Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Recovery of pulmonary structure and exercise capacity by treatment with granulocyte-colony stimulating factor (G-CSF) in a mouse model of emphysema

Emphysema is a chronic obstructive pulmonary disease characterized abnormal dilatation of alveolar spaces, which impairs alveolar gas exchange, compromising the physical capacity of a patient due to airflow limitations. Here we tested the effects of G-CSF administration in pulmonary tissue and exercise capacity in emphysematous mice. C57Bl/6 female mice were treated with elastase intratracheally to induce emphysema. Their exercise capacities were evaluated in a treadmill. Lung histological sections were prepared to evaluate mean linear intercept measurement. Emphysematous mice were treated with G-CSF (3 cycles of 200 μg/kg/day for 5 consecutive days, with 7-day intervals) or saline and submitted to a third evaluation 8 weeks after treatment. Values of run distance and linear intercept measurement were expressed as mean ± SD and compared applying a paired t-test. Effects of treatment on these parameters were analyzed applying a Repeated Measures ANOVA, followed by Tukey's post hoc analysis. p < 0.05 was considered statistically significant. Twenty eight days later, animals ran significantly less in a treadmill compared to normal mice (549.7 ± 181.2 m and 821.7 ± 131.3 m, respectively; p < 0.01). Treatment with G-CSF significantly increased the exercise capacity of emphysematous mice (719.6 ± 200.5 m), whereas saline treatment had no effect on distance run (595.8 ± 178.5 m). The PCR cytokines genes analysis did not detect difference between experimental groups. Morphometric analyses in the lung showed that saline-treated mice had a mean linear intercept significantly higher (p < 0.01) when compared to mice treated with G-CSF, which did not significantly differ from that of normal mice. Treatment with G-CSF promoted the recovery of exercise capacity and regeneration of alveolar structural alterations in emphysematous mice. © 2013.

A high-fat diet increases interleukin-3 and granulocyte colony-stimulating factor production by bone marrow cells and triggers bone marrow hyperplasia and neutrophilia in wistar rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

GM-CSF (granulocyte-macrophage colony-stimulating factor): Modulation der durch UV-B-Strahlung induzierten Hautkarzinogenese

UV-B-Strahlung, die durch die fortschreitende Zerstörung der Ozonschicht zunimmt, ist hauptsächlich für das Entstehen von Basaliomen und Plattenepithelkarzinomen verantwort-lich, an denen jedes Jahr etwa 2-3 Millionen Menschen weltweit erkranken. UV-B indu-zierte Hautkarzinogenese ist ein komplexer Prozess, bei dem vor allem die mutagenen und immunsuppressiven Wirkungen der UV-B-Strahlung von Bedeutung sind. Die Rolle von GM-CSF in der Hautkarzinogenese ist dabei widersprüchlich. Aus diesem Grund wurde die Funktion von GM-CSF in vivo in der UV-B induzierten Hautkarzinogenese mittels zwei bereits etablierter Mauslinien untersucht: Erstens transgene Mäuse, die einen GM-CSF Antagonisten unter der Kontrolle des Keratin-10-Promotors in den suprabasalen Schichten der Epidermis exprimieren und zweitens solche, die unter dem Keratin-5-Promotor murines GM-CSF in der Basalschicht der Epidermis überexprimieren. Eine Gruppe von Tieren wurde chronisch, die andere akut bestrahlt. Die konstitutionelle Verfassung der Tiere mit erhöhter GM-CSF-Aktivität in der Haut war nach chronischer UV-B-Bestrahlung insgesamt sehr schlecht. Sie wiesen deshalb eine stark erhöhte Mortali-tät auf. Dies ist sowohl auf die hohe Inzidenz als auch dem frühen Auftreten der benignen und malignen Läsionen zurückzuführen. Eine verminderte GM-CSF Aktivität verzögerte dagegen die Karzinomentwicklung und erhöhte die Überlebensrate leicht. GM-CSF wirkt auf verschiedenen Ebenen tumorpromovierend: Erstens erhöht eine gesteigerte Mastzell-anzahl in der Haut der GM-CSF überexprimierenden Tiere per se die Suszeptibilität für Hautkarzinogenese. Zweitens stimuliert GM-CSF die Keratinozytenproliferation. Dadurch kommt es nach UV-B-Bestrahlung zu einer prolongierten epidermalen Hyperproliferation, die zur endogenen Tumorpromotion beiträgt, indem sie die Bildung von Neoplasien unter-stützt. Der Antagonist verzögert dagegen den Proliferationsbeginn, die Keratinozyten blei-ben demzufolge länger in der G1-Phase und der durch UV-B verursachte DNA-Schaden kann effizienter repariert werden. Drittens kann GM-CSF die LCs nicht als APCs aktivie-ren und eine Antitumorimmunität induzieren, da UV-B-Strahlung zur Apoptose von LCs bzw. zu deren Migration in Richtung Lymphknoten führt. Zusätzlich entwickeln GM-CSF überexprimierende Tiere in ihrer Haut nach UV-B-Bestrahlung ein Millieu von antago-nistisch wirkenden Zytokinen, wie TNF-a, TGF-b1 und IL-12p40 und GM-CSF, die proinflammatorische Prozesse und somit die Karzinomentwicklung begünstigen. Der Anta-gonist hemmt nach UV-B-Bestrahlung die Ausschüttung sowohl von immunsuppressiven Zytokinen, wie etwa TNF-a, als auch solchen, die die Th2-Entwicklung unterstützen, wie etwa IL-10 und IL-4. Dies wirkt sich negativ auf die Karzinomentwicklung aus.

2010 update of EORTC guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapy-induced febrile neutropenia in adult patients with lymphoproliferative disorders and solid tumours

Chemotherapy-induced neutropenia is a major risk factor for infection-related morbidity and mortality and also a significant dose-limiting toxicity in cancer treatment. Patients developing severe (grade 3/4) or febrile neutropenia (FN) during chemotherapy frequently receive dose reductions and/or delays to their chemotherapy. This may impact the success of treatment, particularly when treatment intent is either curative or to prolong survival. In Europe, prophylactic treatment with granulocyte-colony stimulating factors (G-CSFs), such as filgrastim (including approved biosimilars), lenograstim or pegfilgrastim is available to reduce the risk of chemotherapy-induced neutropenia. However, the use of G-CSF prophylactic treatment varies widely in clinical practice, both in the timing of therapy and in the patients to whom it is offered. The need for generally applicable, European-focused guidelines led to the formation of a European Guidelines Working Party by the European Organisation for Research and Treatment of Cancer (EORTC) and the publication in 2006 of guidelines for the use of G-CSF in adult cancer patients at risk of chemotherapy-induced FN. A new systematic literature review has been undertaken to ensure that recommendations are current and provide guidance on clinical practice in Europe. We recommend that patient-related adverse risk factors, such as elderly age (≥65 years) and neutrophil count be evaluated in the overall assessment of FN risk before administering each cycle of chemotherapy. It is important that after a previous episode of FN, patients receive prophylactic administration of G-CSF in subsequent cycles. We provide an expanded list of common chemotherapy regimens considered to have a high (≥20%) or intermediate (10-20%) risk of FN. Prophylactic G-CSF continues to be recommended in patients receiving a chemotherapy regimen with high risk of FN. When using a chemotherapy regimen associated with FN in 10-20% of patients, particular attention should be given to patient-related risk factors that may increase the overall risk of FN. In situations where dose-dense or dose-intense chemotherapy strategies have survival benefits, prophylactic G-CSF support is recommended. Similarly, if reductions in chemotherapy dose intensity or density are known to be associated with a poor prognosis, primary G-CSF prophylaxis may be used to maintain chemotherapy. Clinical evidence shows that filgrastim, lenograstim and pegfilgrastim have clinical efficacy and we recommend the use of any of these agents to prevent FN and FN-related complications where indicated. Filgrastim biosimilars are also approved for use in Europe. While other forms of G-CSF, including biosimilars, are administered by a course of daily injections, pegfilgrastim allows once-per-cycle administration. Choice of formulation remains a matter for individual clinical judgement. Evidence from multiple low level studies derived from audit data and clinical practice suggests that some patients receive suboptimal daily G-CSFs; the use of pegfilgrastim may avoid this problem.

Favorable effect of priming with granulocyte colony-stimulating factor in remission induction of acute myeloid leukemia restricted to dose-escalation of cytarabine

The clinical value of chemotherapy sensitization of acute myeloid leukemia (AML) with G-CSF priming has remained controversial. Cytarabine is a key constituent of remission induction chemotherapy. The effect of G-CSF priming has not been investigated in relationship with variable dose levels of cytarabine. We randomized 917 AML patients to receive G-CSF (456 patients) or no G-CSF (461 patients) at the days of chemotherapy. In the initial part of the study, 406 patients were also randomized between 2 cytarabine regimens comparing conventional-dose (199 patients) versus escalated-dose (207 patients) cytarabine in cycles 1 and 2. We found that patients after induction chemotherapy plus G-CSF had similar overall survival (43% vs 40%, P = .88), event-free survival (37% vs 31%, P = .29), and relapse rates (34% vs 36%, P = .77) at 5 years as those not receiving G-CSF. However, patients treated with the escalated-dose cytarabine regimen benefited from G-CSF priming, with improved event-free survival (P = .01) and overall survival (P = .003), compared with patients without G-CSF undergoing escalated-dose cytarabine treatment. A significant survival advantage of sensitizing AML for chemotherapy with G-CSF was not apparent in the entire study group, but it was seen in patients treated with escalated-dose cytarabine during remission induction. The HOVON-42 study is registered under The Netherlands Trial Registry (www.trialregister.nl) as #NTR230.

Granulocyte death regulation by naturally occurring autoantibodies

Programmed cell death (PCD) plays a central role in the regulation of granulocytes that are key effector cells of the innate immune system. Granulocytes are produced in high amounts in the bone marrow. A safe elimination of granulocytes by cell death (apoptosis) is essential to maintain the numbers of these cells balanced. In many acute and chronic inflammatory diseases, delayed apoptosis is one mechanism that contributes to accumulation of neutrophil and eosinophil granulocytes at the site of inflammation. On the other hand, a safe elimination of granulocytes by cell death is required to avoid unwanted tissue damage for instance by secretion of toxic products from these cells. Recent evidence shows that humans produce an array of naturally occurring autoantibodies (NAbs) with the capacity to regulate granulocyte death, including agonistic and antagonistic NAbs that bind to the receptors Fas, Siglec-8, and Siglec-9. Together with other factors, these various NAbs exhibit different properties in terms of the form of cell death they induce, the molecular signaling pathways they engage, as well as the efficacy or potency by which they induce cell death. Moreover, several regulatory mechanisms seem to exist that control their biological activity. Novel insights support the concept of granulocyte death regulation by NAbs, which might have important implications for our understanding of the pathogenesis and treatment of inflammatory diseases, including many autoimmune and allergic disorders.

Interleukin-17A stimulates granulocyte-macrophage colony-stimulating factor release by murine osteoblasts in the presence of 1,25-dihydroxyvitamin D(3) and inhibits murine osteoclast development in vitro

OBJECTIVE To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. METHODS Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte-macrophage colony-stimulating factor-knockout (GM-CSF(-/-)) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D(3)(1,25[OH](2)D(3)), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5-6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. RESULTS In both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)(2)D(3) to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF(-/-) mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. CONCLUSION These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

Granulocyte colony-stimulating factor supports liver regeneration in a small-for-size liver remnant mouse model

Experimental partial hepatectomy of more than 80% of the liver weight bears an increased mortality in rodents, due to impaired hepatic regeneration in small-for-size liver remnants. Granulocyte colony-stimulating factor (G-CSF) promotes progenitor cell expansion and mobilization and also has immunomodulatory properties. The aim of this study was to determine the effect of systemically administered G-CSF on liver regeneration and animal survival in a small-for-size liver remnant mouse model. Mice were preconditioned daily for 5 days with subcutaneous injections of 5 microg G-CSF or aqua ad injectabile. Subsequently, 83% partial hepatectomy was performed by resecting the median, the left, the caudate, and the right inferior hepatic lobes in all animals. Daily sham or G-CSF injection was continued. Survival was significantly better in G-CSF-treated animals (P < 0.0001). At 36 and 48 h after microsurgical hepatic resection, markers of hepatic proliferation (Ki67, BrdU) were elevated in G-CSF-treated mice compared to sham injected control animals (P < 0.0001) and dry liver weight was increased (P < 0.05). G-CSF conditioning might prove to be useful in patients with small-for-size liver remnants after extended hepatic resections due to primary or secondary liver tumors or in the setting of split liver transplantation.

Granulocyte colony stimulating factor supports liver regeneration in a surgical small for size liver remnant mouse model

ntense liver regeneration and almost 100% survival follows partial hepatectomy of up to 70% of liver mass in rodents. More extensive resections of 70 to 80% have an increased mortality and partial hepatectomies of >80% constantly lead to acute hepatic failure and death in mice. The aim of the study was to determine the effect of systemically administered granulocyte colony stimulating factor (G-CSF) on animal survival and liver regeneration in a small for size liver remnant mouse model after 83% partial hepatectomy (liver weight <0.8% of mouse body weight). Methods: Male Balb C mice (n=80, 20-24g) were preconditioned daily for five days with 5μg G-CSF subcutaneously or sham injected (aqua ad inj). Subsequently 83% hepatic resection was performed and daily sham or G-CSF injection continued. Survival was determined in both groups (G-CSF n=35; Sham: n=33). In a second series BrdU was injected (50mg/kg Body weight) two hours prior to tissue harvest and animals euthanized 36 and 48 hours after 83% liver resection (n=3 each group). To measure hepatic regeneration the BrdU labeling index and Ki67 expression were determined by immunohistochemistry by two independent observers. Harvested liver tissue was dried to constant weight at 65 deg C for 48 hours. Results: Survival was 0% in the sham group on day 3 postoperatively and significantly better (26.2% on day 7 and thereafter) in the G-CSF group (Log rank test: p<0.0001). Dry liver weight was increased in the G-CSF group (T-test: p<0.05) 36 hours after 83% partial hepatectomy. Ki67 expression was elevated in the G-CSF group at 36 hours (2.8±2.6% (Standard deviation) vs 0.03±0.2%; Rank sum test: p<0.0001) and at 48 hours (45.1±34.6% vs 0.7±1.0%; Rank sum test: p<0.0001) after 83% liver resection. BrdU labeling at 48 hours was 0.1±0.3% in the sham and 35.2±34.2% in the G-CSF group (Rank sum test: p<0.0001) Conclusions: The surgical 83% resection mouse model is suitable to test hepatic supportive regimens in the setting of small for size liver remnants. Administration of G-CSF supports hepatic regeneration after microsurgical 83% partial hepatectomy and leads to improved long-term survival in the mouse. G-CSF might prove to be a clinically valuable supportive substance in small for size liver remnants in humans after major hepatic resections due to primary or secondary liver tumors or in the setting of living related liver donation.

Granulocyte colony-stimulating factor increases hepatic sinusoidal perfusion during liver regeneration in mice

Conditioning with granulocyte colony-stimulating factor (G-CSF) promotes liver regeneration in an experimental small-for-size liver remnant mouse model. The mechanisms involved in this extraordinary G-CSF effect are unknown. The aim of this study was to investigate the influence of G-CSF on the hepatic microvasculature in the regenerating liver. The hepatic sinusoidal microvasculature and microarchitecture of the regenerating liver were evaluated by intravital microscopy in mice. Three experimental groups were compared: (1) unoperated unconditioned animals (control; n = 5), (2) animals conditioned with G-CSF 48 h after 60% partial hepatectomy (G-CSF-PH; n = 6), and (3) animals sham conditioned 48 h after 60% PH (sham-PH; n = 6). PH led to hepatocyte hypertrophy and increased hepatic sinusoidal velocity in the sham-PH and G-CSF-PH groups. Increased sinusoidal diameter and increased hepatic blood flow were observed in the G-CSF-PH group compared to the sham-PH and control groups. Furthermore, there was a strong positive correlation between spleen weight and hepatic sinusoidal diameter in the G-CSF-PH group. The increased hepatic blood flow could explain the observed benefit of G-CSF conditioning during liver regeneration. These results elucidate an unexplored aspect of pharmacological modulation of liver regeneration and motivate further experiments.

Myocardial salvage through coronary collateral growth by granulocyte colony-stimulating factor in chronic coronary artery disease: a controlled randomized trial

BACKGROUND: The efficacy of granulocyte colony-stimulating factor (G-CSF) for coronary collateral growth promotion and thus impending myocardial salvage has not been studied so far, to our best knowledge. METHODS AND RESULTS: In 52 patients with chronic stable coronary artery disease, age 62+/-11 years, the effect on a marker of myocardial infarct size (ECG ST segment elevation) and on quantitative collateral function during a 1-minute coronary balloon occlusion was tested in a randomized, placebo-controlled, double-blind fashion. The study protocol before coronary intervention consisted of occlusive surface and intracoronary lead ECG recording as well as collateral flow index (CFI, no unit) measurement in a stenotic and a > or =1 normal coronary artery before and after a 2-week period with subcutaneous G-CSF (10 microg/kg; n=26) or placebo (n=26). The CFI was determined by simultaneous measurement of mean aortic, distal coronary occlusive, and central venous pressure. The ECG ST segment elevation >0.1 mV disappeared significantly more often in response to G-CSF (11/53 vessels; 21%) than to placebo (0/55 vessels; P=0.0005), and simultaneously, CFI changed from 0.121+/-0.087 at baseline to 0.166+/-0.086 at follow-up in the G-CSF group, and from 0.152+/-0.082 to 0.131+/-0.071 in the placebo group (P<0.0001 for interaction of treatment and time). The absolute change in CFI from baseline to follow-up amounted to +0.049+/-0.062 in the G-CSF group and to -0.010+/-0.060 in the placebo group (P<0.0001). CONCLUSIONS: Subcutaneous G-CSF is efficacious during a short-term protocol in improving signs of myocardial salvage by coronary collateral growth promotion.