dose modification study with the primary endpoint hematological response at 16 weeks. EPAG was administered at 50mg daily and escalated every 2 weeks by 25 mg to the maximal dose of 300mg daily. At response assessment the patient’s hemoglobin (Hgb) improved from 7.0g/dl to 8.9g/dl and platelets from 112K/ul to 171k/ul. She required no further transfusions, and was deemed a responder. EPAG was subsequently continued at the same dose level for an additional 6 months when the peripheral cells counts peaked at Hgb 11.9g/dl, platelets 251K/ul, ANC 3.19 K/ul, and EPAG was discontinued for protocol-defined robust response. After discontinuation of EPAG the patient’s hemoglobin steadily declined over a period of 8 months. EPAG was reinitiated at 300mg per protocol. Peripheral blood cell counts rapidly improved and the EPAG dose was slowly tapered. The patient is currently on EPAG 50mg daily with normal blood cell counts. We have not observed any EPAG related toxicities in our patient, and repeated cytogenetic studies on bone marrow documented a normal karyotype.To the best of our knowledge this is the first report documenting EPAG efficacy in a DBA patient. Trautman et al. (2012) previously reported a DBA patient that failed treatment with low dose EPAG after six months. Our observation warrants further systematic investigation of EPAG treatment for DBA patients within a clinical research study setting. [Display omitted] DisclosuresWinkler:GSK/Novartis: Research Funding. Townsley:Novartis: Research Funding. Desmond:Novartis: Honoraria. Dumitriu:GSK/Novartis: Research Funding. Grasmeder:GSK/Novartis: Research Funding. Young:GSK/Novartis: Research Funding. Dunbar:GSK/Novartis: Research Funding.

Single-Agent MOR208 in Relapsed or Refractory (R-R) Non-Hodgkin's Lymphoma (NHL): Results from Diffuse Large B-Cell Lymphoma (DLBCL) and Indolent NHL Subgroups of a Phase IIa Study

Single-Agent MOR208 in Relapsed or Refractory (R-R) Non-Hodgkin's Lymphoma (NHL): Results from Diffuse Large B-Cell Lymphoma (DLBCL) and Indolent NHL Subgroups of a Phase IIa Study

Overexpression of KDM6B, an Epigenetic and Innate Immune Regulator, Results in Hematopoietic Alterations of Mice Including Changes in Hematopoietic Stem Cells

Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML)aremyeloid neoplasms characterized by abnormal bone marrow hematopoiesis and increased risk of transformation to acute myelogenous leukemia (AML). Epigenetic dysregulation and inflammatory hyper-activation have been recognized as key molecular abnormalities in the bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) of MDS and CMML, which implies that key modulators of epigenetic and inflammatory regulation play an important role in the pathophysiology of these diseases, which could also serve as effective therapeutic targets. We recently identified such a candidate molecule: the histone demethylase KDM6B (JMJD3). We demonstrated that KDM6B is significantly overexpressed in the BM HSPCs of patients with MDS and CMML, and the overexpression of KDM6B mediates aberrant epigenetic activation of innate immune/inflammatory signals and consequent differentiation skewing in BM HSPCs of MDS, which can be reversed by targeting KDM6B. Importantly, systematic analysis of the global transcriptomic and genomic data of patients indicates that, although KDM6B is overexpressed in MDS and CMML, genetic lesions in this gene are very rare, and higher KDM6B expression correlates with TET2 mutation. These results imply that constitutive expression of KDM6B potentially interacts with more common genetic lesions during the development of MDS and CMML.To further investigate the effects of KDM6B overexpression on hematopoiesis and its role in myeloid disorders, we developed a novel hematopoietic KDM6B transgenic (Tg) mouse model that overexpresses KDM6B under the control of the murine hematopoietic specific Vav promoter (Vav-KDM6B). Long-term monitoring of the peripheral blood counts of the mice indicates that, although younger Vav-KDM6B mice display only minor changes in whole white blood cells (WBC), monocytes, and platelets, aged KDM6B mice (>1 year old) have significant increases of WBC (by 22%, p<0.05) and monocyte counts (by 34%, p<0.01). In BM biopsies, the megakaryocytic progenitors of Vav-KDM6B mice specifically possess dysplastic morphology. Analysis of BM HSPCs revealed a tendency of increased numbers of long-term hematopoietic stem cells (LT-HSC) in Vav-KDM6B mice. Because MDS and CMML are associated with a pro-inflammatory BM microenvironment, we applied low-dose lipopolysaccharide (LPS) treatment (6 µg/mouse) for 6 weeks to both Vav-KDM6B and control mice. After chronic immune stimulation, more significant decreases of peripheral red cell (RBC) count and hemoglobin were observed in LPS-treated Vav-KDM6B mice compared to others. In BM biopsies, we noted more dramatic increases of megakaryoblasts in LPS-treated Vav-KDM6B mice than treated wild-type mice. Increases of BM HSPCs were also detected in LPS-treated Vav-KDM6B mice, including LSK cells and LT-HSC populations. Furthermore, the LSK cells isolated from LPS-treated Vav-KDM6B mice consistently demonstrated increased serial plating capacity in methocult-supported colony formation assays. Taken together, these hematopoietic phenotypes observed in the LPS-treated Vav-KDM6B Tg mice indicate that KDM6B overexpression in combination with pro-inflammatory stimulation can accelerate the occurrence of the MDS- and CMML-like abnormalities in BM HSPCs. To gain more insight into the cellular and molecular impacts of KDM6B overexpression on BM HSPCs, detailed analysis, including in vivo repopulating capacity assays as well as gene expression profiling, are being performed. DisclosuresNo relevant conflicts of interest to declare.

Sustained Natural Killer Cell Recovery Post-Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation Predicts Survival in Non-Hodgkin's Lymphoma

AbstractOur group recently reported a Phase III clinical trial (Porrata et al. Biology of Blood and Marrow Transplantation 2016; 22: 1017-1023) demonstrating that the infusion of the autograft-absolute lymphocyte count (A-ALC) and autograft natural killer cells (A-NKC) are survival prognostic factors in non-Hodgkin lymphoma (NHL) patients treated with autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). A limitation of our Phase III clinical trial was the short-term follow-up of just two years. Therefore, we evaluated longer follow-up and assessed the role of the infused A-ALC, infused A-NKC, and peripheral blood absolute natural killer cell count (ANKC) recovery on survival post-APBHSCT. Of the 122 patients that participated in the Phase III trial, 111 patients were able to complete APBHSCT and included in the current study. With a median follow-up of 57.2 months (range: 2.1-84.6 months), superior overall survival (OS) and progression-free survival (PFS) was observed in patients infused with an A-ALC ≥ 0.5 x 109 lymphocytes/kg [OS: HR = 0.429, 95%CI, 0.128-0.828, p < 0.01; and PFS: HR = 0.456, 95% CI, 0.177-0.863, p < 0.01] and an A-NKC ≥ 0.09 x 109 cells/kg [OS: HR = 1.58e-3, 95% CI, 1.046e-6-0.179, p < 0.003; and PFS: HR= 1.58e-3, 95% CI, 7.81e-7-0.064, p < 0.003]. Furthermore, patients that maintained an ANKC ≥ 250 cells/µl at 3 months, 6 months, 9 months and 12 months post-APBHSCT also experienced superior OS (Figure 1A) and PFS (Figure 1B)[OS and PFS times in months were evaluated from infusion date of stem cells]. In the multivariate analysis a sustained ANKC ≥ 250 cells/µl was an independent predictor for OS (HR = 0.013, 95% CI, 0.001-0.063, p < 0.0001) and PFS (HR = 0.014, 95% CI, 0.001-0.067, p < 0.0001). This study is an extension of our Phase III clinical trial showing that sustained innate immunity by measuring the ANKC correlated with superior clinical outcomes; thus providing a platform to develop innate immunity immunotherapeutic strategists directing to improve clinical outcomes post-APBHSCT in NHL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacological Inhibition of Lck is Able to Revert Glucocorticoid Resistance in Pediatric T-ALL

The AIEOP-BFM group has traditionally used the peripheral blood blast cells count after a 7-day glucocorticoids (GC) prephase to classify patients as Prednisone Good Responders (PGR) or Prednisone Poor Responders (PPR). As described by Schrappe M. in 2011, PPR patients tend to have a worse prognosis, despite the fact that all of them are assigned to the High Risk protocol. Little is known about the molecular mechanisms that lead to GC resistance, guiding our research to the identification of new specific molecular targets in order to develop new approaches to improve therapy efficacy in these patients.To this end, we performed a Reverse Phase Protein Analysis (RPPA) of 54 PGR and 33 PPR pediatric T-ALL patients at diagnosis, and studied the activation or expression of 87 proteins involved in key cellular signaling pathways. Interestingly, we found a higher expression of LCK phosphorylated at Y505 (inhibited form) in PGR patients (p=0.001), together with a higher phosphorylation of SRC Y416 (active form) in PPR patients (p=0.01). Total LCK and LCK RNA expression were not differentially expressed in the two subgroups of patients, suggesting an increased activation of LCK in PPR patients. Indeed, in agreement with these results, also LCK downstream target PLCγ, phosphorylated at Y783, resulted hyperactivated in PPR compared to PGR patients (p=0.05), confirmed also by a positive correlation between PLCγ Y783 and SRC Y416 (r=0.51, p=0.01). Taken together, these results indicate a hyperactivation of the LCK pathway in PPR patients compared to PGR ones. LCK is part of the TCR multiprotein complex together with the GC receptor. In normal T lymphocytes, after GC treatment the complex is disrupted, LCK activation is decreased and downstream prosurvival signaling inhibited, thus leading to cell death. In this light, in GC resistant patients hyperactivated LCK might sustain cell survival regardless of GC activity.We then tested if FDA-approved or recently developed LCK inhibitors would revert GC resistance in T-ALL cells. GC resistant cell lines ALL-SIL, T-ALL1 and CEM were treated with Dasatinib, Bosutinib, Nintedanib and WH-4-023 alone or in combination with Dexamethasone (Dex). All four inhibitors alone are able to decrease cell survival, and all of them strongly synergize with Dex, bringing to the sensitization of these cells to GC treatment. We also tested these compounds alone or in combination with Dex in 4 PPR T-ALL patients cells derived from xenograft mice. Also in these cases we observed an enhanced sensitization of cells to GC treatment. Finally, corroborating the crucial role of LCK in GC resistance, we observed a strong decrease in cell viability after specific LCK gene silencing and Dex treatment in ALL-SIL cells, together with an increased GC resistance following LCK hyperactivation in P12-ICHIKAWA GC sensitive cells.Thus, our results strongly suggest that the inhibition of LCK using clinically approved drugs could represent a promising new additional therapeutic strategy to revert drug resistance in high-risk pediatric T-ALL patients. DisclosuresIndraccolo:OncoMed Pharmaceuticals, Inc.: Research Funding.

Effect of A-769662, a direct AMPK activator, on Tlr-4 expression and activity in mice heart tissue.

Objective(s)::TLR-4 activates a number of inflammatory signaling pathways. Also, AMPK could be involved in anti-inflammatory signaling. The aim of this study was to identify whether stimulation of AMPK could inhibit LPS-induced Tlr-4 gene expression in mice hearts.Materials and methods:Heart AMPK activity and/or Tlr-4 expression was stimulated in different mice groups, using respectively IP injection of A-769662 (10 mg/kg) and LPS (2 mg/kg) or a combination of both agents. Moreover, compound-C (20 mg/kg), as an AMPK antagonist, was intraperitoneally co-administrated with both A-769662 and LPS in another group to investigate the role of AMPK activity on Tlr-4 regulation. After 8 hr, in addition to peripheral neutrophil cell count, myocardial p-AMPK, p-ACC as well as MyD88 protein contents and Tlr-4 expression was assessed by Western blotting and real-time qRT-PCR, respectively. TNF-α and IL-6 expression levels were also determined by ELISA.Results:LPS induced heart Tlr-4 expression (P<0.001) associating with an increase in the myocardial MyD88 protein content (P<0.001), elevation of heart TNF-α (P<0.01) and IL-6 (P<0.05) concentrations, and rise in the peripheral neutrophil cell count (P<0.001). Administration of A-769662 decreased LPS-induced Tlr-4 expression (P<0.01) and alleviated peripheral neutrophil cell count (P<0.01). The inhibitory effect of A-769662 on LPS-induced Tlr-4 expression was reversed by antagonizing AMPK with compound-C (P<0.001) which reduced p-AMPK (P<0.05) and p-ACC (P<0.01) myocardial protein contents in the LPS+A-769662 group.Conclusion:This study demonstrated that activation of AMPK, by A-769662 agent, could inhibit Tlr-4 expression and activity, suggesting a link between AMPK and Tlr-4 in heart tissue.

Algorithms utilizing peripheral blood hematopoietic progenitor cell counts in lieu of some CD34+ cell counts predict successful peripheral blood stem cell collections with substantial time and cost savings.

Hematopoietic progenitor cell (HPC) counts from Sysmex hematology analyzers have been shown to correlate with peripheral blood (PB) CD34+ cell counts by flow cytometry. Algorithms utilizing HPC counts to guide stem cell collections have been proposed but rarely tested. This study describes the development and validation of algorithms utilizing HPC and PB CD34+cell counts to predict adequate peripheral blood stem cell (PBSC) collections for chemomobilized and cytokine-mobilized individuals. Utilizing a test set of 83 PB samples from chemomobilized or cytokine-mobilized PBSC collection patients, PB CD34+ counts were correlated with HPC counts and a receiver operating characteristic curve was constructed. Cut-offs of ≤0.5 HPC/μl and ≥7 HPC/μl were established to maximize sensitivity and specificity for using HPC to predict PB CD34+ ≥ 10 cells/μl. These cut-offs were subsequently validated using a separate prospective validation set of 88 HPC/CD34+ cell sample pairs. Using the algorithms, all patients in the prospective validation data set achieved adequate collections of ≥1 × 106 CD34+ cells/kg, and a 67% reduction in the number of CD34+ cell counts performed was achieved. This lead to a direct cost savings of at least $18,700 USD over a 21-month period (88% reduction in direct costs). Use of the algorithms provides significant time and cost savings for the laboratory while accurately predicting (i) timing of PBSC collections to obtain adequate CD34+ product yields for chemomobilized patients and (ii) when to administer plerixafor to cytokine-mobilized patients to improve the likelihood of achieving adequate collections.

Is it feasible to use granulocyte-colony stimulating factor alone to mobilize progenitor cells in multiple myeloma patients induced with a cyclophosphamide, thalidomide and dexamethasone regimen?

BackgroundCyclophosphamide plus thalidomide as induction for multiple myeloma patients eligible for autologous stem cell transplantation may be a limiting factor for cell mobilization. The minimum acceptable mobilized peripheral blood stem cell count to prevent deleterious effects during transplantation is 2.0 × 106 CD34+ cells/kg. Combining other treatments to granulocyte-colony stimulating factor, such as cyclophosphamide, could overcome the mobilization limitation. The objective of this study was to assess the number of CD34+ cells mobilized using granulocyte-colony stimulating factor with and without cyclophosphamide after induction with cyclophosphamide, thalidomide and dexamethasone.MethodsA retrospective study was performed of a cohort of multiple myeloma patients submitted to autologous stem cell transplantations at two Brazilian centers between May 2009 and July 2013. The oral cyclophosphamide and thalidomide induction doses used were 1500 mg/month and 100–200 mg/day, respectively. Mobilization doses were 10–15 mcg/kg granulocyte-colony stimulating factor with 2–4 g/m2 cyclophosphamide, or 15–20 mcg/kg granulocyte-colony stimulating factor alone for 5 days. Collection of >2.0 × 106 CD34+ cells/kg was considered sufficient.ResultsEighty-eight patients were analyzed; only 18 received cyclophosphamide. The median age was 58 years old (range: 51–62) for the granulocyte-colony stimulating factor group and 56.5 years old (range: 54–60) for granulocyte-colony stimulating factor plus cyclophosphamide group. Fifty-two patients were male. Eighty cases (90.9%) were Durie-Salmon Staging System III-A/B and 38 (44.7%) and 20 cases (23.5%) were International Staging System 2 and 3, respectively. The group that received cyclophosphamide collected a higher median number of progenitor cells [3.8 (range: 3.1–4.4) vs. 3.2 (range: 2.3–3.8)] (p-value = 0.008). No correlation was observed between better responses or number of induction cycles and the number of cells collected.ConclusionThe number of cells mobilized with granulocyte-colony stimulating factor plus cyclophosphamide was higher. However, in both groups, the median number of CD34+ cells was sufficient to perform a single autologous stem cell transplantation; no deleterious effects were reported during harvesting.

Cryptococcal Antigen Testing in Newly Diagnosed HIV-Positive Patients

Detection of cryptococcal antigen (CrAg) in serum or plasma is a sensitive predictor of cryptococcal meningitis (CM). Retrospective observations suggest that routine CrAg screening and preemptive antifungal therapy for those who are positive could prevent subsequent CM. To examine the effect of CrAg screening prospectively, 645 antiretroviral therapy (ART)-naive, HIV-infected patients with peripheral blood CD4 cell counts ≤100/µL in two South African clinics were screened for CrAg, first by serum latex agglutination (LA) …

T-cell large granular lymphocyte proliferation in myelodysplastic syndromes: Clinicopathological features and prognostic significance

Inflammatory and immune dysregulation are crucial in the initiation and development of myelodysplastic syndromes (MDS). It is noted that clonal T-cell large granular lymphocyte (T-LGL) proliferation associated with MDS is not uncommon. However, clinicopathological features, and prognostic and predictive value of presence of T-LGL proliferation in MDS patients is not very clear. This study compared 35 MDS patients with T-LGL proliferation with 36 MDS patients without T-LGL proliferation and summarized clinicopathologic features, including peripheral blood LGL cell counts, immunophenotype, T cell receptor gene rearrangement, bone marrow hematopoietic status, and adjuvant immunosuppressive therapy. The peripheral blood CD3+/CD57+ cell counts were significantly different (p<0.01) between the two groups. Notably, on examination of the bone marrow, MDS patients with T-LGL proliferation showed more frequent hypocellularity and/or lineage hypoplasia, particularly erythroid hypoplasia. On survival analysis, no overall difference was noted between MDS patients with T-LGL proliferation and those without T-LGL proliferation, and between the patients who received therapy for LGL and those who did not receive adjuvant therapy for LGL in the same risk group. In conclusion, T-LGL proliferation present in MDS patients can be associated with bone marrow hypocellularity and lineage hypoplasia. Although immunosuppressive therapy to eliminate T-LGL cells is potentially beneficial to the MDS patients with associated T-LGL proliferation, there is no overall survival benefit to the patients who received such treatment.

Peripheral Immune Cell Counts and Advanced Imaging as Biomarkers of Stroke Outcome

Introduction: Circulating neutrophil and lymphocyte levels may be modifiable outcome predictors of ischemic stroke. We sought to compare these immune cell parameters with advanced imaging assessment and the 90-day clinical outcome. Methods: We used a retrospectively collected cohort of consecutive ischemic stroke patients presenting within 4.5 h of symptom onset who had acute CT perfusion and routine blood collection before treatment with intravenous thrombolysis and 24-hour MRI scanning at the John Hunter Hospital. Full blood counts were performed acutely at 24 h and 7 days. Patient outcomes were assed at 90 days after stroke with the modified Rankin Scale (mRS). Results: Overall, 142 patients were assessed during the study period. Patients with a poor outcome (mRS 3-6) had increased neutrophils (44% increase, p = 0.016), decreased lymphocytes (7% decrease, p = 0.491) and an increased lymphocyte-to-neutrophil ratio (196% increase, p < 0.001). Patients with good outcomes (mRS 0-2) did not have significant changes in their full blood counts. There was no relationship between the neutrophil count at 24 h and penumbral volume (r² = 0.217, p = 0.212), reperfusion (r² = 0.111, p = 0.085), or core growth (r² = 0.297, p = 0.107). A backward multivariate analysis containing the 24-hour core volume and 24-hour neutrophil count was strongly related to the 3-month outcome (r² = 0.477, area under the curve = 0.902, p < 0.001). Conclusions: Peripheral neutrophils have potential as a biomarker of outcome when used in conjunction with advanced imaging. Peripherally measured neutrophil counts change significantly over time after stroke and may be potential targets for immunomodulatory therapy in patients with a severe stroke or a large infarct volume.

Correlation of the lung microbiota with metabolic profiles in bronchoalveolar lavage fluid in HIV infection.

BackgroundWhile 16S ribosomal RNA (rRNA) sequencing has been used to characterize the lung’s bacterial microbiota in human immunodeficiency virus (HIV)-infected individuals, taxonomic studies provide limited information on bacterial function and impact on the host. Metabolic profiles can provide functional information on host-microbe interactions in the lungs. We investigated the relationship between the respiratory microbiota and metabolic profiles in the bronchoalveolar lavage fluid of HIV-infected and HIV-uninfected outpatients.ResultsTargeted sequencing of the 16S rRNA gene was used to analyze the bacterial community structure and liquid chromatography-high-resolution mass spectrometry was used to detect features in bronchoalveolar lavage fluid. Global integration of all metabolic features with microbial species was done using sparse partial least squares regression. Thirty-nine HIV-infected subjects and 20 HIV-uninfected controls without acute respiratory symptoms were enrolled. Twelve mass-to-charge ratio (m/z) features from C18 analysis were significantly different between HIV-infected individuals and controls (false discovery rate (FDR) = 0.2); another 79 features were identified by network analysis. Further metabolite analysis demonstrated that four features were significantly overrepresented in the bronchoalveolar lavage (BAL) fluid of HIV-infected individuals compared to HIV-uninfected, including cystine, two complex carbohydrates, and 3,5-dibromo-l-tyrosine. There were 231 m/z features significantly associated with peripheral blood CD4 cell counts identified using sparse partial least squares regression (sPLS) at a variable importance on projection (VIP) threshold of 2. Twenty-five percent of these 91 m/z features were associated with various microbial species. Bacteria from families Caulobacteraceae, Staphylococcaceae, Nocardioidaceae, and genus Streptococcus were associated with the greatest number of features. Glycerophospholipid and lineolate pathways correlated with these bacteria.ConclusionsIn bronchoalveolar lavage fluid, specific metabolic profiles correlated with bacterial organisms known to play a role in the pathogenesis of pneumonia in HIV-infected individuals. These findings suggest that microbial communities and their interactions with the host may have functional metabolic impact in the lung.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0147-4) contains supplementary material, which is available to authorized users.

Up-Regulation of CAR-T Cells after G-CSF and Dexamethasone Treatment in a Defuse Large B Cell Lymphoma Patient Receiving 4SCAR19 T Cell Therapy

A 37-year-old woman was diagnosed with DLBCL, GCB origin, positive with CD20, Bcl-2, Bcl-6, CD10 and negative with CD3, CD5, ,D21, CD23, CyclinD1. PET-CT scan showed that her lymph nodes were broadly involved. Based on PET-CT, the stage was IIIA and aaIPI score was 1. After eight cycles of R-CHOP (rituximab, cyclophosphamide, epirubicin, vincristine, and prednisone), the tumor regressed to a small retroperitoneum lesion with SUV 2.0, and she received another four cycles of rituximab for maintenance. The tumor relapsed one year later with resistance to ESHAP after she had failed multiple alternative treatments including GA101 trial, 3x DICE, MTX1g, 2x GEMOX, and 2x EPOCH. She was enrolled in a CD19-CAR (chimeric antigen receptor) T cell pilot study in January 2015. Her T cells were apheresis collected and transduced with a 4th generation, apoptosis-inducible, safety-engineered lentiviral CAR: CD19- scFv/CD28/CD137/CD27/CD3ζ-iCasp9 (4SCAR19). A personalized conditioning regimen was given based on the patient's history to chemotherapy: cyclophosphamide 500mg/d d1-3, and fludarabine 50mg/d d1-4, 40mg d5. Two days later, she received infusions of a total dose of 1.27×108 of the 4SCAR19 T cells (2x106/kg). At day 7 (D7) after infusion, she developed a fever over 39o C, which lasted for 7 days; this was controlled with NSAIDs (Non-Steroidal Anti-inflammatory Drugs). The tumor in her lymph nodes began to shrink 5 days after CAR-T infusion and she achieved nCR (near complete response) after 30 days. In a follow up PET/CT 3 months after CAR-T infusion, there was only one suspected retroperitoneum lesion (SUV 3.8) in the whole body, but it was indiscernable whether the SUV signal was tumor or T cell related. We monitored the peripheral blood CAR-T cell counts by qPCR and detected 0.03%, 0.07%, 0.3%, 9.3%, 0.01%, 1.57% on D7, D13, D48, D69, D84 and D112, respectively. In attempt to perform an autotransplantation to pursue a cure, she was mobilized twice with G-CSF on D68-D72, D110-112, plus dexamethasone 20mg/d on D101-102 and 15 mg/d on D110-111. Unexpectedly, the CAR-T cells in the patient peripheral blood increased to 9% after the first mobilization, and to 18% after the second mobilization. To this date, the patient has remained in nCR after 4SCAR19 therapy (January 23 to July 23, 2015). To our knowledge, this is the first report of evident CAR-T cell boost associated with G-CSF plus dexamethasone treatment. Further investigation is warranted to understand the molecular mechanisms behind such a favorable CAR-T therapy outcome in a terminal DLBCL disease. DisclosuresKuo:America Yuva Biomed: Employment. Liu:America Yuva Biomed: Employment. Dong:America Yuva Biomed: Consultancy.