[Retracted] MicroRNA­320a suppresses tumour cell proliferation and invasion of renal cancer cells by targeting FoxM1.

Subsequently to the publication of the above paper, the authors drew to the attention of the Editorial Office that they made a couple of errors in terms of the data assembly in Figs. 2 and 4 in their paper; specifically, the Transwell assay data shown for the 'miR-320a+/FoxM1+' panel in Fig. 5D on p. 1923 also appeared as the 'ACTN/NC' data panel in Fig. 4E on the same page (Fig. 4E contained the erroneously duplicated panel). In addition, data featured in Fig. 2D of the above paper were strikingly similar to data that appeared in Fig. 6e of the following paper, published subsequently to this article, written by different authors (although a Dr Shiyue Zhao worked in the molecular biology laboratory of Harbin Medical University from 2017 to 2018, and the research collaboration was conducted with Dr Chenlong Li's research group): Li C, Zheng H, Hou W, Bao H, Xiong J, Che W, Gu Y, Sun H and Liang P: Long non-coding RNA linc00645 promotes. TGF-ß-induced epithelial-mesenchymal transition by regulating miR-205-3p-ZEB1 axis in glioma. Cell Death Dis 10: 17, 2019. Finally, after having conducted an independent investigation of the data in this paper, the Editorial Office noted that one of the Petri dish images in Fig. 2C was also strikingly similar to data that appeared in Fig. 2H of the abovementioned article in the journal Cell Death & Disease. After having considered the authors' request for corrigendum, in view of the problems that were identified with the data, the Editor of Oncology Reports has decided that, owing to a lack of confidence in the presented data, the paper should instead be retracted from the journal. After having informed the authors of this decision, they accepted the decision to retract this paper. The Editor apologizes to the readership for any inconvenience caused.  [Oncology Reports 40: 1917­1926, 2018; DOI: 10.3892/or.2018.6597].

Application of vacuum-sealing drainage in buttonhole-related arteriovenous fistula infection: A case report.

Few studies have reported the application of vacuum-sealing drainage of infected dialysis vascular access wounds. Herein, we present a case of buttonhole-related arteriovenous fistula infection treated with vacuum-sealing drainage. A 53-year-old female hemodialysis patient was hospitalized with an inflamed arteriovenous fistula. The patient underwent non-tunneled catheterization for dialysis and was treated with moxifloxacin and vancomycin for staphylococcal infection. On Day 3, the skin overlying the inflamed fistula was ulcerated, resulting in severe hemorrhage. Emergency surgery was performed along with vacuum-sealing drainage for fistula reconstruction. Vacuum-sealing drainage accelerated the recovery of the wound without complications. No further access complications occurred during over a 3-year follow-up.

Research Progress on Podocyte Pyroptosis in Diabetic Nephropathy.

Pyroptosis is a recently discovered type of lytic-programmed cell necrosis. The process involves cells assembling an inflammasome and cleaving gasdermin (GSDM) to trigger the release of pro-inflammatory cytokines that eventually induce inflammatory cell death. Diabetic nephropathy (DN) is a microvascular complication of diabetes mellitus, which leads to end-stage renal disease. Podocyte damage or loss is an important feature of diabetic kidney injury. Pyroptosis involvement in podocyte injury is closely associated with DN progression, manifesting as increased renal fibrosis, glomerulosclerosis, and tubular injury. The study aims to elucidate the mechanism of pyroptosis and summarize the pathways and potential inhibitors related to pyroptosis activation in DN podocytes. We undertook a search of bibliographic databases for peer-reviewed research literature on various aspects of pyroptosis. Multiple different pathways mediate podocyte pyroptosis to promote DN progression. Inhibition of pyroptosis can reduce podocyte damage and improve renal function in DN, suggesting that pyroptosis may help identify potential new therapeutic targets for DN treatment.

Obesity-related glomerulopathy: recent advances in inflammatory mechanisms and related treatments.

Obesity-related glomerulopathy, which is an obesity-triggered kidney damage, has become a significant threat to human health. Several studies have recently highlighted the critical role of inflammation in obesity-related glomerulopathy development. Additionally, excess adipose tissue and adipocytes in patients with obesity produce various inflammatory factors that cause systemic low-grade inflammation with consequent damage to vascular endothelial cells, exacerbating glomerular injury. Therefore, we conducted a comprehensive review of obesity-related glomerulopathy and addressed the critical role of obesity-induced chronic inflammation in obesity-related glomerulopathy pathogenesis and progression, which leads to tubular damage and proteinuria, ultimately impairing renal function. The relationship between obesity and obesity-related glomerulopathy is facilitated by a network of various inflammation-associated cells (including macrophages, lymphocytes, and mast cells) and a series of inflammatory mediators (such as tumor necrosis factor α, interleukin 6, leptin, adiponectin, resistin, chemokines, adhesion molecules, and plasminogen activator inhibitor 1) and their inflammatory pathways. Furthermore, we discuss a recently discovered relationship between micronutrients and obesity-related glomerulopathy inflammation and the important role of micronutrients in the body's anti-inflammatory response. Therefore, assessing these inflammatory molecules and pathways will provide a strong theoretical basis for developing therapeutic strategies based on anti-inflammatory effects to prevent or delay the onset of kidney injury.

Association of serum Nrf2 protein levels with disease activity and renal impairment in lupus nephritis.

Introduction: We aimed to investigate the relationship between nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression levels, lupus nephritis (LN) disease activity, and the degree of renal injury (based on the estimated glomerular filtration rate [eGFR]) in patients with LN. Methods: We selected 40 healthy control participants and 102 patients with LN who were treated in the Second Hospital of Jilin University, China, for inclusion in this study. Patients with LN were classified into LN with high-eGFR and LN with low-eGFR groups. Nrf2 protein levels were measured in the serum and renal tissues of the participants in both groups to assess the correlation between Nrf2 protein levels and different LN disease states. Results: There was a significantly positive correlation between serum Nrf2 protein levels, the degree of renal injury, and systemic lupus erythematosus disease activity index (SLEDAI) scores in patients with LN. Nrf2 protein levels were higher in the LN with high-eGFR group than in the healthy control and LN with low-eGFR groups. In follow-up patients in the LN high eGFR group, Nrf2 protein levels decreased significantly after remission of disease activity. Conclusion: Nrf2 protein expression has a dual role in patients with LN. Nrf2 protein levels not only correlate with disease activity in patients with LN, but also with the degree of kidney injury. Before implementing targeted therapy for Nrf2, evaluating both Nrf2 protein expression and the disease state in patients with LN is necessary to better identify and place each patient in an appropriate patient group.

Zinc deficiency increases lung inflammation and fibrosis in obese mice by promoting oxidative stress.

BACKGROUND: Zinc deficiency can lead to multiple organ damage. In this study, we investigated the effects of zinc deficiency on obesity-related lung damage. METHODS: C57BL/6 J mice were fed a diet with differing amounts of zinc and fat over a 6-month period. Palmitic acid was used to stimulate A549 cells to construct a high-fat alveolar epithelial cell model. Western blotting and histopathological staining were performed on animal tissues. Nuclear expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was detected in cultured cells. A reactive oxygen species (ROS) assay kit was used to detect intracellular ROS. Furthermore, Nrf2 siRNA was used to examine zinc deficiency effects on A549 cells. RESULTS: Pathological results showed significant damage to the lung structure of mice in the high-fat and low-zinc diet group, with a significant increase in the expression of inflammatory (IL-6, TNF-α) and fibrosis (TGFß1, PAI-1) factors, combined with a decrease in the expression of Nrf2, HO-1 and NQO1 in the antioxidant pathway. In A549 cells, high fat and low zinc levels aggravated ROS production. Western blot and immunofluorescence results showed that high fat and zinc deficiency inhibited Nrf2 expression. After Nrf2-specific knockout in A549 cells, the protective effect of zinc on oxidant conditions induced by high fat was reduced. Phosphorylated Akt and PI3K levels were downregulated on the high-fat and low-zinc group compared with the high-fat group. CONCLUSIONS: Zinc attenuated lung oxidative damage in obesity-related lung injury and Nrf2 activation is one of the important mechanisms of this effect. GENERAL SIGNIFICANCE: Regulating zinc homeostasis through dietary modifications or supplemental nutritional therapy can contribute to the prevention and treatment of obesity-related lung injury.

Immune-mediated inflammatory diseases and leukocyte telomere length: A Mendelian randomization study.

Objective: To elucidate the potential causality of leukocyte telomere length (LTL) with immune-mediated inflammatory diseases (IMIDs), we conducted a Mendelian randomization (MR) study. Methods: The genetically predicted causation between LTL and IMIDs was evaluated using a two-sample MR method. We analyzed 16 major IMIDs, which included systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), ankylosing spondylitis (AS), sicca syndrome (SS), rheumatoid arthritis (RA), type 1 diabetes (T1D), primary sclerosing cholangitis (PSC), idiopathic pulmonary fibrosis (IPF), atopic dermatitis (AD), sarcoidosis, hypothyroidism, hyperthyroidism, psoriasis, and childhood asthma. The random-effects inverse-variance weighted (IVW) method was performed as the main analytical approach in MR. Various sensitivity analyses, including MR-Egger, MR robust adjusted profile score (MR-RAPS), weighted median, MR pleiotropy residual sum and outlier (MR-PRESSO) methods, weighted mode, radial plot, and radial regression, were used to guarantee the robustness of the results and detect horizontal pleiotropy. Cochran's Q value was calculated to check for heterogeneity, and the MR Steiger approach was used to test the causal direction. Results: The MR results indicated significant inverse associations of LTL with risks of psoriasis (OR: 0.77, 95% CI: 0.66-0.89, and p = 3.66 × 10-4), SS (OR: 0.75, CI: 0.58-0.98, and p = 0.03), RA (OR: 0.77, 95% CI: 0.68-0.88, and p = 9.85 × 10-5), hypothyroidism (OR: 0.84, 95% CI: 0.78-0.91, and p = 7,08 × 10-6), hyperthyroidism (OR: 0.60, 95% CI: 0.44-0.83, and p = 1.90 × 10-3), sarcoidosis (OR: 0.67, 95% CI: 0.54-0.83, and p = 2.60 × 10-4), and IPF (OR: 0.41, 95% CI: 0.29-0.58, and p = 4.11 × 10-7) in the FinnGen study. We observed that longer LTL was associated with an increased risk of AS susceptibility (OR: 1.51, 95% CI: 1.18-1.94, and p = 9.66 × 10-4). The results of the IVW method showed no causal relationship between TL and SLE (OR: 0.92, 95% CI: 0.62-1.38, and p = 0.69) in the FinnGen study; however, a significantly positive correlation was shown between LTL and SLE in another larger GWAS (OR: 1.87, 95% CI: 1.37-2.54, and p = 8.01 × 10-5). Conclusion: Our findings reveal that abnormal LTL has the potential to increase the risk of IMIDs. Therefore, it could be treated as a predictor and may provide new potential treatment targets for IMIDs. However, the change of LTL may not be the direct cause of IMIDs. Further studies should aim at the pathogenic mechanism or potential protective effects of LTL in IMIDs.

SS-31, a Mitochondria-Targeting Peptide, Ameliorates Kidney Disease.

Mitochondria are essential for eukaryotic cell activity and function, and their dysfunction is associated with the development and progression of renal diseases. In recent years, there has been a rapid development in mitochondria-targeting pharmacological strategies as mitochondrial biogenesis, morphology, and function, as well as dynamic changes in mitochondria, have been studied in disease states. Mitochondria-targeting drugs include nicotinamide mononucleotide, which supplements the NAD+ pool; mitochondria-targeted protective compounds, such as MitoQ; the antioxidant coenzyme, Q10; and cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. However, traditional drugs targeting mitochondria have limited clinical applications due to their inability to be effectively absorbed by mitochondria in vivo and their high toxicity. Recently, SS-31, a mitochondria-targeting antioxidant, has received significant research attention as it decreases mitochondrial reactive oxygen species production and prevents mitochondrial depolarization, mitochondrial permeability transition pore formation, and Ca2+-induced mitochondrial swelling, and has no effects on normal mitochondria. At present, few studies have evaluated the effects of SS-31 against renal diseases, and the mechanism underlying its action is unclear. In this review, we first discuss the pharmacokinetics of SS-31 and the possible mechanisms underlying its protective effects against renal diseases. Then, we analyze its renal disease-improving effects in various experimental models, including animal and cell models, and summarize the clinical evidence of its benefits in renal disease treatment. Finally, the potential mechanism underlying the action of SS-31 against renal diseases is explored to lay a foundation for future preclinical studies and for the evaluation of its clinical applications.

Administration of mesenchymal stem cells in diabetic kidney disease: mechanisms, signaling pathways, and preclinical evidence.

Diabetic kidney disease (DKD) is a serious microvascular complication of diabetes. Currently, the prevalence and mortality of DKD are increasing annually. However, with no effective drugs to prevent its occurrence and development, the primary therapeutic option is to control blood sugar and blood pressure. Therefore, new and effective drugs/methods are imperative to prevent the development of DKD in patients with diabetes. Mesenchymal stem cells (MSCs) with multi-differentiation potential and paracrine function have received extensive attention as a new treatment option for DKD. However, their role and mechanism in the treatment of DKD remain unclear, and clinical applications are still being explored. Given this, we here provide an unbiased review of recent advances in MSCs for the treatment of DKD in the last decade from the perspectives of the pathogenesis of DKD, biological characteristics of MSCs, and different molecular and signaling pathways. Furthermore, we summarize information on combination therapy strategies using MSCs. Finally, we discuss the challenges and prospects for clinical application.

Immunomodulatory Activity of Mesenchymal Stem Cells in Lupus Nephritis: Advances and Applications.

Lupus nephritis (LN) is a significant cause of various acute and chronic renal diseases, which can eventually lead to end-stage renal disease. The pathogenic mechanisms of LN are characterized by abnormal activation of the immune responses, increased cytokine production, and dysregulation of inflammatory signaling pathways. LN treatment is an important issue in the prevention and treatment of systemic lupus erythematosus. Mesenchymal stem cells (MSCs) have the advantages of immunomodulation, anti-inflammation, and anti-proliferation. These unique properties make MSCs a strong candidate for cell therapy of autoimmune diseases. MSCs can suppress the proliferation of innate and adaptive immune cells, such as natural killer cells (NKs), dendritic cells (DCs), T cells, and B cells. Furthermore, MSCs suppress the functions of various immune cells, such as the cytotoxicity of T cells and NKs, maturation and antibody secretion of B cells, maturation and antigen presentation of DCs, and inhibition of cytokine secretion, such as interleukins (ILs), tumor necrosis factor (TNF), and interferons (IFNs) by a variety of immune cells. MSCs can exert immunomodulatory effects in LN through these immune functions to suppress autoimmunity, improve renal pathology, and restore kidney function in lupus mice and LN patients. Herein, we review the role of immune cells and cytokines in the pathogenesis of LN and the mechanisms involved, as well as the progress of research on the immunomodulatory role of MSCs in LN.

Tofacitinib combined with leflunomide for treatment of psoriatic arthritis with IgA nephropathy: a case report with literature review.

Psoriasis is a systemic inflammatory disease that is associated with increased risk of several diseases, such as psoriatic arthritis (PsA), inflammatory bowel disease, and cardiovascular diseases. About 20 to 30% patients with psoriasis subsequently develop PsA. IgA nephropathy is the most common primary glomerular disease world-wide. Psoriasis and IgA nephropathy appear to be associated, but the mechanism underlying this connection is unclear. Tofacitinib and leflunomide are common treatments for psoriatic arthritis. We administered tofacitinib combined with leflunomide to a 38-year-old female patient who presented with PsA and IgA nephropathy. After treatment, she experienced significant reductions in the psoriatic lesions, pain in the right knee joint, and proteinuria. Administration of tofacitinib combined with leflunomide for treatment of a patient who had PsA complicated with IgA nephropathy led to significant resolution of the symptoms of both conditions. These results suggest similarities in the pathogenesis of PsA and IgA nephropathy and a possible new treatment for IgA nephropathy.

Cellular crosstalk of glomerular endothelial cells and podocytes in diabetic kidney disease.

Diabetic kidney disease (DKD) is a serious microvascular complication of diabetes and is the leading cause of end-stage renal disease (ESRD). Persistent proteinuria is an important feature of DKD, which is caused by the destruction of the glomerular filtration barrier (GFB). Glomerular endothelial cells (GECs) and podocytes are important components of the GFB, and their damage can be observed in the early stages of DKD. Recently, studies have found that crosstalk between cells directly affects DKD progression, which has prospective research significance. However, the pathways involved are complex and largely unexplored. Here, we review the literature on cellular crosstalk of GECs and podocytes in the context of DKD, and highlight specific gaps in the field to propose future research directions. Elucidating the intricates of such complex processes will help to further understand the pathogenesis of DKD and develop better prevention and treatment options.

Relationship between hyperuricemia and serositis in patients with lupus nephritis.

PURPOSE: To investigate the clinical and pathological characteristics of lupus nephritis (LN) patients with serositis and analyse the relationship between serositis and hyperuricemia (HUA) in LN patients in northeast China. METHODS: The data of patients with LN diagnosed by renal biopsy in our hospital from April 2013 to May 2020 were retrospectively analyzed. The differences between the non-serositis and serositis groups were compared by t tests and Chi-square test. Factors with P < 0.05 in the univariate analyses were investigated further using binary logistic regression analysis to investigate the independent risk factors of serositis in patients with LN. RESULTS: LN patients with serositis were more likely to have fever, hypertension, neuropsychiatric and hematological involvement than those without serositis (P < 0.05). Compared with the non-serositis group, LN patients with serositis were prone to have HUA, high D-dimer, high triglycerides, and had significant differences in the levels of plasma albumin (Alb), estimated glomerular filtration rate (eGFR), erythrocyte sedimentation rate, C-reactive protein, complement C3, 24-h urinary protein, pathological types, pathological score and SLEDAI score. Logistic regression analysis showed that HUA was one of risk factors for serositis in LN patients. The rate of complete remission in LN patients with serositis was significantly lower (P < 0.05) and the rate of no remission and mortality were significantly higher (P < 0.05) than LN patients without serositis. CONCLUSION: LN patients with serositis had more severe clinical and pathological manifestations, higher systemic lupus erythematosus (SLE) activity and worse prognosis. Hyperuricemia is associated with serositis in LN patients.

FFNT25 ameliorates unilateral ureteral obstruction-induced renal fibrosis.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) patients who progress to end-stage renal disease (ESRD). With the increasing incidence of CKD, it is of importance to develop effective therapies that blunt development of renal fibrosis. FFNT25 is a newly developed molecular compound that could be used to prevent fibrosis. In this study, we administered FFNT25 to rats following unilateral ureteral obstruction (UUO) to investigate its anti-fibrosis mechanism. Thirty-two Sprague-Dawley rats were randomly divided into four groups: (1) control (normal rats), (2) sham-operated, (3) UUO-operated + vehicle, and (4) UUO-operated + FFNT25. Two weeks after UUO, the rats were gavaged with either FFNT25 (20.6 mg/kg/day) or vehicle for two weeks. Serum, urine, and kidney samples were collected at the end of the study. FFNT25 reduced levels of renal fibrosis and decreased mRNA and protein levels of extracellular matrix (ECM) markers α-smooth muscle actin (α-SMA) and plasminogen activator inhibitor-1 (PAI-1) following UUO compared to vehicle treatment (n = 8, p<.05). The current results indicate that FFNT25 can affect both the production and degradation of collagen fibers to reduce fibrosis.

Elabela protects against podocyte injury in mice with streptozocin-induced diabetes by associating with the PI3K/Akt/mTOR pathway.

Diabetic nephropathy is a common complication of diabetes characterized by an increased rate of protein excretion in urine and kidney function loss. Elabela is a newly discovered peptide whose role in the regulation of diabetes is the major focus of this research. We established an in vivo model of Type 1 diabetes mellitus by injecting mice intraperitoneally with streptozotocin. The treatment group was administered Elabela for 6 months. In the present study, Elabela administration under diabetic conditions was found to reduce renal inflammation and fibrosis markers, leading to improvement in renal pathology and kidney dysfunction. Furthermore, Elabela acts through the phosphoinositide 3-kinase /Akt/mammalian target of rapamycin signaling pathway and decreases podocyte apoptosis, thereby exhibiting a nephroprotective effect against diabetic nephropathy. Our findings provide the first evidence that Elabela has a potential renoprotective effect in patients of diabetes.

Molecular dynamics based improvement of the solubilizing self-cleavable tag Zbasic-ΔI-CM application in the preparation of recombinant proteins in Escherichia coli.

Zbasic-ΔI-CM is a novel intein-based self-cleavable tag we developed to accelerate the soluble expression of recombinant proteins in Escherichia coli (E. coli). Previously we found that intein activity could be interfered by its flanking exteins, and thus reducing the production efficiency and final yield. In this work, we used CXC-chemokine 9 (CXCL9) as a model C-extein, which fusion with Zbasic-ΔI-CM showed high intein activity. When the fusion protein got soluble expression, CXCL9 was released immediately and purified directly from cell lysis supernatant. The results demonstrated that Zbasic-ΔI-CM tag had successfully mediated the efficient production of high-quality CXCL9 with reduced time and resources consumption in comparison with inclusion bodies expression. Molecular dynamics simulations suggested that the improved cleavage activity of Zbasic-ΔI-CM upon fusion with CXCL9 may be due to the higher dynamics of the first half loop and stabilization of the second half loop of intein. Our results proved that the self-cleavable Zbasic-ΔI-CM mediated soluble expression could be a feasible process for cytokines like CXCL9, thus of attractive potentials for production of therapeutic proteins using E. coli expression system.

Clinical and Pathological Analysis of 4910 Patients Who Received Renal Biopsies at a Single Center in Northeast China.

PURPOSE: To identify the epidemiology and pathological types of kidney diseases and their changes during the past decade, in a population from Northeast China. METHODS: We retrospectively analysed clinical and renal pathological data from 4910 patients who received renal biopsies in the Second Hospital of Jilin University from 2008 to 2017. RESULTS: Males received more renal biopsies than females (p < 0.001). The average age (p < 0.001) and percentage of elderly patients (p < 0.001) increased over time. The pathological types were primary glomerulonephritis (PGN, 73.2%), secondary glomerulonephritis (SGN, 23.7%), tubular-interstitial nephropathy (TIN, 2.8%), and hereditary nephropathy (HN, 0.3%). The most common forms of PGN were membranous nephropathy (MN, 37.2%) and IgA nephropathy (IgAN, 29.9%). Over time, the prevalence of IgAN decreased, but the prevalence of MN increased. MN was more common in middle-aged and elderly patients, but IgAN was most common in young adults. Analysis of SGN data indicated that lupus nephritis (LN, 34.0%), Henoch-Schönlein purpura glomerulonephritis (HSPN, 17.9%), and diabetic nephropathy (DN, 11.7%) were the most common forms. Over time, the prevalence of DN (p = 0.003), hypertension-associated renal damage (p = 0.005), and systemic vasculitis-associated nephritis (SVARD, p < 0.001) increased, but the prevalence of HSPN (p < 0.001) and hepatitis B virus-associated glomerulonephritis (HBV-GN, p = 0.001) decreased. Nephrotic syndrome was the main clinical manifestation of PGN. CONCLUSION: From 2008 to 2017, renal biopsies were increasingly performed in the elderly. There were notable changes in the epidemiology and pathological types of kidney disease among renal biopsy patients at our centre.

A general platform for efficient extracellular expression and purification of Fab from Escherichia coli.

Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.

Development of a recombinant human IL-15·sIL-15Rα/Fc superagonist with improved half-life and its antitumor activity alone or in combination with PD-1 blockade in mouse model.

Recombinant human interleukin-15 (IL-15) is a potent cancer immunotherapeutic candidate due to its excellent immune stimulating effects. Previous work demonstrated that IL-15 appeared with short half-life in circulation system, while the complex with its receptor can prolong the half-life as well as benefit its activities in vivo. Therefore, IL-15 complex was more favorably considered for clinical development. Herein we developed IL-15·sIL-15Rα/Fc, a complex comprising of IL-15 and the extracellular region of its receptor alpha subunit which fused to Immunoglobulin G (IgG1) Fc to further prolong the half-life in plasma. Through transient gene expression in HEK293 cells, we expressed the superagonist by co-transfection of plasmids encoding IL-15 and sIL-15Rα/Fc respectively, yielding 36 mg/L of product after purification. Pharmacokinetic study demonstrated that the combination profoundly prolonged the half-life of IL-15 to 13.1 h in mice, about 18 folds longer than that of IL-15 monomer which is around 0.7 h. The bioactivity of the superagonist was characterized by CTLL-2 cells proliferation assay in vitro, showing its capability of stimulating the expansion of memory CD8+ T cells (cluster of differentiation) in mouse spleen. Using a HT-29 xenograft NOD-SCID mouse model, we observed tumor growth inhibition in all groups that received the superagonist, indicating its anti-tumor efficacy via stimulating infused human immune cells. In addition, combo cancer treatment by IL-15·sIL-15Rα/Fc and programmed death-1 (PD-1) antibody have shown stronger inhibitory effects as compared with treatment with either single molecule. Therefore, we developed IL-15·sIL-15Rα/Fc to be a long half-life potential cancer immunotherapy candidate that can be applied alone or in synergy with PD-1/PD-L1 blockade.

A PhoA-STII Based Method for Efficient Extracellular Secretion and Purification of Fab from Escherichia coli.

In comparison with full-length IgGs, antigen binding fragments (Fabs) are smaller in size and do not require the complexed post-translational modification. Therefore, Fab can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, the disulfide-bonds containing exogenous protein, including Fab, tend to form insoluble inclusion bodies in E. coli, which has been the bottleneck for exogenous protein expressions using this system. The secretory expression of proteins in periplasm or extracellular medium are promising strategies to prevent the formation of inclusion bodies to improve the efficiency to produce Fabs from E. coli. The extracellular expression is of particularly interest since it releases the product into the medium, while periplasmic expression yield is limited to the periplasm space. In addition, the extracellular expression allows for the direct harvesting of proteins from the culture supernatant, sparing the procedures of cell lysis and reducing contamination of host cell protein or DNA. Using anti-VEGF Fab as an example, here we provide a protocol based on the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence. Using phosphate starvation to induce the secretory expression, the protocol could be generally used for the efficient production of Fabs.