Clinico-Pathological and Prognostic Significance of a Combination of Tumor Biomarkers in Iranian Patients With Breast Cancer.

PURPOSE: Breast cancer is one of the most common cancers in the world. It is a multifaceted malignancy with different histopathological and biological features. Molecular biomarkers play an essential role in accurate diagnosis, classification, prognosis, prediction of treatment response, and cancer surveillance. This study investigated the clinico-pathological and prognostic significance of HER3 and ROR1 in breast cancer samples. METHODS: Tissue microarrays (TMA) were constructed using tissue blocks of 444 Iranian breast cancer patients diagnosed with breast cancer. Overall survival (OS) and disease-free survival (DFS) were assessed after 5 years follow-up. TMA slides were stained with monoclonal antibodies against ROR1, HER3, ER, PR, Ki67, P53, HER2 and CK5/6 using IHC and correlation between the investigated tumor markers and the clinico-pathological parameters of patients were analyzed. RESULTS: Our results showed a significant correlation between ROR1 and ER, PR, HER3, and CK5/6 expression. Additionally, there was a significant correlation between HER3 and ER, PR, HER2, and Ki67 expression. Ki67 was also correlated with HER2 and P53 expression. HER3 expression was significantly correlated with tumor stage, lymph node metastasis, perineural invasion, and multifocal tumors. Furthermore, ROR1 expression was significantly associated with tumor metastasis, lympho-vascular invasion, and perineural invasion. While HER2-HER3 coexpression was significantly associated with poor OS, HER3-ROR1 coexpression was associated with lymph node invasion, lymph node metastasis, and distant metastasis. CONCLUSION: ROR1 and HER3 displayed significant association with different clinic-pathological features and in addition to the other tumor biomarkers could be considered as diagnostic and prognostic biomarkers in breast cancer patients.

Comparison of different transient gene expression systems for the production of a new humanized anti-HER2 monoclonal antibody (Hersintuzumab).

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.

Cross-Reactivity of HBe Antigen-Specific Polyclonal Antibody with HBc Antigen.

Hepatitis B virus (HBV) infection is a major health problem worldwide and causes almost one million deaths annually. The HBV core gene codes for two related antigens, known as core antigen (HBcAg) and e-antigen (HBeAg), sharing 149 residues but having different amino- and carboxy-terminals. HBeAg is a soluble variant of HBcAg and a clinical marker for determining the disease severity and patients' screening. Currently available HBeAg assays have a shortcoming of showing cross-reactivity with HBcAg. In this study, for the first time, we evaluated whether HBcAg-adsorbed anti-HBe polyclonal antibodies could specifically recognize HBeAg or still show cross-reactivity with HBcAg. Recombinant HBeAg was cloned in pCold1 vector and successfully expressed in Escherichia coli and after purification by Ni-NTA resin was used to generate polyclonal anti-HBe antibodies in rabbit. Purified HBeAg was further characterized by assessing its reactivity with anti-HBe in the sera of chronically infected patients and HBeAg-immunized rabbit. Sera from patients with chronic HBV infection, containing anti-HBe, specifically reacted with recombinant HBeAg, implying antigenic similarity between the prokaryotic and native HBeAg in the serum of HBV-infected patients. In addition, the designed enzyme-linked immunosorbent assay (ELISA) with rabbit anti-HBe polyclonal antibodies could detect recombinant HBeAg with high sensitivity, while high cross-reactivity with HBcAg was observed. It is noteworthy that HBcAg-adsorbed anti-HBe polyclonal antibodies still showed high cross-reactivity with HBcAg, implying that due to the presence of highly similar epitopes in both antigens, HBcAg-adsorbed polyclonal antibodies cannot differentiate between the two antigens.

Novel Monoclonal Antibodies Specific for Human Ki67 and P53 Tumor Markers in Breast Cancer Tissue Samples.

Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test. Objective: To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes. Methods: Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs. Results: Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer. Conclusion: The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.

Physicochemical properties of intact fungal cell wall determine vesicles release and nanoparticles internalization.

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.

The frequency of psychiatric disorder co-morbidities in patients with fibromyalgia: A cross-sectional study in Iran.

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.

A Novel Fc-Engineered Anti-HER2 Bispecific Antibody With Enhanced Antitumor Activity.

Human epidermal growth factor receptor 2 (HER2) overexpression has been demonstrated in a variety of cancers. Targeted therapy with anti-HER2 monoclonal antibodies (mAbs) has been approved as a therapeutic modality. Despite the efficacy of mAbs in tumor treatment, many patients do not benefit from this therapeutic platform. Fragment crystallizable (Fc) engineering is a common approach to improve the efficacy of therapeutic mAbs. Five Fc-engineered mAbs have so far been approved by FDA. We have recently developed an anti-HER2 bispecific mAb, BiHT, constructed from variable domains of trastuzumab, and our novel humanized anti-HER2 mAb, hersintuzumab. BiHT displayed promising antitumor activity as potently as the combination of the parental mAbs. Here, we aimed to modify the Fc of BiHT to improve its therapeutic efficacy. The Fc-engineered BiHT (MBiHT) bound to recombinant HER2 and its subdomains with an affinity similar to BiHT. It also recognized native HER2 on different cell lines, inhibited their proliferation, downregulated HER2 expression, and suppressed downstream signaling pathways similar to BiHT. Compared with BiHT, MBiHT displayed enhanced antibody-dependent cellular cytotoxicity activity against various tumor cell lines. It also inhibited the growth of ovarian xenograft tumors in nude mice more potently than BiHT. Our findings suggest that MBiHT could be a potent therapeutic candidate for the treatment of HER2-overexpressing cancer types.

Evaluation of Clinical, Laboratory, and Radiologic Findings of Pulmonary and Extrapulmonary Involvement in Sarcoidosis.

Background: Sarcoidosis is a systemic disease with unknown etiology that is characterized by the presence of granuloma in various organs with diverse pulmonary and extrapulmonary manifestations. Regarding differences in the presentation of sarcoidosis in different geographical areas, the present study aimed to determine clinical, laboratory, and radiologic findings of patients with sarcoidosis in the north of Iran. Materials and Methods: In a cross-sectional study, patients with sarcoidosis were enrolled, and demographic data in addition to disease manifestations including clinical, laboratory, and imaging findings were recorded. Results: A total of 58 patients with sarcoidosis were enrolled in the study. The mean age and disease duration were 51.10±10.2 and 3.07±2.7 years, respectively. 62.1% of patients were female. Clinical manifestations were: cough and dyspnea (55.2%), fever and weight loss (11%), arthritis (15.5%), dermatologic presentation (15.5%), and ophthalmic involvement (17.2 %). Abnormalities in liver, renal, and calcium levels are found in approximately 1-8% of cases. The ACE level was increased in 56.9 % of patients, especially in those who presented in summer and autumn. Chest CT abnormalities were found in 94.8 % of patients, more predominantly hilar and paratracheal lymphadenopathy in 84.5% and 74.1%, respectively. Conclusion: Although sarcoidosis presents with varying clinical, radiological, and laboratory features, knowledge of its epidemiology and the incidence of these features in different populations can aid in its diagnosis in a particular geographic area.

Evaluation of Antiphospholipid Antibodies in COVID-19 Patients with Coagulopathy.

Background: Due to the importance of recent published studies regarding the thrombotic events in COVID-19 patients, the purpose of this study was to evaluate the frequency of antiphospholipid antibodies in COVID-19 patients with coagulopathy. Materials and Methods: The present cross-sectional study was conducted on COVID-19 patients with coagulopathy admitted to Imam Khomeini Hospital in Sari, Iran, between June and September in 2020. Later on, the levels of anti-phospholipid antibodies (aPL-ab) and biochemical factors were measured. Results: This study was performed on 40 patients. Individuals who were positive for at least one of the aPL-ab were classified in the group of aPL-ab positive; according to which 29 patients (72.5%) had no positive aPL-ab and 11 patients (27.5%) had at least one positive aPL-ab. 8 patients were only positive for lupus anticoagulant (LA) assay, one patient had B2GPI- IgM, one patient had aCL-IgG and only one patient had two positive simultaneous tests for LA and aCL-IgG. Thrombotic events have been found in 7 patients (17.5%) of which, three patients with deep vein thrombosis, one patient with pulmonary embolism, two patients with stroke, and one patient with myocardial infarction. The values of aPTT for the screening of Lupus anticoagulant assay were significantly different between the two groups, although there was no significant difference between the two groups in the co-morbidities, disease severity, death and laboratory tests (P> 0.05). Conclusion: Despite the high incidence of thrombotic complications reported in COVID-19 patients in the current study, the levels of antiphospholipid antibodies had no significant correlation with the occurrence of thromboembolic events and disease outcome in COVID-19 patients with coagulopathy.

Generation and Characterization of Novel Diagnostic Mouse Monoclonal Antibodies Against Human Estrogen Receptor Alpha and Progesterone Receptor.

BACKGROUND: Estrogen and progesterone regulate the growth and development of several human cells and tissues. Their corresponding receptors (ER and PR) are important diagnostic and prognostic indicators for cancers of the breast and reproductive organs. Immunohistochemical analysis of ER and PR is the current standard method for evaluating the expression of these receptors in different cancers. Nonetheless, there is a significant lack of reproducibility of IHC results in laboratories worldwide, necessitating to develop more sensitive and specific antibodies for ER and PR IHC staining. METHODS: ER and PR-specific monoclonal antibodies (MAbs) were generated by immunizing mice with synthetic peptides from ERα and PR. The isotypes and affinity constants of the selected MAbs were determined, and their specificities were assessed by peptide-specific ELISA, IHC, Western-blot analysis, and flow cytometry. In addition, the reactivity of generated MAbs was compared with that of the commercially-available anti-ER and anti-PR antibodies in IHC using normal and cancerous tissue sections. Moreover, 200 breast cancer tissue samples were stained using the newly generated MAbs along with commercial antibodies by IHC, and the sensitivity, specificity and accuracy of our MAbs were evaluated. RESULTS: Among different MAbs generated in this study, two anti-ER and one anti-PR MAbs specifically detected the target antigens in normal and cancerous tissues in IHC. Further analyses confirmed the specificity of the MAbs in Western blotting and flow cytometry using a panel of ER and PR positive cell lines. The sensitivity, specificity and accuracy calculated for clone 1B9 (anti-ER) were 92.3%, 94.8% and 93%, and for clone 3D6 (anti-PR) were 93.0%, 94.3% and 93.5%, respectively. CONCLUSION: Our novel anti-ER and PR MAbs could be considered as suitable tools for diagnostic and research purposes.

Epitope mapping of severe acute respiratory syndrome coronavirus 2 neutralizing receptor binding domain-specific monoclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the outbreak led to the coronavirus disease 2019 (COVID-19) pandemic. Receptor binding domain (RBD) of spike (S) protein of SARS-CoV-2 is considered as a major target for immunotherapy and vaccine design. Here, we generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology. Epitope mapping was performed using a panel of 20-mer overlapping peptides spanning the entire sequence of the RBD protein from wild-type (WT) Wuhan strain by enzyme-linked immunosorbent assay (ELISA). Several hybridomas showed reactivity toward restricted RBD peptide pools by Pepscan analysis, with more focus on peptides encompassing aa 76-110 and 136-155. However, our MAbs with potent neutralizing activity which block SARS-CoV-2 spike pseudovirus as well as the WT virus entry into angiotensin-converting enzyme-2 (ACE2) expressing HEK293T cells showed no reactivity against these peptides. These findings, largely supported by the Western blotting results suggest that the neutralizing MAbs recognize mainly conformational epitopes. Moreover, our neutralizing MAbs recognized the variants of concern (VOC) currently in circulation, including alpha, beta, gamma, and delta by ELISA, and neutralized alpha and omicron variants at different levels by conventional virus neutralization test (CVNT). While the neutralization of MAbs to the alpha variant showed no substantial difference as compared with the WT virus, their neutralizing activity was lower on omicron variant, suggesting the refractory effect of mutations in emerging variants against this group of neutralizing MAbs. Also, the binding reactivity of our MAbs to delta variant showed a modest decline by ELISA, implying that our MAbs are insensitive to the substitutions in the RBD of delta variant. Our data provide important information for understanding the immunogenicity of RBD, and the potential application of the novel neutralizing MAbs for passive immunotherapy of SARS-CoV-2 infection.

Visual Landing Based on the Human Depth Perception in Limited Visibility and Failure of Avionic Systems.

This paper introduces a novel visual landing system applicable to the accurate landing of commercial aircraft utilizing human depth perception algorithms, named a 3D Model Landing System (3DMLS). The 3DMLS uses a simulation environment for visual landing in the failure of navigation aids/avionics, adverse weather conditions, and limited visibility. To simulate the approach path and surrounding area, the 3DMLS implements both the inertial measurement unit (IMU) and the digital elevation model (DEM). While the aircraft is in the instrument landing system (ILS) range, the 3DMLS simulates more details of the environment in addition to implementing the DOF depth perception algorithm to provide a clear visual landing path. This path is displayed on a multifunction display in the cockpit for pilots. As the pilot's eye concentrates mostly on the runway location and touch-down point, "the runway" becomes the center of focus in the environment simulation. To display and evaluate the performance of the 3DMLS and depth perception, a landing auto test is also designed and implemented to guide the aircraft along the runway. The flight path is derived simultaneously by comparison of the current aircraft and the runway position. The Unity and MATLAB software are adopted to model the 3DMLS. The accuracy and the quality of the simulated environment in terms of resolution, the field of view, frame per second, and latency are confirmed based on FSTD's visual requirements. Finally, the saliency map toolbox shows that the depth of field (DOF) implementation increases the pilot's concentration resulting in safe landing guidance.

Differential tumor inhibitory effects induced by HER3 extracellular subdomain-specific mouse monoclonal antibodies.

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 (DI+II and DIII+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-DI+II and six anti-DIII+IV MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-DI+II and four anti-DIII+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.

Immunologic markers, vasculitis-associated autoantibodies, and complement levels in patients with COVID-19.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Contraceptive Prevalence and Consulting Service in Women with Systemic Lupus Erythematosus: A Cross-Sectional Study.

BACKGROUND: 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. METHODS: 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). RESULTS: 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). CONCLUSION: 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.

Influence of Pattern Recognition Receptor Ligands on Induction of Innate Immunity and Control of Hepatitis B Virus Infection.

Failure of current therapies to cure chronic hepatitis B has led to renewed interest in therapies that stimulate the host immune system. APOBEC3 (A3) family enzymes have been shown to induce mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) leading to inhibition of HBV transcription and replication. Pattern recognition receptor (PRR) agonists have been reported to suppress HBV, but it is unclear whether these agonists induce A3 gene expression in hepatocytes. We, therefore, evaluated whether PRR signaling activates the expression of A3 genes and other innate immunity genes and restricts HBV infection. HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were infected with HBV and treated with various PRR agonists. The level of HBV infection was subsequently assessed by measurement of HBV biomarkers, including HBV DNA, cccDNA, HBs, and HBe antigens in infected hepatocytes. Among all tested PRR ligands, only Poly(I:C)-HMW/LyoVec and Poly(I:C)-HMW significantly inhibited hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), HBV DNA, and cccDNA, whereas R848 and lipopolysaccharide (LPS) only showed significant inhibition on HBsAg and HBeAg, but not virus DNA. CpG and Pam3CSK4, on the other hand, had no significant inhibitory effect on any of the HBV infection parameters. Moreover, Poly(I:C)-HMW/LyoVec and Poly(I:C)-HMW were the only ligands that significantly increased IL-8 secretion. Interestingly, HBV infection reduced IL-8 secretion induced by Poly(I:C)-HMW and to a lesser extent Poly(I:C)-HMW/LyoVec. Poly(I:C)-HMW/LyoVec had a significant effect on increasing the expression level of A3F, A3G, A3H, TLR3, RIG-1, and MDA5 genes. Our data suggest that PRR agonists may control HBV infection through different mechanisms. The RIG-1 and MDA5 agonist, Poly(I:C)-HMW/LyoVec, seems to downregulate HBV infection through induction of A3 genes.

A novel tumor inhibitory hybridoma monoclonal antibody with dual specificity for HER3 and HER2.

BACKGROUND: The human epidermal growth factor receptor (HER/ErbB) family-targeted therapies result in a significant improvement in cancer immunotherapy. Monoclonal antibodies (MAb) against HER2 demonstrated a survival benefit for patients; however, drug resistance unavoidably occurs due to the overexpression of HER3, which leads to treatment failure. Effective inhibition of HER3 besides HER2 is thought to be required to overcome resistance and enhance therapeutic efficacy. OBJECTIVE: The present study describes the production and characterization of a novel MAb, designated 1G5D2, which acts as a natural bispecific antibody targeting extracellular domains (ECD) of both HER2 and HER3. METHODS: In this study, 1G5D2 was produced by hybridoma technology against HER3-ECD, and its structural and functional characteristics were studied by various methodologies, including enzyme linked-immunosorbent assays, flow cytometry, immunoblotting, cell signaling, and cell proliferation assays. RESULTS: 1G5D2 specifically binds to both HER2 (subdomain III + IV) and HER3 (subdomain I + II) expressed on tumor cells, and these receptors compete with each other for binding to this MAb. Competition flow cytometry experiments demonstrated that 1G5D2 does not compete with heregulin and recognizes an epitope out of HER3 ligand-binding site. Evaluation of 1G5D2 inhibitory effects in tumor cell lines co-expressing HER2 and HER3 showed that 1G5D2 synergizes with trastuzumab to inhibit both PI3K/AKT and MAPK/ERK pathways and potently downregulates the proliferation of these tumor cells more efficiently than each MAb alone. CONCLUSION: 1G5D2 is the first reported hybridoma antibody, which acts as a natural HER2/HER3 bispecific antibody. It might potentially be a suitable therapeutic candidate for HER2/HER3 overexpressing cancer types.

Potent synergistic anti-tumor activity of a novel humanized anti-HER2 antibody hersintuzumab in combination with trastuzumab in xenograft models.

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.

Heerfordt-Waldenström Syndrome, A Rare Presentation of Sarcoidosis in a Patient with Old Ocular Toxoplasmosis.

BACKGROUND: There are similarities between the ophthalmic presentation of toxoplasmosis and sarcoidosis, and there are some concerns of immunosuppressive treatments for sarcoidosis, which may lead to T. gondii reactivation. We report a rare case with acute sarcoidosis (Heerfordt- Waldenström syndrome) with a history of ocular toxoplasmosis from the North of Iran. CASE PRESENTATION: The patient was a 36-year-old woman with left painful eye and swollen parotid, right facial paresis, maculopapular rash in left eyebrow and erythema nodosa on both legs. Anti-Toxoplasma IgG antibody was positive, and IgM was not detectable. Radiographic findings on the chest revealed bilateral hilar lymphadenopathy. The initial treatment was sulfamethoxazole- trimethoprim to prevent the recurrence of retinal toxoplasmosis and corticosteroid and mycophenolate mofetil for sarcoidosis. The patient showed clinical and vision improvement without recurrences during three months follow-up. DISCUSSION: Ophthalmological examinations and laboratory tests to rule out toxoplasmosis could be considered in known cases of sarcoidosis, particularly in ocular sarcoidosis status. To the best of our knowledge, this is the first report of comorbidity of ocular toxoplasmosis/sarcoidosis from Iran and possibly the world.

A Novel Anti-HER2 Bispecific Antibody With Potent Tumor Inhibitory Effects In Vitro and In Vivo.

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.