Human Granulocyte Research Articles

Synthetic pro-peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae.

Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony‐stimulating factor (hG‐CSF) in S. cerevisiae, a secretion‐enhancing peptide cassette including an hIL‐1β‐derived propeptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters—N‐glycosylation, net negative charge balance, and glycine‐rich flexible linker—were investigated in batch cultures of S. cerevisiae. Using a three‐stage design involving screening, selection, and optimization, the production and secretion of hG‐CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG‐CSF produced by the optimized propeptide after the final stage increased by 190% compared to that of the original propeptide. Although hG‐CSF was used as the model protein in the current study, this strategy applies to the enhanced production of other heterologous proteins, using S. cerevisiae as the host.

Preparation and Characterization of Site-Specific Fatty Chain-Modified Recombinant Human Granulocyte Colony Stimulating Factor.

The clinical use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is limited by its short serum half-life. In this study, a long-acting strategy for site-specific modification of rhG-CSF with 1-pentadecyl-1H-pyrrole-2,5-dione (C15 fatty chain-maleimide, C15-MAL) was studied in mixed DMSO-aqueous solutions. The factors influencing the conjugation reaction were investigated and optimized, and a high yield of the desired product (C15-rhG-CSF) was achieved. Subsequently, C15-rhG-CSF product was efficiently purified using preparative liquid chromatography, and further characterized. Circular dichroism spectroscopy analysis showed that the secondary structure of C15-rhG-CSF had no significant difference from unmodified rhG-CSF. C15-rhG-CSF retained 87.2% of in vitro bioactivity of unmodified rhG-CSF. The pharmacokinetic study showed that the serum half-life of C15-rhG-CSF in mice was 2.08-fold longer than that of unmodified rhG-CSF. Furthermore, C15-rhG-CSF by single-dose subcutaneous administration showed better in vivo efficacy than those of both PEG10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration. This study demonstrated the potential of C15-rhG-CSF being developed into a novel drug candidate as well as an efficient process for the development of long-acting protein and peptide drugs.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), β-casein (Csn2), whey acidic protein (WAP), or β-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a “precise” integration approach, placing the human granulocyte–macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2–11 µg/ml hGMCSF). This was probably caused by the absence of the 3’-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30–40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2–0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals’ health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

Safety and efficacy of pegylated recombinant human granulocyte colony-stimulating factor during concurrent chemoradiotherapy for small-cell lung cancer: a retrospective, cohort-controlled trial

ObjectiveTo investigate pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) safety and efficacy in preventing hematological toxicity during concurrent chemoradiotherapy (CCRT) for small-cell lung cancer (SCLC).MethodsWe retrospectively assessed 80 SCLC patients treated with CCRT from January 2013 to December 2018 who received PEG-rhG-CSF within 48 hours after the end of chemotherapy, defined as prophylactic use, as the experimental group. An additional 80 patients who were not treated with PEG-rhG-CSF were matched 1:1 by the propensity score matching method and served as the control group. The main observations were differences in hematological toxicity, neutrophil changes, febrile neutropenia (FN) incidence and adverse reactions. Progression-free survival (PFS) and overall survival (OS) were analyzed with regular assessment and follow-up.ResultsThe leukocyte, neutrophil, erythrocyte, and platelet counts and hemoglobin level decreased after CCRT, but the experimental group had slightly higher leukocyte and neutrophil counts than the control group (P < 0.05). The incidences of grade III-IV leukopenia (18.75% vs. 61.25%) and neutropenia (23.75% vs. 67.5%) in the experimental group were significantly lower than those in the control group (P < 0.05). The absolute neutrophil count was 4.17 ± 0.79 (× 109/L) on day 1 and peaked 6.81 ± 2.37 (× 109/L) on day 10 in the experimental group; the value in the control group was 2.81 ± 0.86 (× 109/L) on day 1. It decreased significantly and reached the minimum 0.91 ± 0.53 (× 109/L) on day 10 (P < 0.05). The experimental group had a lower FN incidence than the control group (P < 0.05). There was also no significant acute esophagitis or pulmonary toxicity. The treatment had no significant effect on PFS (11.4 months vs. 8.7 months, P = 0.958) or OS (23.9 months vs. 17.3 months, P = 0.325) over an 18.6-month median follow-up time.ConclusionPEG-rhG-CSF has good efficacy and safety in preventing hematological toxicity in SCLC patients during CCRT and has no significant effects on PFS or OS.

Yeast-Mycelial Dimorphism in Pichia pastoris SMD1168 Is Triggered by Nutritional and Environmental Factors

This study reports, for the first time, morphological transition from yeast-like to filamentous form, normally associated with pathogenicity/increased protein secretion, in Pichia pastoris SMD1168 strain. The response was recorded in response to nutritional and environmental cues. The factors affecting this switch were extracellular pH (under nitrogen starvation conditions), carbon and nitrogen source under nitrogen- and carbon-limiting conditions respectively. Under nitrogen-limiting conditions, addition of fructose and sucrose in the culture medium induced filamentous morphology in a segregated form whereas addition of galactose led to a mixture of yeast and the filamentous form of the cells. Under carbon-limiting conditions, isoleucine and proline forced a filamentous form whereas glycine, valine, alanine and phenylalanine promoted yeast-like morphology. Similar dimorphic shift was also displayed by a recombinant methanol slow utilizing (Muts) strain (SMD-GCSF Muts) producing human granulocyte colony-stimulating factor in response to change in the initial inoculum level. Analysis of the extracellular metabolome by GC-MS indicated that several amino acids (leucine, proline, tyrosine), carboxylic acids (phenylacetic-, propanoic acid), alcohols and butylamine were present at different levels in the culture broth of the two morphological forms. High accumulation of proline and butylamine was seen in the extracellular culture filtrate of the filamentous form of the yeast. Presence of quorum-sensing molecules (phenylethyl alcohol, dodecanol) suggested complex network of pathways involved in this morphological transition.

Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome

ObjectivePolycystic ovary syndrome (PCOS) is characterized by follicular dysplasia. An insufficient glycolysis-derived energy supply of granulosa cells (GCs) is an important cause of follicular dysplasia in PCOS. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been proven to regulate the function of GCs. In this study, exosomes extracted from clinical FF samples were used for transcriptome sequencing (RNA-seq) analysis, and a human ovarian granulocyte tumour cell line (KGN cells) was used for in vitro mechanistic studies.Methods and resultsIn FF exosomal RNA-seq analysis, a decrease in glycolysis-related pathways was identified as an important feature of the PCOS group, and the differentially expressed miR-143-3p and miR-155-5p may be regulatory factors of glycolysis. By determining the effects of miR-143-3p and miR-155-5p on hexokinase (HK) 2, pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate, lactate and apoptosis in KGN cells, we found that upregulated miR-143-3p expression in exosomes from the PCOS group inhibited glycolysis in KGN cells; knockdown of miR-143-3p significantly alleviated the decrease in glycolysis in KGN cells in PCOS. MiR-155-5p silencing attenuated glycolytic activation in KGN cells; overexpression of miR-155-5p significantly promoted glycolysis in KGN cells in PCOS. In this study, HK2 was found to be the mediator of miR-143-3p and miR-155-5p in FF-derived exosome-mediated regulation of glycolysis in KGN cells. Reduced glycolysis accelerated apoptosis of KGN cells, which mediated follicular dysplasia through ATP, lactate and apoptotic pathways.ConclusionsIn conclusion, these results indicate that miR-143-3p and miR-155-5p in FF-derived exosomes antagonistically regulate glycolytic-mediated follicular dysplasia of GCs in PCOS.9LsDb7oAvmV44Q9GUxhugtVideo

LF-EMF Compound Block Type Signal Activates Human Neutrophilic Granulocytes In Vivo.

This research aims to demonstrate in a randomized, placebo‐controlled crossover design study that a nominal 5 μT low‐frequency electromagnetic field (LF‐EMF) signal for 30 min activates neutrophils in vivo in humans. Granularity of neutrophils was measured in blood samples of healthy human volunteers (n = 32) taken before and after exposure for both the exposure and control sessions. A significant decrease in the granularity, indicative of neutrophil activation, was observed both in the exposure measurements and the exposure minus control measurements. Earlier EMF publications show immune function increase in isolated cells and more effective immune responses in animals with infections. This result, therefore, supports the thesis that the exposure can activate the innate immune system in humans, speed up the innate immune response, and may have potential beneficial effects in infectious disease. © 2022 Bioelectromagnetics Society.

A wearable gamma radiation-responsive granulocyte colony-stimulating factor microneedle system protecting against ionizing radiation-induced injury

Exposure to a nuclear accident or a radiological attack may cause serious death events due to ionizing radiation-induced injury and acute radiation syndrome (ARS). Recombinant human granulocyte colony-stimulating factor (G-CSF) is now used for the treatment of ARS. However, the current injection formulation might not ensure treatment as early as possible after a nuclear accident, resulting in a decrease in therapeutic efficiency. In the present study, we have developed a G-CSF wearable system (GWS) consisting of a commercial microchip, a temperature sensor, a gamma-ray detection sensor, a flexible heater, and a G-CSF temperature-sensitive microneedle (GTSMN) patch. G-CSF-containing hyaluronic acid solutions were cast into the mold to obtain G-CSF microneedles (GMNs), which were coated with a temperature-sensitive layer of dodecanoic acid-cetylamine salt to obtain GTSMNs. The flexible heater was prepared by jet printing Ag nanoparticle inks. The GWS and its components are explored and optimized in the aspects of electronics, mechanics, heat transfer and drug diffusion. The γ radiation signal is sensitively monitored by the GWS. The wearable G-CSF system immediately releases G-CSF into the body in response to signal feedback and provides maximal protection against ionizing radiation-induced injury. Therefore, the GWS is a promising wearable system against emergent ionizing radiation injury. Statement of significanceIonizing radiation-induced injury is always the very important public health problem all the global people care. Some medicines have been applied to protect the body from the injury. Unfortunately, sometimes the injuries accidently happen and the medicines cannot be administered in time, leading to serious acute radiation syndrome. Here, we design a wearable system loading G-CSF that has been approved by FDA to protect the body from ionizing radiation-induced injury. This system consists of a commercial microchip, a temperature sensor, a Gamma-ray detection sensor, a flexible heater, and a G-CSF temperature-sensitive microneedle patch. It can monitor γ radiation and immediately release G-CSF into the body to protect the body to the maximal extent. Therefore, the system is a promising wearable medical device against emergent ionizing radiation injury.

Effects of Prophylactic Administration of Granulocyte Colony-Stimulating Factor on Peripheral Leukocyte and Neutrophil Counts Levels After Chemotherapy in Patients With Early-Stage Breast Cancer: A Retrospective Cohort Study.

BackgroundBoth chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) frequently occur and can lead to dose-limiting toxicity and even fatal chemotherapy side effects. The prophylactic use of recombinant human granulocyte colony-stimulating factor (rhG-CSF), including pegylated rhG-CSF (PEG-rhG-CSF), significantly reduces the risks of CIN and FN during chemotherapy in early-stage breast cancer (ESBC) patients. However, whether the prophylactic use of granulocyte colony-stimulating factor (G-CSF), especially PEG-rhG-CSF, can influence white blood cell (WBC) counts and absolute neutrophil counts (ANCs) after finishing the chemotherapy remains unknown. Therefore, exploring the development and recovery tendency of WBC counts and ANCs during and after chemotherapy is crucial.ObjectiveWe aimed to investigate the variation tendency and recovery of WBC counts and ANCs during and after chemotherapy and evaluate the independent factors influencing leukopenia and neutropenia lasting longer after chemotherapy. We also aimed to provide individualized prophylactically leukocyte elevation therapy for breast cancer patients.MethodsThis single-center retrospective cohort study evaluated 515 ESBC patients who received rhG-CSF or PEG-G-CSF for prophylaxis after adjuvant or neoadjuvant chemotherapy. Blood test reports were analyzed during chemotherapy, and on a 12-month follow-up period after finishing the chemotherapy. The WBC counts and ANCs were measured to assess their variation tendency characteristics and to identify independent factors that influenced the occurrence of leukopenia and neutropenia lasting longer than 12 months after chemotherapy.ResultsProphylaxis with rhG-CSF or PEG-rhG-CSF kept the mean values of WBC counts and ANCs within the normal range during chemotherapy, but a significant difference in WBC levels was detected before the end of the last chemotherapy compared to the prechemotherapy period (baseline) (p < 0.001). During the 12-month follow-up after the end of the last chemotherapy, WBC counts and ANCs gradually recovered, but the group that used only PEG-rhG-CSF (long-acting group, p WBC = 0.012) or rhG-CSF (short-acting group, p WBC = 0.0005) had better leukocyte elevation effects than the mixed treatment group (PEG-rhG-CSF mixed rhG-CSF). Besides, the short-acting group had a better neutrophil elevation effect than the longer-acting (p ANC = 0.019) and mixed (p ANC = 0.002) groups. Leukopenia was still present in 92 (17.9%) patients and neutropenia in 63 (12.2%) 12 months after the end of the last chemotherapy. The duration of leukopenia over 12 months was closely associated with the baseline WBC level (p < 0.001), G-CSF types (p = 0.027), and surgical method (p = 0.041). Moreover, the duration of neutropenia over 12 months was closely related to the baseline ANC (p < 0.001), G-CSF types (p = 0.043), and molecular typing (p = 0.025).ConclusionThe prophylactic application of G-CSF effectively stabilized the WBC counts and ANCs during chemotherapy in ESBC patients. Nevertheless, the recovery of WBC counts and ANCs after chemotherapy varied between different G-CSF treatment groups. The risk of leukopenia and neutropenia persisting for more than 12 months after chemotherapy was associated with G-CSF types, the baseline level of WBC count/ANCs, surgical method, and molecular typing.

A retrospective study of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in preventing neutropenia during definitive concurrent chemoradiotherapy in patients with esophageal squamous carcinoma

ObjectiveTo compare the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) for preventive or delayed treatment in neutropenia, completion rate of concurrent chemoradiotherapy and hospitalization rate in patients with esophageal squamous carcinoma during definitive concurrent chemoradiotherapy. MethodsA total of 70 patients with esophageal squamous carcinoma in Peking University Cancer Hospital from January 2019 to December 2020, who received PEG-rhG-CSF during concurrent chemoradiotherapy, were enrolled in this retrospective analysis. There were 32 patients in the preventive group, and 38 patients in the delayed group. The incidence of neutropenia, completion rate of concurrent chemoradiotherapy and neutropenia-related hospitalization rate were compared between PEG-rhG-CSF preventive group and delayed group. ResultsThe incidence of severe neutropenia (Grades 3–4) in all patients was 31.4%. Comparison between preventive group and delayed group showed that the incidence of severe neutropenia was 6.3% and 39.4% (χ2 ​= ​10.428, P ​= ​0.001), respectively. In preventive group, the incidence of severe neutropenia was 3.7% and 20.0%, respectively, for primary prevention and secondary prevention of PEG-rhG-CSF (χ2 ​= ​12.321, P ​= ​0.001). The completion rate of concurrent chemoradiotherapy was 93.8% in the preventive group and 63.2% in the delayed group (χ2 ​= ​9.220, P ​= ​0.002). The incidence of treatment interruption was 25.7% in the whole group, 12.5% in the preventive group and 36.8% in the delayed group (χ2 ​= ​5.389, P ​= ​0.020). Seven patients (7/70, 10.0%) were hospitalized and treated with intravenous antibiotics for neutropenia, including 1 in the preventive group and 6 in the delayed group (P ​= ​0.078). ConclusionsProphylactic use of PEG-rhG-CSF during concurrent chemoradiotherapy for patients with esophageal squamous carcinoma can effectively reduce the incidence of neutropenia, ensure the safety of treatment, and improve the completion rate of concurrent chemoradiotherapy.

Therapy-related acute myeloid leukemia: A case series.

Patients with primary cancer receiving chemotherapy and/or radiotherapy may develop therapy-related acute leukemia (t-AL). Therapy-related acute myeloid leukemia (t-AML) accounts for the majority of these cases and is frequently associated with a variety of cytogenetic and molecular abnormalities. The aim of the present study was to explore the clinical characteristics, treatments and prognosis of patients with t-AML. A total of 272 cases of AML treated at our institution between 2016 and 2020 were reviewed, among which nine cases of t-AML were identified for analysis. All patients had received alkylating or topoisomerase II inhibitor chemotherapy drugs for primary cancer treatment and three patients had received radiotherapy. A total of nine patients had been administered recombinant human granulocyte colony-stimulating factor (G-CSF). The median latency period for the nine patients with t-AML was 25 months (range, 10–240 months). The molecular cytogenetic abnormalities included t(15:17)(q22:q21), inv(16)(p13q22), del(5)(q22), CBFB/MYH11(+), FLT3(+), NARS(+), IDH(+), TET2(+), and TP53(+). Out of nine patients with t-AML, eight received chemotherapy, two of whom underwent HSCT. The median survival time of the nine patients with t-AML was 10 months and the 2-year-survival rate was 44.4%. Greater clarity around the diagnosis and treatment is required to improve the outcomes of patients with t-AML.

The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli.

The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 °C and then recombinant protein production at 42 °C. Since the heat shock response in E. coli is triggered above 34 °C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 °C) on the growth of E. coli W3110 producing the human granulocyte–macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a λpL/pR-cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 °C showed a ~ 69% increase in the specific growth rate compared to cultures grown at 30 °C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 °C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew at 34 °C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 °C. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs.Key Points• Pre-induction temperature determines inclusion bodies architecture• In pre-induction (above 34 °C), the heat shock response increases recombinant protein production• Inclusion bodies at higher pre-induction temperature show a lower amyloid contentSupplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-11908-z.

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed exvivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. Invivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic invivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NPs), which could also be used for invivo administration, for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34+ cells. NP-mediated exvivo delivery showed no toxicity, and the efficiency was directly correlated with the charge of the NPs. In a side-by-side comparison with electroporation, NP-mediated gene editingallowed for a 3-fold reduction in the amount of reagents, with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.

Modulating Myeloid Immune Cell Migration Using Multivalently Presented Monosaccharide Ligands for Advanced Immunotherapy

Due to their importance for the outcome of the inflammatory response, the motile myeloid cells are a focus of novel treatment options. The interplay of selectins and their ligands with leukocytes and endothelial cells, which mediate endothelial attachment and transmigration of immune cells, can be modulated by selectin-binding structures. Here, a library of selectin-targeting ligands coupled to either gold, silver, iron oxide nanospheres, or quantum dots of 5–10 nm in size is used to systematically study their impact on immune cell motility. The multivalent presentation of the carbohydrate mimetics results in very low sub-nanomolar binding to L-selectin. Using human primary monocytes, granulocytes, lymphocytes, and macrophages, it is shown that the ligands exhibit only minor effects on uptake, whereas the motility of leukocytes is critically affected as observed in migration assays evaluated by flow cytometry. The carbohydrate mimetic ring structure, sulfation, in particular, and the degree of ligand presentation, are constituents which cohere in this process. Specific carbohydrate ligands can thus selectively regulate leukocyte subsets. These data form the basis for advanced immunotherapy which inhibits the amplification of inflammation by restricting leukocyte influx to injured tissue sites. Furthermore, the targeting ligands may complement existing treatment options for inflammatory diseases.

Abstract P5-18-10: Mecapegfilgrastim for primary prophylaxis of neutropenia in 355 HER2+ breast cancer patients treated with neoadjuvant docetaxel in combination with trastuzumab and/or pyrotinib: Exploratory analysis from randomized, double-blind, phase 3 PHEDRA study

Abstract Background: A dose relationship may exist for both antitumor activity and toxicity of docetaxel in breast cancer (BC) patients, while 86% grade 4 neutropenia and 12% febrile neutropenia (FN) were reported when pretreated advanced breast cancer (ABC) patients received 100 mg/m2 docetaxel monotherapy without hematopoietic support. PHEDRA was a randomized, double-blind, multicenter, phase 3 study comparing the efficacy and safety of adding pyrotinib to trastuzumab and docetaxel as neoadjuvant treatment in women with HER2+ early or locally ABC (ClinicalTrials.gov: NCT03588091). We conducted this exploratory analysis to evaluate the effectiveness of mecapegfilgrastim, a long-acting recombinant human granulocyte colony-stimulating factor (rhG-CSF), as primary prophylaxis for neoadjuvant chemotherapy-induced neutropenia in BC patients. Methods: Patients with HER2-positive early or locally ABC were randomly assigned (1:1) to pyrotinib arm receiving 4 neoadjuvant cycles of docetaxel (100 mg/m2 iv d1 q3w), trastuzumab (8 mg/kg iv, cycle 1 d1, then 6 mg/kg d1 q3w), and pyrotinib (400 mg po qd, d1-21, q3w) or placebo arm with placebo, trastuzumab and docetaxel. Per protocol, patients were required to receive a single, 6-mg fixed dose of mecapegfilgrastim on Day 2 of each cycle. Other G-CSF was permitted if mecapegfilgrastim was unavailable at the local center or patients occurred mecapegfilgrastim intolerance. The incidence of neutropenia, FN, time to first neutropenia onset, duration per neutropenia event and cumulative neutropenia duration during neoadjuvant treatment period; and the incidences of grade 3/4 neutropenia, FN and decreased WBC count in Cycle 1 to 4 (C1-4) were presented. The data cutoff date was April 30, 2021. Results: Between July 23, 2018 and January 8, 2021, 355 patients were randomized (pyrotinib arm, n=178; placebo arm, n=177). Among them, 291 (82.0%) patients received a single, 6-mg fixed dose of mecapegfilgrastim in Cycle 1 and 270 (76.1%) patients received mecapegfilgrastim in each of the 4 cycles. Grade 3/4 neutropenia was reported in 33 (18.5%) patients in the pyrotinib arm and 36 (20.3%) patients in the placebo arm. Five (2.8%) patients in the pyrotinib arm and 2 (1.1%) patients in the placebo arm developed FN (5 FN occurred in C1; 2 FN occurred in C2). Median duration of grade 3/4 neutropenia was 3 days in the pyrotinib group and 3 days in the placebo group. Median cumulative duration of grade 3/4 neutropenia was 4 days and 3 days in the pyrotinib group and the placebo group, respectively. Grade 3/4 neutropenia mainly occurred during the first cycle of treatment for both pyrotinib (13.5%) and placebo arm (15.8%), reduced in the second cycle (5.9% vs 4.0%) and thereafter (C3: 1.8% vs 3.4%; C4: 2.4% vs 1.7%). Similar trends were observed for grade 3/4 WBC count decreased in Cycle 1 to 4. No grade 4 infection occurred. Overview of neutropenia, FN and WBC count decreased was summarized in Table 1. Consistent findings were observed in 291 mecapegfilgrastim treated patients. Conclusion: The exploratory analysis demonstrated 6-mg fixed dose of mecapegfilgrastim was effective when administrated as primary prophylaxis for neoadjuvant chemotherapy-induced neutropenia, which could be considered as a new treatment option for its advantage of once-per-cycle dosing and convenient dose management. Table 1.Overview of neutropenia, febrile neutropenia and WBC count decrease during neoadjuvant treatment period.Docetaxel+Trastuzumab+Pyrotinib(N=178)Docetaxel+Trastuzumab+Placebo (N=177)All randomized patients(N=355)Neutropenia, n (%)Any grade57 (32.0)54 (30.5)111 (31.3)Grade 16 (3.4)5 (2.8)11 (3.1)Grade 218 (10.1)13 (7.3)31 (8.7)Grade 315 (8.4)20 (11.3)35 (9.9)Grade 418 (10.1)16 (9.0)34 (9.6)Median time to first onset (IQR), days7 (6-63)6 (6-49)7 (6-53)Median duration per grade 3 or higher neutropenia, days (range)3 (1-16)3 (2-12)3 (1-16)Median cumulative duration of grade 3 or higher neutropenia, days (range)4 (2-16)3 (2-14)3 (2-16)FN, n (%)5 (2.8)2 (1.1)7 (2.0)Grade 3 or higher neutropenia, n (%) *Cycle 124 (13.5)28 (15.8)52 (14.6)Cycle 210 (5.9)7 (4.0)17 (4.9)Cycle 33 (1.8)6 (3.4)9 (2.6)Cycle 44 (2.4)3 (1.7)7 (2.1)Grade 3 or higher FN, n (%) *Cycle 12 (1.1)2 (1.1)4 (1.1)Cycle 22 (1.2)02 (0.6)Cycle 3000Cycle 4000Grade 3 or higher WBC count decreased, n (%) *Cycle 120 (11.2)20 (11.3)40 (11.3)Cycle 28 (4.7)2 (1.1)10 (2.9)Cycle 32 (1.2)1 (0.6)3 (0.9)Cycle 44 (2.4)2 (1.1)6 (1.8)Note: IQR, interquartile range; FN, febrile neutropenia; WBC, white blood cell.*The denominator indicates number of patients with mecapegfilgrastim for prophylaxis use in this cycle. Citation Format: Min He, Benlong Yang, Jiong Wu, Zhenzhen Liu, Hongjian Yang, Jinhai Tang, Kun Wang, Yunjiang Liu, Haibo Wang, Peifen Fu, Shuqun Zhang, Qiang Liu, Zefei Jiang, Shusen Wang, Jian Huang, Chuan Wang, Shu Wang, Yongsheng Wang, Linlin Zhen, Xiaoyu Zhu, Shulin Liu, Ping Yan, Jianjun Zou. Mecapegfilgrastim for primary prophylaxis of neutropenia in 355 HER2+ breast cancer patients treated with neoadjuvant docetaxel in combination with trastuzumab and/or pyrotinib: Exploratory analysis from randomized, double-blind, phase 3 PHEDRA study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-18-10.

Periodontal inflamed surface area in oral cavity associated with febrile neutropenia in patients with hematologic malignancy undergoing chemotherapy

Febrile neutropenia (FN) is an infectious complication that develops during chemotherapy. Although the oral cavity can be an important infection route, it is unknown whether the oral environment is associated with FN. The present study examined the relationship between the oral environment using periodontal inflamed surface area (PISA), a new periodontal disease parameter, and FN in hematologic cancer patients undergoing chemotherapy. In this retrospective cohort study, 157 patients were divided into FN onset during chemotherapy (n = 75) and the FN negative groups (n = 82). The associations of risk factors related to the intraoral environment were assessed. Logistic regression analysis showed that types of blood cancer (odds ratio 1.98; P < 0.01), use of a high-risk regimen (odds ratio 4.44; P < 0.05), prophylaxis treatment with human granulocyte colony-stimulating factor (G-CSF) (odds ratio 4.15; P < 0.01) and PISA (odds ratio 1.02; P < 0.01) were independent factors associated with FN onset. Finally, propensity score matching was performed between two groups; 37 matched pairs were generated. PISA was significantly higher in the FN group than the FN negative group. There was a significant relationship between PISA and FN onset (P = 0.035). The present findings indicate that periodontitis treatment before starting cancer treatment is recommended as supportive care for preventing FN onset during chemotherapy.

Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance.

Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

Cost-Effectiveness Analysis of PEG-rhG-CSF as Primary Prophylaxis to Chemotherapy-Induced Neutropenia in Women With Breast Cancer in China: Results Based on Real-World Data.

Background: Pegylated recombinant human granulocyte colony-stimulating factors (PEG-rhG-CSFs) are more commonly and widely used than recombinant human granulocyte colony-stimulating factors (rhG-CSFs) in preventing chemotherapy-induced neutropenia in patients with stage II-IV breast cancer. To reduce the financial burden on these patients, the corresponding medical insurance directory needs to be revised. Objectives: To evaluate the cost-effectiveness of PEG-rhG-CSF versus rhG-CSF in patients with stage II-IV breast cancer in central China. Methods: Two Markov models, a chemotherapy model and a post-chemotherapy model, were developed to study the effects and costs, with a time horizon of 12 weeks and 35 years, respectively. Cost and probability input data were primarily obtained from a retrospective real-world study conducted in five tertiary hospitals. Propensity score matching was adopted to overcome retrospective bias. Other parameters were extracted from literature as well as advice from clinical experts. Univariate and probabilistic sensitivity analyses were conducted. Results: In the first chemotherapy model, PEG-rhG-CSF was associated with fewer episodes of febrile neutropenia (FN) (N = 19 per 1000 patients treated), infections (N = 24 per 1000 patients treated) and deaths (N = 2 per 1000 patients treated), but higher costs (¥36 more per patient treated). The post-chemotherapy model indicated that PEG-rhG-CSF led to higher gains in quality-adjusted life years (QALYs) (11.695 versus 11.516) in comparison to rhG-CSF. Sensitivity analysis showed that the cost of PEG-rhG-CSF had the greatest impact on the incremental costs, and incremental QALYs were very sensitive to the risk of RDI <85%. The probability of PEG-rhG-CSF being cost-effective compared to rhG-CSF was 66% at the willingness to pay (WTP) thresholds of ¥72,371 per QALY gained. Conclusion: According to this economic evaluation based on real-world data, PEG-rhG-CSF may be considered as a more cost-effective strategy relative to rhG-CSF for stage II-IV breast cancer patients in central China. However, to reflect a national perspective, further evidence is needed using data from larger-scale studies.

Use Intein Cleavable Polyhydroxyalkanoate Synthase Fusions to Improve Protein Solubility.

Recombinant E. coli producing intein-cleavable polyhydroxyalkanoate synthase fusions mediates the intracellular formation of polyhydroxyalkanoate (PHA) particles densely coated with intein-cleavable target protein fusion. These PHA particles can be efficiently purified from lysed cells. The self-cleaving intein performs as a bio-linker between the PHA synthase and the target protein. The tagless target protein can be released as pure soluble protein from the PHA particles by a simple pH reduction to 6.0. Here we describe that PHA particles serve as bioseparation resin for purification of soluble target proteins with pharmaceutical grade purity, similar to commercial affinity separation technologies. This cost-effective technique does not involve multiple complicated protein purification procedures, and we have exploited this approach to purify six target proteins: green fluorescent protein (GFP) from A. victoria, antigen Rv1626 from M. tuberculosis, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).

Serum chitotriosidase and YKL-40 in acute pancreatitis: Reliability as prognostic marker for disease severity and correlation with inflammatory markers

Chitotriosidase and YKL-40, also called chitinase 3-like protein 1, are homologs of family 18 glycosyl hydrolases, secreted by human macrophages and granulocytes under inflammatory conditions. Although increased levels of chitotriosidase and YKL-40 are linked with several inflammatory diseases, the physiological utility of these two enzymes is still not fully characterized. This study aims to analyse the serum YKL-40 and chitotriosidase levels of acute pancreatitis patients to assess whether their activity correlates with acute pancreatitis and its severity. Chitotriosidase and YKL-40 levels, along with routine laboratory parameters, were determined from the serum samples of 41 acute pancreatitis patients, at both onset and remission (male/female: 22/19), and 39 healthy subjects (male/female: 19/20). The Modified Glasgow Prognostic Score was used to predict the severity of the disease, and a correlation analysis was performed between study variables. A statistically significant increase in both chitotriosidase and YKL-40 levels was observed in acute pancreatitis patients compared to healthy controls (P < 0.001). Higher levels of YKL-40, chitotriosidase and C-reactive protein were found in patients with acute pancreatitis at onset than in remission. The correlation analysis showed a statistically significant association between YKL-40 and chitotriosidase (p = 0.039, r = 0.323). The cut-off point for YKL-40, for detecting acute pancreatitis, was 60.3 with a sensitivity and specificity of 84.9% and 84.6% (AUC: 0.890). The optimum cut-off points for chitotriosidase, for detecting acute pancreatitis, was 33.5 with a sensitivity and specificity of 79.5% and 78.4% (AUC: 0.899). Elevated YKL-40 and chitotriosidase levels in acute pancreatitis patients demonstrate the importance of possible macrophage involvement in the pancreatic microenvironment during acute pancreatitis progression.