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Aplastic anemia (AA) is the most serious non-malignant blood disorder in Pakistan, ranked second in prevalence, after thalassemia. We investigated various epidemiological, clinical, and genetic factors of AA in a Pakistani cohort of 214 patients reporting at our hospital between June 2014 and December 2015. A control group of 214 healthy subjects was included for comparison of epidemiological and clinical features. Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between ages 10 and 29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence, and high rate of consanguineous marriages. Serum granulocyte colony-stimulating factor and thrombopoietin levels were significantly elevated in AA patients, compared to healthy controls (P < 0.0001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exons 3 and 7 of TERT gene. Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.

Purpose: Granulocytopenia is the major toxicity associated with cisplatin treatment. Atrial natriuretic peptide (ANP) is a cardiac hormone used clinically for the treatment of acute heart failure in Japan. ANP exerts a wide range of protective effects on various organs, including the heart, blood vessels, lungs, and kidneys. This study's objective was to investigate the protective effects of ANP on cisplatin-induced granulocytopenia in mice., Methods: The mice were divided into two groups: cisplatin with vehicle and cisplatin with ANP. ANP (1.5 μg/kg/min via osmotic pump, subcutaneously) or vehicle administration was started 1 day before cisplatin injection until the mice were killed. At 0, 2, 4, 8, and 14 days after cisplatin injection (16 mg/kg, intraperitoneally as a single dose), the white blood cell, red blood cell, and platelet counts were measured in the peripheral blood in both groups. The numbers of total and live cells and colony-forming unit-granulocyte-macrophage (CFU-GM) colonies in the bone marrow of the mice were also examined. In addition, at 0, 0.5, 1, and 2 days after cisplatin injection, serum granulocyte colony-stimulating factor (G-CSF) levels were measured., Results: ANP significantly attenuated the white blood cell count decrease in the peripheral blood 2 and 4 days after cisplatin injection. ANP also attenuated the decrease in the number of live cells and CFU-GM colonies in bone marrow 2, 4, and 8 days after cisplatin injection. ANP significantly increased serum G-CSF levels 1 day after cisplatin injection., Conclusions: ANP has protective effects in cisplatin-induced granulocytopenia, with increased G-CSF production.

Leukocytosis associated with secretion of granulocyte colony-stimulating factor (G-CSF) has been reported in various tumors, primarily poorly differentiated epithelial tumors, but is extremely rare in bone tumors. An 84-year-old woman experienced swelling and pain in the shoulder for 1 month. Leukocytosis and elevated serum G-CSF were observed, but resolved following tumor resection. A diagnosis of leiomyosarcoma of the bone with expression of G-CSF was confirmed immunohistochemically. Histological diagnosis of leiomyosarcoma showed it to be differentiated, which is unusual for G-CSF-secreting tumors.

Objective: To investigate the effects of exogenous recombinant human granulocyte colony-stimulating factor (rhG-CSF; filgrastim) application on the neutrophils of patients at risk of sepsis following major trauma or operation., Design: Randomized controlled trial., Setting: Surgical intensive care unit and research laboratory of a university hospital., Patients: Twenty-seven patients with systemic inflammatory response syndrome (SIRS)., Interventions: Thirteen patients were treated with filgrastim (1 micro g.kg.24 h) for 10 days as a continuous infusion. Fourteen patients served as controls., Measurements and Results: Surface expression of FcgammaR type I (CD64), phagocytosis of E. coli, and the E. coli-induced oxidative burst of neutrophils were tested by flow cytometry. On the first postoperative/posttraumatic day, endogenous G-CSF plasma concentrations were <300 pg/ml in seven controls (subgroup 1) and nine filgrastim patients (subgroup 3), and were already elevated with >500 pg/ml in seven controls (subgroup 2) and four filgrastim patients (subgroup 4). G-CSF values ( P=0.0026, subgroup 1/3; P=0.0167, 2/4), neutrophil counts ( P=0.0026, 1/3; P=0.0167, 2/4), and CD64 expression ( P=0.0013, 1/3) were higher in filgrastim-treated than non-treated subgroups, but not phagocytic and burst activities. From day zero to day 1, phagocytosis decreased in subgroups 1 (5/7 patients) and 3 (5/9), but increased in subgroups 2 (5/7) and 4 (3/4), and respiratory burst activity decreased in subgroup 3 (8/9)., Conclusions: Besides activation of neutrophil maturation, low-dose rhG-CSF application in postoperative patients with SIRS has different effects on neutrophil functions, in part depending on already endogenously produced G-CSF.

Unlabelled: In acute-phase Kawasaki disease, neutrophils cause injury to the coronary arterial endothelium through the production of elastase. Previous research has demonstrated the modulation of neutrophil function and kinetics, such as development and maturation, by granulocyte colony-stimulating factor (G-CSF). To examine the correlation between G-CSF and cardiac complications in Kawasaki disease, functional activity of serum G-CSF and cytokines was measured by enzyme-linked immunosorbent assay in 30 patients with acute-phase Kawasaki disease aged 2 months to 5 years. The mean serum G-CSF was higher in the 1st week of Kawasaki disease than during weeks 2 to 4, and G-CSF was significantly higher in patients with coronary artery dilatation (CAL) than in those without. There was no significant difference in the activity of other cytokines studied or white blood cell counts between the patients with CAL., Conclusion: granulocyte colony-stimulating factor is correlated with coronary artery dilatation in acute-phase Kawasaki disease and increased neutrophil function may contribute to the pathogenesis of coronary arterial endothelial injury in these patients.

Objective: To elucidate the diagnostic accuracy of granulocyte colony-stimulating factor (G-CSF), interleukin-8 (IL-8), and interleukin-1 receptor antagonist (IL-1ra) in identifying patients with sepsis among critically ill pediatric patients with suspected infection., Design and Setting: Nested case-control study in a multidisciplinary neonatal and pediatric intensive care unit (PICU) PATIENTS: PICU patients during a 12-month period with suspected infection, and plasma available from the time of clinical suspicion (254 episodes, 190 patients)., Measurements and Results: Plasma levels of G-CSF, IL-8, and IL-1ra. Episodes classified on the basis of clinical and bacteriological findings into: culture-confirmed sepsis, probable sepsis, localized infection, viral infection, and no infection. Plasma levels were significantly higher in episodes of culture-confirmed sepsis than in episodes with ruled-out infection. The area under the receiver operating characteristic curve was higher for IL-8 and G-CSF than for IL-1ra. Combining IL-8 and G-CSF improved the diagnostic performance, particularly as to the detection of Gram-negative sepsis. Sensitivity was low (<50%) in detecting Staphylococcus epidermidis bacteremia or localized infections., Conclusions: In this heterogeneous population of critically ill children with suspected infection, a model combining plasma levels of IL-8 and G-CSF identified patients with sepsis. Negative results do not rule out S. epidermidis bacteremia or locally confined infectious processes. The model requires validation in an independent data-set.

Objective: To clarify the changes in total leukocyte counts, CD64 neutrophil receptor expression and serum granulocyte colony-stimulating factor (G-CSF) concentrations in critically ill patients without infection and sepsis and in patients with septic shock., Design: Prospective study., Setting: Intensive care unit (ICU) and research laboratory of a university hospital., Patients: Eleven critically ill patients without infections and 22 patients with proven infections in septic shock for the first time and of at least 3 days' duration., Measurements and Results: Over a 6month period, a longitudinal analysis of expression of the monomeric Fc receptor type I (CD64, FcgammaRI) on neutrophils was performed by flow cytometric analysis on a daily basis in all postoperative/post-traumatic patients admitted to the ICU until discharge from the ICU or death. Out of 273 patients, 11 patients without sepsis had organ failure and 22 patients with proven infections had septic shock for the first time and of at least 3 days' duration. Ten out of the 22 patients survived, 12 died. CD64 expression was greater in patients with septic shock than in patients without sepsis. Moreover, CD64 expression was only initially and transiently elevated in most survivors (9/10) and non-survivors (8/12) of septic shock. In survivors, G-CSF serum concentrations were markedly decreased in the 2nd week., Conclusions: Decreased neutrophil CD64 expression in an acutely ill population with septic shock may reflect the development of a non-responsive state as well as the early downregulation of neutrophil activation prior to the resolution of an ongoing infection.

We investigated the effects of recombinant G-CSF (Filgrastim) on the function of neutrophils and the rate of infectious complications in an open-label, nonrandomized study of patients with esophageal cancer undergoing esophagectomy. In this single-center phase-II trial 20 sequential patients (19 evaluable) received Filgrastim at standard doses (300 microg or 480 microg) subcutaneously for 2 days prior to and up to 7 days after surgery. The phagocytotic activity of neutrophils and the oxidative burst in the study group and in an experimental control group (n=27) were measured on days -2, 2, and 10. Neutrophil function was enhanced in the Filgrastim-treated group by factor 1.2 for phagocytosis (p=0.016) and 1.4 for oxidative burst (p)=0.154). Leukocyte counts increased from 7.6 x 10(9)/l (day -2) to a maximum of 45 x 10(9)/l on day 6. No infection was reported in the study group (mean age 59.7 years; 13 men, seven women) up to 10 days after surgery. In contrast, 23 patients (29.9%) in a historical control group (mean age 56 years; 67 men, ten women) treated at the same center developed infections within the first 10 days (p = 0.008). In addition, no postoperative deaths occurred in the study group, compared with 9.1% in the group of historical controls. Thus, in this study, administration of Filgrastim stimulated neutrophil function in patients undergoing esophagectomy, and it might be effective in reducing infectious complications related to the surgical procedure.

The pattern of changes in leukocyte counts and the blood concentration of G-CSF were observed in 15 patients undergoing aortocoronary bypass surgery. Myelopoietic function was assessed by examining the myelogram and performing flow cytometry to identify human leukocyte differentiation antigens on bone marrow aspirates obtained from the sternum when opening and closing the sternotomy. The blood concentration of G-CSF increased gradually after removal of the aortic cross clamp and peaked on the first postoperative day (232 +/- 98 ngml). The white blood cell count also increased during the operation and peaked on the second postoperative day, demonstrating a threefold increase (15,800 +/- 2700). Granulocytes represented most of the increase, while lymphocytes and monocytes showed no significant changes. The myelogram showed that the percentages of myeloblasts, promyelocytes, and metamyelocytes did not change; however, the percentage of myelocytes increased significantly during surgery (14.0 +/- 2.5% vs. 17.3 +/- 3.5%, p < 0.05). The number of mature myelocytes (LFA-1 beta and Leu-15 positive) decreased significantly (p < 0.01 and p < 0.05) during surgery. With the two-color method, the ratio of immature myelocytes (MCS-2 negative and Leu-15 negative) increased significantly (p < 0.01). The ratio of myeloblasts (Leu-11 and HLA-DR positive) and the ratio of stem cells (CD 34 and MY-9 positive) did not increase significantly during the operation. G-CSF concentrations increase substantially during aortocoronary bypass surgery and may be responsible for the rise in granulocyte and total leukocyte counts, as well as for the increase in immature myelocytes seen on bone marrow examination.

Unlabelled: We measured serum granulocyte colony stimulating factor (GCSF) concentration and absolute neutrophil count in four groups of infants: (1) 15 healthy term newborn infants; (2) 21 healthy preterm newborn infants, with mean (SD) birth weight 1583 (533) g, and gestational age 32.0 (3.8) weeks; (3) 5 infected newborn infants; (4) 22 6-month-old control infants. Median (range) serum GCSF concentration was 132.2 (41.5-176.0) pg/ml in term infants, 51.5 (1.8-175.7) pg/ml in preterm infants and 138.9 (54.1-449.8) pg/ml in 6-month-old control infants, with a significant reduction in preterm infants, as compared to term and control infants. GCSF levels were significantly higher in the infected infants, as compared to healthy neonates., Conclusion: A significant positive relationship was found in term and preterm infants between serum GCSF concentration and gestational age or birth weight. No relationship was found between serum GCSF concentration and neutrophil count. The low GCSF baseline levels may contribute to the increased incidence and severity of infection in preterm infants.

Objective: To evaluate the effect and safety of a low dose Filgrastim treatment in surgical intensive care patients., Design: Prospective, clinical study., Setting: Surgical intensive care unit (ICU) in a university hospital., Patients: Ten patients with the systemic inflammatory response syndrome (SIRS) and ten patients with sepsis were included in the study., Interventions: Filgrastim was given intravenously at 1.0 microgram/kg for 3 days, followed by 0.5 microgram/kg for 4 days., Measurements and Results: Filgrastim treatment increased leukocyte counts and plasma levels of G-CSF. Cytokine levels (IL-6 and IL-8) decreased in the first 3 days of treatment. None of the SIRS patients developed sepsis or multiple organ failure and none of the patients died. In the sepsis group four patients died. No adverse side effects were observed, especially no attenuation of lung injury., Conclusions: Low-dosage Filgrastim treatment in ICU patients is safe. Whether the observed changes of the inflammatory response can be attributed to Filgrastim has to be clarified in further randomized trials.

Plasma levels of IL-1, IL-6, IL-8, IL1-RA, TNF alpha, and G-CSF were prospectively studied during 23 chemotherapy cycles of 20 patients suffering from acute myelogenous leukemia. Increased plasma levels of IL-6, IL-8, and G-CSF were observed in patients with febrile neutropenia and/or major infection. Plasma levels of IL-6, IL-1, TNF alpha and IL-1-RA measured 1 day before and 1 day after the onset of febrile episodes did not accurately predict the development of major infection. In contrast, IL-8 plasma levels were significantly higher in those patients who subsequently developed major infection. The question whether IL-8 plasma levels identify high risk or low risk patients with sufficient specificity and sensitivity has to be answered in large scale clinical trials.

The inhibitory effect of murine interferon gamma (muIFN gamma) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). muIFN gamma was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN gamma nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN gamma into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN gamma suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.

A patient with chronic idiopathic neutropenia, who had been suffering from repeated infections, was successfully treated with recombinant granulocyte stimulating factor (rhG-CSF). Subcutaneous injection of 30 micrograms/m2 rhG-CSF every two days was sufficient to maintain the neutrophil count at approximately 1,000/microliter. The patient has lived without any evidence of infection for the last 10 months using that treatment. There were no side effect caused by rhG-CSF and antibodies against G-CSF were not detected in the patient's plasma.