Purpose
The therapeutic potential of targeting the human epidermal growth factor receptor-3 (ErbB3/HER3) has long been ignored due to impaired tyrosine kinase function and low expression level in ...tumor cells compared with EGFR and HER2. Although recent investigations have explored the potential benefit of HER3 targeting and several anti-HER3 agents have been developed, there is still a critical need to design and produce more efficient therapeutics. This study was designed to develop tumor inhibitory monoclonal antibodies (MAbs) against different extracellular subdomains of HER3.
Methods
Distinct extracellular subdomains of HER3 (D
I+II
and D
III+IV
) were utilized to produce MAbs by hybridoma technology. Biochemical and functional characteristics of these MAbs were then investigated by various methodologies, including immunoblotting, flow cytometry, cell proliferation, cell signaling, and enzyme-linked immunosorbent assays.
Results
Four anti-D
I+II
and six anti-D
III+IV
MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-D
I+II
and four anti-D
III+IV
MAbs recognized the native form of HER3 by immunoblotting. Four MAbs recognized the membranous HER3 by flow cytometry leading to induction of different levels of receptor internalization and subsequent degradation. Results of cell proliferation assays using these MAbs indicated that they differentially inhibited proliferation of HER3-expressing cancer cells and showed considerable synergistic effects in combination with trastuzumab. Selected MAb with the highest inhibitory effect significantly inhibited the phosphorylation of AKT and ERK1/2 molecules.
Conclusion
Some of the anti-HER3 MAbs produced in this study displayed tumor inhibitory function and may be considered promising candidates for future HER3-targeted cancer therapy.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Our previous microscopic observations on the wet mount of cultured Candida yeast showed release of large extracellular vesicles (EVs) that contained intracellular bacteria (∼500–5000 nm). We used ...Candida tropicalis, to examine the internalization of nanoparticles (NPs) with different properties to find out whether the size and flexibility of both EVs and cell wall pores play role in transport of large particles across the cell wall.
Candida tropicalis was cultured in N-acetylglucoseamine-yeast extract broth (NYB) and examined for release of EVs every 12 h by the light microscope. The yeast was also cultured in NYB supplemented with of 0.1%, 0.01% of Fluorescein isothiocyanate (FITC)-labelled NPs; gold (0.508 mM/L and 0.051 mM/L) (45, 70 and 100 nm), albumin (0.0015 mM/L and 0.015 mM/L) (100 nm) and Fluospheres (0.2 and 0.02%) (1000 and 2000 nm). Internalization of NPs was recorded with fluorescence microscope after 30 s to 120 min. Release of EVs mostly occurred at 36 h and concentration of 0.1% was the best for internalization of NPs that occurred at 30 s after treatment. Positively charged 45 nm NPs internalized into >90% of yeasts but 100 nm gold NPs destroyed them. However, 70 nm gold and 100 nm negatively-charged albumin were internalized into <10% of yeasts without destroying them. Inert Fluospheres either remained intact on the surface of yeasts or became degraded and internalized into ∼100% of yeasts. Release of large EVs from the yeast but internalization of 45 nm NPs indicated that flexibility of EVs and cell wall pores as well as physicochemical properties of NPs determine transport across the cell wall.
•Nanoparticles properties determine their internalization into yeast cell.•Size and flexibility of yeast’s cell wall pores determine nanoparticles entry.•Flexibility of extracellular vesicles play role in their release from yeast cell.•Fungal cell wall has evolved as a dynamic structure for efficient uptake and export.•Cell wall pores and extracellular vesicles play critical roles in fungal survival.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Overexpression of HER2 has been reported in many types of cancer, making it a perfect candidate for targeted immunotherapy. The combination of two FDA approved monoclonal antibodies (mAbs), ...trastuzumab and pertuzumab, has more robust anti-tumor activity in patients with HER2-overexpressing breast cancer. We recently produced a new humanized anti-HER2 mAb, hersintuzumab, which recognizes a different epitope than trastuzumab and pertuzumab on HER2. This mAb, in combination with trastuzumab, exhibits more potent anti-tumor activity than each parental mAb alone. Here we have developed a novel bispecific anti-HER2 antibody (BsAb) designated as trasintuzumab, composed of trastuzumab and hersintuzumab, using dual variable domain immunoglobulin (DVD-Ig) technology. Both variable domains of trasintuzumab are fully functional and have similar affinities to the parental mAbs and are also able to bind to natural HER2 on the surface of several HER2-expressing cell lines. Trasintuzumab was found to inhibit the growth of different types of tumor cell lines through suppression of the AKT and ERK signaling pathways as efficiently as the combination of the parental mAbs. It also induced tumor regression as potently as the combination of the two mAbs in nude mice bearing ovarian and gastric cancer xenografts. Our data suggest that trasintuzumab may be a promising BsAb therapeutic candidate for the treatment of HER2-overexpressing cancers.
Summary
Immunotherapy of HER2-overexpressing cancers by FDA approved monoclonal antibodies (mAbs) such as trastuzumab and pertuzumab has shown promising results. We have recently produced a novel ...humanized anti-HER2 mAb, hersintuzumab, which did not sterically inhibit binding of trastuzumab and pertuzumab to HER2, thus recognizing a distinct epitope on subdomain I + II of HER2. In this study, we assessed the in vitro and in vivo anti-tumor activity of this mAb individually and in combination with trastuzumab. Different HER2-overexpressing human cancer cell lines, including SKOV3, NCI-N87 HCC1954 and BT-474 were cultured and binding reactivity of Hersintuzumab to these cell lines was analyzed by flow cytometry. In addition, the inhibitory effect of different concentrations of hersintuzumab, trastuzumab and their combination on tumor cells growth was assessed by XTT assay. For Assessment of tumor growth inhibition in xenograft model, Balb/c athymic nude mice were subcutaneously injected with NCI-N87 and SKOV3 tumor cells and then treated intravenously with these mAbs. Our results showed that hersintuzumab could bind to all HER2-overexpressing cell lines similar to trastuzumab. In vitro experiments showed that both hersintuzumab and trastuzumab individually and in combination inhibited growth of all cell lines with the exception of HCC-1954.Inhibitory effect of the combination of mAbs was significantly higher than that of each mAb alone. Similar results were obtained in the gastric (NCI-N87) and ovarian (SKOV-3) tumor xenograft models. Hersintuzumab in combination with trastuzumab induces synergic anti-tumor effects on HER2-overexpressing cells in vitro and in vivo and is potentially a therapeutic tool for treatment of HER2-overexpressing cancers.
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CEKLJ, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Introduction: Rheumatoid Arthritis (RA) is the most common inflammatory rheumatic disease and there are some concerns about the association of Metabolic Syndrome (MetS) with this disorder. Aim: To ...evaluate the association between MetS and its components with RA disease activity and chronicity. Materials and Methods: The present investigation was a cross-sectional study on 200 consecutive RA patients (30-60- year-old, all of them female) according to the American College of Rheumatology/European League Against Rheumatism (ACR/ EULAR) 2010. MetS was diagnosed according to the definition of 2005 National Cholesterol Education Program Adult Treatment Panel III (NCEP/ATP) III and International Diabetes Federation (IDF). Disease activity was measured by the 28 joint count of Disease Activity Score (DAS 28), and functional status was evaluated by Health Assessment Questionnaire (HAQ). Disease chronicity was defined as early or established RA, based on disease duration of less or more than six months. Statistical analysis was performed in SPSS version 19 by Student’s t-test and chi-square test and ANOVA tests and results were considered significant at p<0.05. Results: The mean age of RA patients was 50.29±6.2 years, and all were female. MetS according to NCEP/ATP III and IDF criteria was present in 109 (54.5%) and 112 (56%) patients. Hyperglycaemia was significantly higher in patients with established active RA (p=0.003), but other MetS components were not different between groups. MetS had more related active disease as assessed by DAS28 and greater disability as assessed by the HAQ (p<0.001). Conclusion: The frequency of MetS was not significantly different in subgroups of RA according to disease activity or duration, but it was found in more than half of RA patients. Comparison of MetS components showed more frequency of hyperglycaemia in patients with active disease.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Aim
This study aimed to investigate psychiatric disorders in Iranian female patients with fibromyalgia (FM).
Design
Female patients, newly diagnosed with FM, were interviewed by a psychiatrist for ...psychiatric assessments during a 2‐year period.
Methods
The diagnosis of the psychiatric disorders was based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM‐5), and sleep quality was evaluated using the Pittsburgh Sleep Quality Index (PSQI).
Results
In total, 159 patients with the mean age of 42.15 ± 9.89 were recruited in this study. Over 92% of the cases were also diagnosed with at least one type of psychiatric disorder. Sleep disorders (SDs, 90.57%), mood disorders (MDs, 52.83%), personality disorders (PDs, 40.25%) and anxiety disorders (ADs, 16.98%) were the most prevalent diagnoses among these patients. The logistic regression results correspondingly showed that suffering from Cluster‐B PDs was associated with a higher prevalence rate of somatic symptom disorders (SSDs), with a p‐value of 0.019 and an odds ratio (OR) of 2.7.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK, VSZLJ
Background
Producing therapeutic proteins can be done quickly and on a large scale through Transient Gene Expression (TGE). Chinese hamster ovary (CHO) cell lines are commonly used to achieve this. ...Although there are few comparative studies, TGE has been observed in suspension-adapted CHO cells.
Objectives
We tested TGE’s effectiveness in DG-44, CHO-S, and ExpiCHO-S cell lines with four transfection reagents.
Methods
A design of experiments (DoE) was followed to optimize transfection using a recombinant monoclonal antibody (mAb) construct. To evaluate the efficacy, flow cytometry and ELISA were used. Feeding strategies and temperature shifts were implemented to enhance transfection effectiveness. The quality of the mAb was assessed through ELISA, SDS-PAGE, and proliferation inhibition assays.
Results
We adapted all cell lines to grow in suspension using a serum-free medium. Our findings from flow cytometry and ELISA tests indicate that PEI and Pmax reagents had a higher rate of transfection and mAb production than the ExpiCHO commercial transfection reagent. While DG-44 cells had better transfection efficiency than CHO-S and ExpiCHO-S, there was no significant difference between CHO-S and ExpiCHO-S. Our TGE system was more productive at 32 °C than at 37 °C. In the optimized TGE of Pmax-based transfection in DG-44 at 37 and 32 °C, the production level of mAb was more than half of the amount of the commercial ExpiCHO-S expression system. Still, the number of transfected cells was three times higher, making it more efficient. The purified mAb from all transfected cell lines had similar structural and functional properties under different conditions.
Conclusion
Our research shows that using Pmax and DG-44 cells in the TGE system is a cost-effective and efficient way to produce humanized monoclonal antibodies. We discovered that this method outperforms the ExpiCHO-S kit.
Graphical abstract
The cause of coronavirus disease 2019 (COVID-19) is a virus which can lead to severe acute respiratory syndrome-CoV-2 (SARS-COV-2). There are evidences of involvement of immune system in pathogenesis ...of this disease. We investigated the presence of various vasculitis-associated autoantibodies and complement levels in patients with COVID-19.
Patients with severe or critical type of COVID-19 were evaluated for symptoms, signs, and laboratory tests of vasculitis syndromes including rheumatoid factor (RF), antinuclear antibody (ANA), anti-double-stranded DNA, c and p anti-neutrophilic cytoplasmic antibody (c ANCA and P ANCA), and complement levels.
The study was performed in forty patients with severe or critical illness. The mean age of the participants was 48.5 ± 9.8 years. All patients had pulmonary involvement in lung computed tomography scans. Vasculitis laboratory test results included RF in two patients, ANA in three patients, and ANCA in one patient. Seventeen (42.5%) patients had hypocomplementemia in one or more complement tests. Four patients expired, of whom three had a decrease in complement level.
Decrease in complement levels may predict a critical state of COVID-19 disease. Therefore, measuring its levels may be of great benefit in making earlier decisions to initiate disease-suppressing treatments including corticosteroids.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Correction to: Efficacy of Stretching Exercises on Fibromyalgia Impact and Quality of Life in Patients under Medical Treatment: A Randomized Clinical Trial, 2021 Zahra Madani1,2, Sahar Bagheri1, ...Forozan Elyasi3,4, Maryam Mobini5, Jamshid Yazdani Charati6, Faeze MalekLou7, Hanieh Ahmadi8 1 Assistant Professor, Diabetes Research Center, Mazandaran University of Medical Sciences, Sari, Iran 2 Assistant Professor, Department of Sports Medicine, Traditional and Complementary Medicine Research Center, Mazandaran University of Medical Sciences, Sari, Iran 3 Associate Professor, Psychiatry and Behavioral SciencesResearch Center, Sexual and ReproductiveHealth Research Center, AddictionInstitute, Mazandaran University ofMedical Sciences, Sari, Iran 4 Department of Psychiatry, Faculty ofMedicine, Mazandaran University of Medical Sciences, Sari, Iran 5 Associate Professor, Diabetes Research Center, Imam Khomeini Hospital, Mazandaran University of Medical Sciences, Sari, Iran 6 Professor, Department of Biostatistics, Faculty of Health, Mazandaran University of Medical Sciences, Sari, Iran 7 MD, Sport Medicine Specialist, Tehran , Iran 8 Assistant Professor, Department of Sports Medicine, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran In the article published in volume 33, issue 219, 2023, the affiliations of Forozan Elyasi and Faeze MalekLou were published incorrectly, which are now corrected.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Systemic lupus erythematosus (SLE), often affects women of childbearing age. Family planning consultation is a major aspect of medical care in these patients because of the risk of disease activation ...and poor pregnancy and fetal outcomes. The aim of the present study was to evaluate contraceptive prevalence and consulting service in women with SLE.
In a cross-sectional study, a total of 144 female patients with SLE, ages 15-50, who were presented to rheumatology clinics in Sari, north of Iran, were evaluated. The study was conducted between March 2019 and May 2020. Patients' clinico-demographic profile and fertility information were obtained. Disease activity and damage were assessed by the systemic lupus erythematosus disease activity (SLEDAI) and Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index (SDI).
One hundred and forty-four SLE patients of childbearing age participated in this study. From 102 patients with the possibility of pregnancy, 36(35.2%) received contraceptive consultations in last year. Withdrawal was the most prevalent contraceptive method (41.7%), followed by permanent (11.8%), and barrier methods (9%). There were no significant differences in age, disease duration, marriage duration, SDI or SLEDAI scores between the women who received or not received contraceptive counseling (P>0.05).
Many SLE patients did not receive adequate information about contraception, and it may be associated with many adverse effects on disease activity and pregnancy outcomes. Therefore, contraceptive consultation as an important aspect of patient's management is strongly suggested.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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