Reduction In Neutrophil Count Research Articles

COVID-19 infection causes a reduction in neutrophil counts in patients taking clozapine.

BackgroundMonitoring of white cell counts during clozapine treatment leads to cessation of therapy if levels fall below pre-determined values. Reductions in white cell counts, driven by lower levels of lymphocytes, have been observed with coronavirus disease 2019 (COVID-19). Neutropenia during COVID-19 has not been reported. We present data for 56 patients who were taking clozapine and had COVID-19.MethodsWe included patients who were taking clozapine at the time they tested positive for COVID-19. We compared absolute neutrophil counts, lymphocyte counts and white cell counts between baseline and the first week of infection, and baseline and the second week of infection.ResultsWe observed reductions in absolute neutrophil counts (p = 0.005), lymphocyte counts (p = 0.003) and white cell counts (p < 0.001) between baseline and the first 7 days of COVID-19. All cell counts had returned to baseline levels by days 8 to 14. Six patients experienced neutropenia (absolute neutrophil counts < 2.0 × 109/L) and of those, 4 underwent mandatory cessation of clozapine. For 3 patients, clozapine treatment had been established for more than 6 months with no previous neutropenia, neutrophil levels returned to baseline within 2 weeks and no further neutropenia was observed on restarting treatment.LimitationsThis was a retrospective chart review; larger cohorts are required. Clozapine plasma levels were largely not measured by clinicians.ConclusionThese data strongly suggest that mild neutropenia in the acute phase of COVID-19 in patients who are well established on clozapine is more likely to be a consequence of the virus than of clozapine treatment.

Short-term outcomes of preoperative chemoradiotherapy with S-1 for locally advanced rectal cancer.

The purpose of the present study was to evaluate the short-term results of preoperative chemoradiation therapy with S-1 for locally advanced rectal cancer. A total of 32 patients with advanced rectal cancer who had been treated with preoperative chemoradiotherapy with S-1 and underwent surgical resection between May 2012 and December 2019 were analyzed. Advanced rectal cancer of clinical stage II and III was diagnosed in 13 (41%) and 19 (59%) patients, respectively. Therapeutic toxicities of anemia (24 patients; 75%), anal pain (22 patients; 69%) and skin and subcutaneous tissue disorders (19 patients; 59%) were frequently observed in all grades. Grade ≥3 leukopenia, anemia, neutrophil count reduction, platelet count reduction and diarrhea were identified in 2 (6%), 1 (3%), 1 (3%), 1 (3%) and 1 (3%) patients, respectively. A total of 29 patients (91%) completed this therapy without any change to the protocol or dosage. R0 resection was performed in 100% of the patients, and no postoperative mortality was observed. Pathological complete response was observed in 9 cases (28.1%). This therapy can be considered for cases of locally advanced rectal cancer due to its acceptable toxicity and relatively high antitumor effect.

Neutrophil-to-Lymphocyte Ratio: A Biomarker to Monitor the Immune Status of Astronauts.

A comprehensive understanding of spaceflight factors involved in immune dysfunction and the evaluation of biomarkers to assess in-flight astronaut health are essential goals for NASA. An elevated neutrophil-to-lymphocyte ratio (NLR) is a potential biomarker candidate, as leukocyte differentials are altered during spaceflight. In the reduced gravity environment of space, rodents and astronauts displayed elevated NLR and granulocyte-to-lymphocyte ratios (GLR), respectively. To simulate microgravity using two well-established ground-based models, we cultured human whole blood-leukocytes in high-aspect rotating wall vessels (HARV-RWV) and used hindlimb unloaded (HU) mice. Both HARV-RWV simulation of leukocytes and HU-exposed mice showed elevated NLR profiles comparable to spaceflight exposed samples. To assess mechanisms involved, we found the simulated microgravity HARV-RWV model resulted in an imbalance of redox processes and activation of myeloperoxidase-producing inflammatory neutrophils, while antioxidant treatment reversed these effects. In the simulated microgravity HU model, mitochondrial catalase-transgenic mice that have reduced oxidative stress responses showed reduced neutrophil counts, NLR, and a dampened release of selective inflammatory cytokines compared to wildtype HU mice, suggesting simulated microgravity induced oxidative stress responses that triggered inflammation. In brief, both spaceflight and simulated microgravity models caused elevated NLR, indicating this as a potential biomarker for future in-flight immune health monitoring.

Bioinspired DNase-I-Coated Melanin-Like Nanospheres for Modulation of Infection-Associated NETosis Dysregulation.

The current outbreak of the beta‐coronavirus (beta‐Cov) severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) began in December 2019. No specific antiviral treatments or vaccines are currently available. A recent study has reported that coronavirus disease 2019 (COVID‐19), the disease caused by SARS‐CoV‐2 infection, is associated with neutrophil‐specific plasma membrane rupture, and release excessive neutrophil extracellular traps (NETs) and extracellular DNAs (eDNAs). This mechanism involves the activation of NETosis, a neutrophil‐specific programmed cell death, which is believed to play a crucial role in COVID‐19 pathogenesis. Further progression of the disease can cause uncontrolled inflammation, leading to the initiation of cytokine storms, acute respiratory distress syndrome (ARDS), and sepsis. Herein, it is reported that DNase‐I‐coated melanin‐like nanospheres (DNase‐I pMNSs) mitigate sepsis‐associated NETosis dysregulation, thereby preventing further progression of the disease. Recombinant DNase‐I and poly(ethylene glycol) (PEG) are used as coatings to promote the lengthy circulation and dissolution of NET structure. The data indicate that the application of bioinspired DNase‐I pMNSs reduce neutrophil counts and NETosis‐related factors in the plasma of SARS‐CoV‐2 sepsis patients, alleviates systemic inflammation, and attenuates mortality in a septic mouse model. Altogether, the findings suggest that these nanoparticles have potential applications in the treatment of SARS‐CoV‐2‐related illnesses and other beta‐CoV‐related diseases.

The Effect of Antenatal Steroid Therapy on Early Inflammation Markers and Leukocyte Counts in Premature Infants

Objective We investigated the effect of antenatal steroid therapy(AST) on white blood cell (WBC) and neutrophil counts and the inflammatory markers C-reactive protein(CRP), interleukin 6(IL-6), interleukin 10(IL-10), and beta-2 microglobulin(ß2M) in preterm infants. Materials Method Neonates born at ≤34 weeks of gestation and admitted at hospital between May and November 2018 were included. The neonates were divided into three groups based on AST dose administered: 24 mg betamethasone (full course), 12 mg betamethasone (incomplete course), and no AST. 170 infants were analyzed. Results Of these, 45.2% (n = 77) received a full course of AST, 38.8% (n = 66) received an incomplete course of AST, and 15.8% (n = 27) did not receive AST. WBC, CRP, IL-6, IL-10, and ß2M levels were similar between the three groups, whereas neutrophil count was significantly lower in full course AST group. Conclusion Consistent with the literature data, AST was associated with reduced neutrophil count but did not affect the other inflammatory markers studied.

Activation of GPR35 protects against cerebral ischemia by recruiting monocyte-derived macrophages

Pamoic acid is a potent ligand for G protein Coupled Receptor 35 (GPR35) and exhibits antinociceptive property. GPR35 activation leads to increased energy utilization and the expression of anti-inflammatory genes. However, its role in brain disorders, especially in stroke, remains unexplored. Here we show in a mouse model of stroke that GPR35 activation by pamoic acid is neuroprotective. Pharmacological inhibition of GPR35 reveals that pamoic acid reduces infarcts size in a GPR35 dependent manner. The flowcytometric analysis shows the expression of GPR35 on the infiltrating monocytes/macrophages and neutrophils in the ischemic brain. Pamoic acid treatment results in a preferential increment of noninflammatory Ly-6CLo monocytes/macrophages in the ischemic brain along with the reduced neutrophil counts. The neuroprotective effect of GPR35 activation depends on protein kinase B (Akt) and p38 MAPK. Together we conclude that GPR35 activation by pamoic acid reprograms Ly-6CLo monocytes/macrophages to relay a neuroprotective signal into the ischemic brain.

Phase Ib trial to evaluate safety and anti-tumor activity of the AKT inhibitor, ipatasertib, in combination with endocrine therapy and a CDK4/6 inhibitor for patients with hormone receptor positive (HR+)/HER2 negative metastatic breast cancer (MBC) (TAKTIC).

1066 Background: The cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), with an anti-estrogen, are the standard of care for HR+/HER2- MBC. Insights from patient biopsies and preclinical analysis suggest that AKT1 activation can provoke CDK4/6i resistance. We hypothesized that targeting AKT1 following CDK4/6i progression may provide clinical benefit. Methods: TAKTIC is an open-label phase Ib trial exploring the combination of the AKT1 inhibitor, ipatasertib (ipat), with an aromatase inhibitor (Arm A), fulvestrant (Arm B), or the triplet combination (Arm C) of fulvestrant + ipat + palbociclib (palbo). The primary objective is to evaluate the safety and tolerability of ipat in combination with endocrine therapy +/- CDK4/6i. Key inclusion criteria include unresectable HR+/HER2- MBC; at least 1 prior therapy for MBC including any CDK4/6i; up to 2 prior lines of chemotherapy for MBC (no limit on prior endocrine therapy). Here, we present an interim analysis from the triplet combination (Arm C). Results: As of 1/31/2020, 25 pts have enrolled, including 12 on Arm C, all of whom received prior CDK4/6i (median no of prior lines = 5.5, range 2-7). Along with fulvestrant, 3 pts received ipat at 200mg + 125mg palbo, 7 pts received 300mg + 125mg palbo, and 2 pts received 400mg + 100mg palbo. To date, 8/12 pts remain on treatment including 2 with partial response, 3 with stable disease, 3 with restaging studies pending and 4 with progressive disease. The triplet combination was well tolerated. Grade 3 toxicities included reduced WBC (8/12), reduced neutrophil count (11/12), reduced lymphocyte count (2/12) and single instances of transaminitis, rash, and reduced platelet count. The only grade 4 toxicity was reduced neutrophil count (4/12). There were no DLTs observed and no discontinuations due to toxicity. Mean steady state pharmacokinetic parameters for ipat were similar to historical data from single agent trials suggesting that combined treatment with palbo + fulvestrant did not affect the pharmacokinetics of ipat. Updated analysis will be presented at the meeting. Conclusions: The triplet combination of endocrine therapy with CDK 4/6i and AKTi appears to be well tolerated in heavily pre-treated pts, with a subset demonstrating signs of clinical benefit. The trial demonstrates how insights into the molecular mechanisms of CDK4/6i resistance could be leveraged into actionable therapeutic regimens for HR+/HER2- MBC. Clinical trial information: NCT03959891 .

Atezolizumab (in Combination with Nab-Paclitaxel): A Review in Advanced Triple-Negative Breast Cancer.

Atezolizumab (Tecentriq®), an immune checkpoint inhibitor against programmed death ligand 1 (PD-L1), is the first immunotherapy agent to be approved (for use in combination with nab-paclitaxel) in the USA, the EU (as first-line) and Japan for the treatment of advanced triple-negative breast cancer (TNBC). Approval was based on the results of the phase III IMpassion130 trial in patients with unresectable locally advanced or metastatic TNBC, in which atezolizumab plus nab-paclitaxel significantly prolonged progression-free survival (PFS) when compared to placebo plus nab-paclitaxel in the intent-to-treat (ITT) population and the PD-L1+ subgroup. Statistically significant overall survival (OS) benefits were not seen in two interim analyses and final OS data are awaited. The tolerability and safety profile of atezolizumab plus nab-paclitaxel was consistent with those of each individual drug. The most common treatment-related adverse events included neutropenia, peripheral neuropathy and reduced neutrophil count. Adverse events of special interest occurred with higher frequency in patients who received atezolizumab plus nab-paclitaxel than placebo plus nab-paclitaxel, and were mostly immune-related (e.g. immune-related rash, hypothyroidism and hepatitis). Health-related quality of life was not significantly impacted by the addition of atezolizumab to nab-paclitaxel therapy. Thus, atezolizumab plus nab-paclitaxel is a useful immunochemotherapy option for patients with unresectable locally advanced or metastatic TNBC, including those whose tumours have PD-L1 expression ≥ 1%.

Targeting myeloid-cell specific integrin α9β1 inhibits arterial thrombosis in mice

AbstractEvidence suggests that neutrophils contribute to thrombosis via several mechanisms, including neutrophil extracellular traps (NETs) formation. Integrin α9β1 is highly expressed on neutrophils when compared with monocytes. It undergoes affinity upregulation on neutrophil activation, and stabilizes adhesion to the activated endothelium. The role of integrin α9 in arterial thrombosis remains unexplored. We generated novel myeloid cell-specific integrin α9−/− mice (α9fl/flLysMCre+) to study the role of integrin α9 in arterial thrombosis. α9fl/fl littermates were used as controls. We report that α9fl/flLysMCre+ mice were less susceptible to arterial thrombosis in ferric chloride (FeCl3) and laser injury-induced thrombosis models with unaltered hemostasis. Neutrophil elastase-positive cells were significantly reduced in α9fl/flLysMCre+ mice concomitant with reduction in neutrophil count, myeloperoxidase levels, and red blood cells in the FeCl3 injury-induced carotid thrombus. The percentage of cells releasing NETs was significantly reduced in α9fl/flLysMCre+ mouse neutrophils stimulated with thrombin-activated platelets. Furthermore, we found a significant decrease in neutrophil-mediated platelet aggregation and cathepsin-G secretion in α9fl/flLysMCre+ mice. Transfusion of α9fl/fl neutrophils in α9fl/flLysMCre+ mice restored thrombosis similar to α9fl/fl mice. Treatment of wild-type mice with anti-integrin α9 antibody inhibited arterial thrombosis. This study identifies the potential role of integrin α9 in modulating arterial thrombosis.

Neutrophil count kinetics during the first cycle of chemotherapy predicts the outcome of patients with locally advanced or metastatic pancreatic cancer.

Neutrophil count reduction after chemotherapy has been related with longer survival of patients with metastatic pancreatic adenocarcinoma, but there is not a standardized measurement for this phenomenon. Some parameters related to the change in neutrophil count between the first and the second cycle of chemotherapy or between the baseline count and the nadir have been evaluated among patients with advanced pancreatic cancer at a single institution. A Cox regression model was built which included, in addition to the common prognostic variables, some variables related to the change of the neutrophil count after chemotherapy. One hundred patients were selected. Two neutrophil kinetics related variables predicted overall survival independently, such as the neutrophil count growth rate (hazard ratio [HR] = 1.245; confidence intervals [CIs], 1.077-1.440) and the chemotherapy-induced neutropenia after one cycle (HR = 0.499; CIs, 0.269-0.927). The kinetics of neutrophil count after chemotherapy is an early and independent prognostic factor, which appears to be simple to measure at the start of the second cycle of chemotherapy by means of the neutrophil count growth rate.

Evaluation of Methanol-Dichloromethane Extract of Stemonocoleus micranthus Harms (Fabaceae) Stem Bark for Anti-Inflammatory and Immunomodulatory Activities.

Background The stem bark decoction of Stemonocoleus micranthus Harms (Fabaceae) is most widely used traditionally as a remedy for various diseases such as malaria and boil. In this study, the anti-inflammatory and immunomodulatory activities of the methanol-dichloromethane extract (MDE) from the stem bark of the plant in rodents were evaluated. Methods The carrageenan-induced rat paw oedema, cotton pellet-induced granuloma in rat, and xylene-induced ear oedema in mice were used to study the anti-inflammatory activity of methanol-dichloromethane extract of Stemonocoleus micranthus (MDESm) (100, 200, and 400 mg/kg). The effects of MDESm (100, 200, and 400 mg/kg) on cyclophosphamide-induced immunosuppression, neutrophil adhesion, carbon clearance, and haematological and biochemical parameters were carried out to study its immunomodulatory activity in mice. Result MDESm (100 mg/kg, p.o.) significantly (p < 0.05) inhibited carrageenan-induced oedema by 57.1% at 5th h posttreatment compared with control. At 100 mg/kg, p.o., MDESm significantly (p < 0.05) reduced cotton pellet-induced granuloma by 39.28% and nonsignificantly reduced xylene-induced ear oedema by 34.1%. Treatment with MDESm (100 and 400 mg/kg) nonsignificantly abolished the neutropenia caused by cyclophosphamide with a percentage neutrophil reduction of 0 and −14.86%, respectively, while MDESm (200 mg/kg) and levamisole (50 mg/kg) had a nonsignificant reduction in neutrophil count (10.16 and 31.40%), respectively, all compared to the distilled water-treated group with a neutrophil count of −9.82%. MDESm at doses of 100 and 200 mg/kg increased phagocytic index by 0.0447 ± 0.00762 and 0.0466 ± 0.00703, respectively, although not significantly when compared to the control group with a value of 0.0226 ± 0.02117. There was a decrease in WBC and lymphocyte counts in MDESm- (200 mg/kg) treated group, suggesting immunosuppressive potential at this dose. MDESm caused a dose-dependent decrease in ALT and core liver enzymes, suggesting a hepatoprotective effect. The acute toxicity test revealed that MDESm is safe in mice with an oral lethal dose (LD50) of >5 g/kg. Conclusion The methanol-dichloromethane extract of Stemonocoleus micranthus Harms possesses mild anti-inflammatory and immunomodulatory activities which may be more pronounced upon fractionation and purification. Therefore, more investigations are needed to explore these activities further.

Neutrophils remain detrimentally active in hydroxyurea-treated patients with sickle cell disease.

Neutrophilia is a feature of sickle cell disease (SCD) that has been consistently correlated with clinical severity and has been shown to remain highly activated even at steady state. In addition to induction of fetal hemoglobin (HbF), hydroxyurea (HU) leads to reduction in neutrophil count and their adhesion properties, which contributes to the clinical efficacy of HU in SCD. Although HU reduces the frequency and severity of acute vaso-occlusive crises (VOCs) and chest syndrome, HU therapy does not abolish these acute clinical events. In this study we investigated whether neutrophils in SCD patients whilst on HU therapy retained features of detrimental pro-inflammatory activity. Freshly isolated neutrophils from SCD patients on HU therapy at steady state and from ethnic-matched healthy controls were evaluated ex vivo for their degranulation response and production of neutrophil extracellular traps (NETs). Unstimulated SCD patient neutrophils already produced NETs within 30 minutes, compared to none for healthy neutrophils, and by 4 hours, these neutrophils produced significantly more NETs than the control neutrophils (P = 0.0079**). Higher numbers of neutrophils from SCD patients also showed higher degree of degranulation-related intracellular features compared to healthy neutrophils, including rough-textured cellular membranes (P = 0.03*), double-positivity for F-Actin and CD63 (P = 0.02*) and re-located CD63 within cytoplasm more efficiently than their healthy counterparts (P = 0.02*). The neutrophils from SCD donors released more myeloperoxidase (P = 0.02*) in the absence of any trigger. Our data showed that neutrophils from patients with SCD at steady state remained active during hydroxyurea treatment and are likely to be able to contribute to the SCD pro-inflammatory environment.

Infectious Complications Following Venetoclax-Proteasome Inhibitor Based Regimens in Relapsed Myeloma: A Single Center Retrospective Analysis

Introduction: Venetoclax is an oral BCL2 inhibitor, effective in CLL and under investigation in multiple clinical trials to treat relapsed or refractory multiple myeloma (RRMM) in combination with a proteasome inhibitor (VPI) - either carfilzomib or bortezomib (Moreau P, 2017; Kumar S, 2017). VPI has shown promising initial results in a phase 2 trial with an overall response rate of 78% and a very good partial response or better (≥VGPR) rate of 56% (Costa L J, 2018; Terpos E, 2019). However, a separate phase 3 trial evaluating bortezomib given with venetoclax or placebo found improved response rates and progression free survival but a decrease in overall survival rate in the venetoclax arm of the study (data not published). The primary association with increased mortality was infection. Better characterizing infections in patients treated with VPI may give insight into the pathophysiology and suggest strategies for safe and effective use of this therapy in RRMM. Methods: We retrospectively reviewed charts of patients treated with VPI for infectious complications from initiation of treatment until one month after progression treated at the Hospital of the University of Pennsylvania. Infections were classified by site, pathogen and severity of infection. Additional data collected included demographics, blood cell counts, quantitative immunoglobulins, SPEP M-spike, light chain analysis, cytogenetics, prior lines of therapy, prophylactic antibiotics and IVIG use. We determined non-monoclonal IgG (NM-IgG) via the difference between serum M-spike and IgG for IgG patients. The two-sample Wilcoxon rank-sum was used to compare different subgroups within our study population. Results: We identified 18 patients treated with a VPI combination regimen of which 78% were male. The median age was 64.5 years (range 47-76) and a median of 3 (1-10) prior regimens. 14 were treated with carfilzomib and 4 with bortezomib combinations. 4 patients progressed by the time of data collection. 8 patients were positive for t(11;14). 11 patients experienced 35 discrete infectious episodes resulting in 4 hospitalizations. Respiratory infections predominated (29/35) with 24 upper respiratory infections or sinusitis. 4 were lower respiratory infections and comprised all the hospitalizations. Among respiratory infections, viruses were the only pathogens identified, including influenza, rhinovirus, coronavirus and RSV. Other sites of infection were gastrointestinal (including recurrent C. difficile) and urinary tract. No CNS, blood, or intra-abdominal infections were identified. Reductions in lymphocyte counts and non-monoclonal IgG were near universal but significant reductions in neutrophil counts were not observed. A greater proportional reduction of NM-IgG was noted in good-responders, defined as CR and VGPR (≥VGPR, n=9), with a mean of 78% decrease compared to 47% in poor-responders (PR/MR/SD/PD, n=4, p=0.045). Importantly, treatment of those with t(11;14) positive status demonstrated no significant difference in NM-IgG. Additionally, there was a relative risk ratio favoring a reduction in infections among those with t(11;14). Conclusion: Patients with RRMM treated with VPI experience frequent infectious complications, some severe, with sustained reductions in serum IgG and lymphocyte counts, but without neutropenia. Reduction in NM-IgG is associated with improved response depth. This phenomenon could explain the paradoxical improvement of response rate and progression free survival with concomitant reduction in overall survival due to infections noted by other reports. Interestingly, the presence of t(11;14) in this analysis was not associated with as great a drop in NM-IgG, as was seen in responders as whole, indicating a more differential effect. The absence of significant reduction in NM-IgG is a potential mechanism for relative reduction of infections in this subgroup. Suggestions of survival benefit among t(11;14) positive subgroups in other reports may be related to this preservation of NM-IgG. Replenishing NM-IgG with IVIG is a hypothetical mitigating strategy that could be of benefit in other subgroups treated with this combination of therapy. Disclosures Cohen: Poseida Therapeutics, Inc.: Research Funding. Garfall:Surface Oncology: Consultancy; Novartis: Patents &amp; Royalties: inventor on patents related to tisagenlecleucel (CTL019) and CART-BCMA, Research Funding; Janssen: Research Funding; Amgen: Research Funding; Tmunity: Honoraria, Research Funding. Vogl:Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Active Biotech: Consultancy. Mangan:janssen: Speakers Bureau; celgene: Speakers Bureau; takeda: Speakers Bureau; amgen: Speakers Bureau. Stadtmauer:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Research Funding; Tmunity: Research Funding; Abbvie: Research Funding. OffLabel Disclosure: venetoclax for myeloma

Cathelicidin Antimicrobial Peptide Expression Is Inversely Associated with Absolute Neutrophil Counts in Pediatric HCT Recipients

BACKGROUND: Neutropenia is a major risk factor for infectious complications in hematopoietic cell transplantation (HCT) recipients. However, much less is understood about neutrophil function in the posttransplant period. Cathelicidins are multifunctional host defense peptides. The sole human homolog, Cathelicidin Antimicrobial Peptide (CAMP, OMIM * 600474) is secreted by invading leukocytes, especially neutrophils and plays an important and pleiotropic role in host defense. It acts both as a direct bactericidal agent, but also affects the activity of many immune cells, including prolonging the lifespan of neutrophils. Another critical step in the microbicidal function of neutrophils is the generation of reactive oxygen species. OBJECTIVE: To characterize neutrophil function and CAMP expression over time in children following HCT. METHODS: A prospective study of children undergoing HCT was conducted. Blood samples were collected at 3 months, 6 mo., and 1 y post-HCT timepoints. White blood cells were purified using a PolymorphPrep gradient (Axis-Shield Diagnostics Ltd., Scotland). CAMP expression was measured using Clone H7 antibody (Santa Cruz Biotechnology Inc., USA) in fixed and permeabilized cells. Neutrophil activation was measured as reduction in membrane potential following stimulation with phorbol 12-myristate 13-acetate (PMA) using fluorescence staining with 3,3'-dipentyloxacarbocyanine iodide (DiOC5). Neutrophil hydrogen peroxide generation was measured by oxidation, and resultant increased staining of the probe 2',7' dichlorohydrofluorescein diacetate (DCFH-DA) following PMA stimulation. STATISTICS: Categorical variables were summarized by frequency and percent, and continuous variables were summarized by median and inter-quartile range (IQR). Distributions of continuous variables were examined and were log-transformed if skewed. Linear mixed-effects model was used to evaluate temporal trends and correlations between pairs of variables across time. points. The Bayesian Information Criterion was used to identify the optimal model that balanced model goodness-of-fit and complexity. RESULTS: Sixty-three patients were enrolled, median age 6.1 y (0.4 - 20.5 y). A majority of transplants (69%) were allogeneic. Patient and transplant characteristics are described in Table 1. CAMP was expressed in 95.8% of white blood cells (IQR 80.3 - 99.0%) (Figure 1). CAMP expression in neutrophils, measured as fold-increase above background staining, showed a median value of 474 (IQR 355 - 623). CAMP expression was inversely corelated with absolute neutrophil counts, with 37 folds increase (95% CI 11 to 63 folds, p = 0.006; Figure 2) for every 106 per milliliter reduction in neutrophil counts. Neutrophil activation measured as reduction in membrane potential was expressed as % decrease in DiOC5 staining following PMA stimulation with a median value of 41% (IQR 31 - 56%). Peroxide generation expressed as % increase in DCFH-DA staining following PMA stimulation showed a median value of 304% (IQR 170 - 630%). The reduction in membrane potential was correlated with peroxide generation. For every 1% reduction in membrane potential, hydrogen peroxide production increased by 2% (95% CI 1 to 3%, Figure 3). CAMP expression in neutrophils was not associated with neutrophil function measured by change in membrane potential or peroxide generation. None of the covariates examined (age, transplant type, and timepoints posttransplant) was found to be associated with CAMP expression or measures of neutrophil function. CONCLUSIONS: We have demonstrated that neutrophils, as characterized by changes in membrane potential and reactive oxygen species generation in response to PMA stimulation, are functional in children as early as 3 months following HCT. Of the parameters assessed in this study the increase in CAMP expression in neutrophils in patients with lower numbers of neutrophils in circulation after HCT stands out and this finding may potentially be of importance. Further studies will be necessary to determine if this is specific to HCT setting or if this can also be observed in normal individuals. It will also be important to study if this is reflected in the serum levels of the CAMP-derived active peptides. Disclosures No relevant conflicts of interest to declare.

Diagnosis and Treatment Management in Patients with Autoimmune Neutropenia

Autoimmune neutropenia (AIN) is the frequent cause of neutropenia in infants and children. AIN is associated with a reduced neutrophil count, which is due to aberrant cell-mediated or humoral immune response. In this review, we will discuss the available diagnostic approaches and management of the diseases. We collected data from PubMed, Google Scholar, Medline, Web of Science databases, using a group of key words, such as neutropenia, autoimmune, diagnosis and management from 2000 until 2019. The most important aspects of primary assessment in the affected children were family history and physical examinations. Diagnostic methods in this disease are granulocyte indirect agglutination test (GAT) and granulocyte immunofluorescence test (GIFT). However, the sensitivity and specificity of these tests are low. In these patients, injection of granulocyte-colony stimulating factor (G-CSF), is the first line of treatment. Despite low prevalence, autoimmune neutropenia is a clinically significant disease and it is critical to identify it and pursue effective treatment in these patients.

Prolonged Administration of Leaf Extract of Ocimum gratissimum Reduces RBC, PCV and Platelet Count but Increases Total WBC and Lymphocyte Counts in Rats

Background: Ocimum gratissimum is an ethnopharmacological plant with numerous healing potentials. But there is paucity in scientific literature on its impact on the blood cells.&#x0D; Aim and Objective: The present study was intended to evaluate the dose and time-dependent effect of aqueous leaf extract of Ocimum gratissimum on some haematological in albino Wistar rats.&#x0D; Methods: Twenty four rats were randomly assigned to four equal groups. Group 1 was control, groups 2, 3 and 4 were given 450 mg/kg, 800 mg/kg and 1800 mg/kg respectively of the extract daily for 30 days. All rats had free access to water and rat chow.&#x0D; Results: The result showed a significantly lower RBC count in groups 3 and 4 when compared with control (p&lt;0.05 and p&lt;0.01 respectively). PCV was significantly lower in group 4 than control (p&lt;0.05). Platelet count was significantly lower in group 4 than control (p&lt;0.05) and group 2 (p&lt;0.05). A significant increase in WBC count was observed in groups 2 and 3 when compared with control (p&lt;0.05 and p&lt;0.01 respectively). A significant reduction in neutrophil count was observed in groups 2, 3 and 4 compared with control (p&lt;0.05; p&lt; 0.05 and p&lt;0.01 respectively). There were significant increases in lymphocyte counts in groups 2, 3 and 4 when compared with control (p&lt;0.05, p&lt;0.01 and p&lt;0.01 respectively). There were no significant changes in MCV, MCH and MCHC in the groups.&#x0D; Conclusion: In conclusion, aqueous leaf extract of Ocimum gratissimum causes reduction in RBC count, PCV, platelet count and neutrophil count but causes increases in total WBC count and lymphocyte count.

Anti-inflammatory activity of Theobroma cacao L. stem bark ethanol extract and its fractions in experimental models

Ethnopharmacological relevanceThe stem bark of Theobroma cacao L. have been used for the treatment of inflammation, toothache, measles and malaria in ethnomedicine. However, the anti-inflammatory activity of Theobroma cacao stem bark has not been fully elucidated. AimThe anti-inflammatory activity of Theobroma cacao stem bark ethanol extract and its fractions was investigated in this study. Materials and MethodsThe anti-inflammatory effect of ethanol extract of Theobroma cacao stem bark (EETc) and its dichloromethane (DCMF), ethylacetate (EAF) and aqueous (AQF) fractions was investigated in erythrocytes membrane stabilizing assay and carrageenan-induced paw oedema. The anti-inflammatory activity of the EAF and EETc was investigated in carrageenan induced-granuloma air pouch models. ResultsThe extract and fractions showed significant membrane stabilizing action on rat erythrocytes cell membrane. The oral administration of DCMF, EAF and AQF (250 mg/kg) significantly inhibited paw oedema induced by carrageenan (41.3%, 55.0% and 45.0%, respectively) compared to control group. The EAF (62.5, 125 and 250 mg/kg) and EETc (250 mg/kg) significantly inhibited exudates formation in carrageenan air pouch by (63.8, 71.5, 74.5, 64.3%) at 24 h and by (69.4%, 75.7%, 77.1% and 68.4%) at 72 h respectively. The EETc and EAF significantly reduced neutrophil counts, protein, nitrite, Tumor necrosis factor (TNF-α) and malondialdehyde (MDA) but increased reduced glutathione (GSH) levels compared to control in pouch exudates. The HPLC fingerprint of EAF revealed presence of caffeic acid, rutin, ferulic acid and morin. ConclusionEthanol extract of Theobroma cacao and its ethylacetate fraction demonstrated anti-inflammatory activity partly by reducing neutrophil migration and inflammatory mediator production.

Transcriptional Regulation of Emergency Granulopoiesis in Leukemia.

Neutropenic conditions are prevalent in leukemia patients and are often associated with increased susceptibility to infections. In fact, emergency granulopoiesis (EG), a process regulating neutrophil homeostasis in inflammatory conditions and infections, may occur improperly in leukemic conditions, leading to reduced neutrophil counts. Unfortunately, the mechanisms central to dysfunctional EG remain understudied in both leukemia patients and leukemic mouse models. However, despite no direct studies on EG response in leukemia are reported, recently certain transcription factors (TFs) have been found to function at the crossroads of leukemia and EG. In this review, we present an update on TFs that can potentially govern the fate of EG in leukemia. Transcriptional control of Fanconi DNA repair pathway genes is also highlighted, as well as the newly discovered role of Fanconi proteins in innate immune response and EG. Identifying the TFs regulating EG in leukemia and dissecting their underlying mechanisms may facilitate the discovery of therapeutic drugs for the treatment of neutropenia.

Prediction of neutrophil reduction using plasma paclitaxel concentration after administration in patients with uterine, ovarian, or cervical cancers in an outpatient clinic.

Plasma paclitaxel (PTX) concentration 24h or later after PTX administration may predict myelosuppression. Here, we explored predictive markers for neutropenia induced by intravenous administration of PTX in an outpatient clinic. Thirty women suffering from uterine, ovarian or cervical cancer were enrolled in this study. PTX (mean dose: 167mg/m2) was intravenously infused and followed by carboplatin. Plasma samples were obtained 4h after PTX administration. Genotyping was carried out for CYP3A5*3, ABCB1 1236 C>T, 2677 G>T/A, and 3435 C>T. There was no significant relationship between genotype and reduced neutrophil count. Neutrophil reduction rate correlated with the patient's height, neutrophil count on the day of administration, and plasma PTX concentration. Multiple regression analysis with those three indices explained 47.7% of the interindividual variability of the neutrophil reduction rate. The model with plasma PTX concentration, patient's height, and plasma 6-α-hydroxy-paclitaxel /PTX concentration ratio also explained 30.0% of the interindividual variability for the neutrophil nadir count after PTX administration. These results indicate that neutrophil reduction after PTX administration can be partially predicted by multiple regression analysis involving plasma concentration data collected at outpatient clinics.

The effect of omalizumab treatment on IgE and other immunoglobulin levels in patients with chronic spontaneous urticaria and its association with treatment response.

IntroductionSuppression of free immunoglobulin E (IgE) levels and an increase in total IgE levels are observed during omalizumab treatment. However, whether omalizumab has any effect on other immunoglobulins is unknown.AimTo investigate the effect of omalizumab treatment on serum IgE and other immunoglobulins, and demonstrate any association with response to treatment in patients with chronic spontaneous urticaria (CSU).Material and methodsThe study included 41 patients diagnosed with CSU. Baseline and post-12-week-treatment total IgE, IgA, IgM, and IgG levels and blood eosinophil, neutrophil, lymphocyte and platelet levels were compared. Patients were grouped based on weekly urticaria activity score (UAS-7) responses and these parameters were compared.ResultsThere was a significant increase in baseline and post-12-week-treatment total IgE levels, while there was no significant difference in other immunoglobulin levels. A significant reduction was found in neutrophil counts after the treatment, whereas there was no significant difference in eosinophil, lymphocyte and platelet levels. There was no difference in these parameters between groups with complete response and without complete response.ConclusionsOmalizumab treatment can also be used in patients with immunoglobulin deficiency. Due to the observed reduction in neutrophil counts after the treatment, patients must be closely followed for whole blood parameters.