Discordant rearrangement of primary and anamnestic CD8+ T cell responses to influenza A viral epitopes upon exposure to bacterial superantigens: Implications for prophylactic vaccination, heterosubtypic immunity and superinfections.

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vß makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.

The nano-based theranostics for respiratory complications of COVID-19.

High morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made coronavirus disease 2019 (COVID-19) the leading challenge for health experts all over the world. Currently, there is no specific treatment for COVID-19; however, thanks to worldwide intense attempts, novel vaccines such as mRNA-1273 (Moderna TX, Inc.) and BNT162b2 (Biontech/Pfizer) were developed very fast and FDA approved them for emergency use. Nanomedicine-based drug delivery can be an advanced therapeutic strategy to deal with clinical complications of COVID-19. Given the fact that SARS-CoV-2 typically affects the respiratory tract, application of inhalable nanoparticles (NPs) for targeted drug delivery to the alveolar space appears to be an effective and promising therapeutic strategy. Loading the medicinal components into NPs enhances the stability, bioavailability, solubility and sustained release of them. This approach can circumvent major challenges in efficient drug delivery such as solubility and any adverse impact of medicinal components due to off-targeted delivery and resulting systemic complications. Inhalable NPs could be delivered through nasal sprays, inhalers, and nebulizers. NPs also could interfere in virus attachment to host cells and prevent infection. Moreover, nanomedicine-based technologies can facilitate accurate and rapid detection of virus compared to the conventional methods. In this review, the nano-based theranostics modalities for the management of respiratory complications of COVID-19 were discussed.

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression.

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vß-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.

HIV-1 Drug Resistance Mutations among Antiretroviral Drug-Experienced Patients in the South of Iran.

BACKGROUND: The therapeutic effect of antiretroviral therapy (ART) is adversely influenced by antiretroviral drug resistance, mainly due to mutations (DRMs) in the human immunodeficiency virus (HIV) genome. These mutations are commonly associated with HIV protease and reverse-transcriptase genes. We sought to determine the frequency of DRMs in a population of ART-experienced patients in the South of Iran. METHOD: A total of 44 HIV-1-positive participants under ART were selected from April 2016 to March 2017. Their DRMs, antiretroviral resistance status, and viral subtypes were determined. RESULTS: At least one DRM was detected in 61.4% of the participants. The highest frequency was related to nucleotide reverse-transcriptase inhibitor (NRTI) mutations (45.45%). In contrast, major protease inhibitor (PI) mutations had the lowest frequency (6.81%). M184V (40.9%) and K103N (25%), respectively related to NRTI and nonnucleoside reverse-transcriptase inhibitor (NNRTI), were the mutations with the highest frequencies. Susceptibility to PI drugs was higher compared to NRTIs and NNRTIs, which was consistent with the results of genotypic DRMs. CONCLUSION: The highest frequency of antiretroviral DRMs was related to NRTIs and NNRTIs. In contrast, PI resistance mutations had the lowest frequency. Laboratory-guided ART to avoid the expansion of mutants as well as investigating DRMs in other viral regions, such as integrase, are recommended.

PD-1 Blockade Promotes Epitope Spreading in Anticancer CD8+ T Cell Responses by Preventing Fratricidal Death of Subdominant Clones To Relieve Immunodomination.

The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8+ T cell (TCD8) responses, and PD-1-based "checkpoint inhibitors" have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1-targeted therapies reverse TCD8 exhaustion/anergy. However, whether they alter the epitope breadth of TCD8 responses remains unclear. This is an important question because subdominant TCD8 are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of TCD8 responses to well-defined epitopes of a clinically relevant oncoprotein, large T Ag. We found that unlike other coinhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant TCD8, which correlated with their propensity to favorably respond to PD-1/PD-1 ligand-1 (PD-L1)-blocking Abs. PD-1 blockade increased the size of subdominant TCD8 clones at the peak of their primary response, and it also sustained their presence, thus giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC class I-restricted cytotoxicity. The selective increase in subdominant TCD8 clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T Ag or epitope mingenes, and tumor cells expressing T Ag variants revealed that anti-PD-1 invigorates subdominant TCD8 responses by relieving their lysis-dependent suppression by immunodominant TCD8 To our knowledge, our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination.

Prophylactic DNA vaccine targeting Foxp3+ regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model.

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.

Prevalence of HIV-1 pre-treatment drug resistance in a southern province of Iran, 2016-2017.

HIV-1 transmitted drug resistance (TDR) occurs when primary viruses bear drug resistance mutations (DRMs). TDR causes first-line antiretroviral (ARV) therapy (ART) failure and is becoming more pronounced due to the widespread use of ART. With the absence of routine individual-level drug resistance testing, the World Health Organization (WHO) recommends the tracking of TDR mutations and optimizing the first-line ART following pre-treatment drug resistance (PDR) surveys. Here, we report the PDR frequency for the first time in Hormozgan, a southern province of Iran. In this study, 41 blood samples from HIV-1-positive ART-candidate volunteers were collected across the province between April 2016 and March 2017. Phylogenetic analysis of the sequenced protease (PR) and reverse transcriptase (RT) regions showed that 39 out of 41 samples (95%) were CRF35_AD and the two remaining cases were subtype B (2.5%) and C (2.5%). D67G (2.4%), a mutation that reduces susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs) was the only detectable TDR mutation in this population. Two other DRMs, including E138A (9.7%) and V179T (4.9%), which confer resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), were also identified. Although no major protease inhibitor (PI) resistance mutations were detected, the minor mutations L10F and L33F (2.5% each) as well as several highly frequent polymorphic mutations were identified. Our results show a PDR frequency of 17% in infected individuals from Hormozgan, classified further as 2.4% NRTIs and 14.6% NNRTIs. These results suggest that first-line ART should be practiced carefully in Hormozgan province, and alternative regimens may become necessary for all starters.

Correction to: Prevalence of HIV-1 transmitted drug resistance in recently infected, treatment-naïve persons in the Southwest of Iran, 2014-2015.

The author would like to correct the error in the online published article. The correct details are given below for your reading.

HIV-1 genetic diversity and transmitted drug resistance frequency among Iranian treatment-naive, sexually infected individuals.

In recent years, the patterns of human immunodeficiency virus 1 (HIV-1) transmission in Iran have been changing gradually from drug injection to unprotected sexual contact. This study sought to investigate the phylogenetic trends and characteristics of transmitted drug resistance (TDR) mutations of HIV-1 in a population that is mainly infected through homo/heterosexual contacts. Sixty newly diagnosed antiretroviral-naive individuals with HIV infection living in Tehran were recruited to this survey, and among them, 42 subjects were established to be infected through sexual intercourse. Following amplification and sequencing of the main part of the HIV-1 pol region, phylogenetic and drug-resistance mutation (DRM) analysis was successfully performed on these 42 patients. Phylogenetic analysis showed that the majority of the subjects were infected with subtype CRF35_AD (88%), followed by subtype B, with 7.1%, and subtype CRF01_AE, with 4.7%. A total of 7.1% of the subjects were found to be infected with HIV-1 variants with surveillance drug-resistant mutations (SDRMs) according to the last world health organisation (WHO) algorithm. All of the identified SDRMs belonged to the non-nucleoside reverse transcriptase inhibitors (NNRTIs) class, including K103 N and V106A, which were found in three patients. Two minor HIV protease-inhibitor-related mutations (L10I and G73S) were detected in two patients, but these mutations are not included in the WHO SDRMs list. The dominance of HIV-1 subtype CRF35_AD was observed among subjects of this study who were infected through sexual contact. The moderate prevalence of SDRMs (7.1%) in this population emphasises the fact that the risk of treatment failure in HIV-infected individuals might increase in the future, and preventive measures should be considered by health authorities.

Prevalence of HIV-1 transmitted drug resistance in recently infected, treatment-naïve persons in the Southwest of Iran, 2014-2015.

The emergence and transmission of drug resistant HIV mutants is a major concern, especially in resource-limited countries with expanding antiretroviral therapy. Studies have recently reported the prevalence of HIV-1 transmitted drug resistance (TDR) mutations in certain Iranian cities; however, no information is currently available about the level of TDR, as well as the nature of the circulating HIV-1 subtypes, in the Southwestern bordering province of Iran, Khuzestan. Herein, we used a WHO-recommended TDR survey method to classify the prevalence of TDR in indigenous people of Khuzestan province. For this purpose, between March 2014 and February 2015, blood samples were collected from 52 newly diagnosed, antiretroviral treatment-naïve, HIV-1 infected persons aged from 18 to 30 years. TDR mutations were determined by sequencing the protease (PR) and reverse transcriptase (RT) genes and interpreted using the WHO drug resistance mutations surveillance list. HIV-1 subtypes were characterized by sequencing the PR-RT, C2-V5, and p17 regions of the pol, env and gag genes, respectively. Two participants had non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations, specifically K103N in one individual and K101EK/K103KN/G190AG in the other. No nucleoside reverse transcriptase inhibitor (NRTI) or major protease inhibitor (PI) mutations were identified. HIV-1 subtyping revealed that all participants were infected with HIV-1 CRF35_AD. According to the WHO sequential sampling method, the prevalence of HIV-1 TDR in the sampling area (Khuzestan province) was classified as moderate for NNRTIs and low for NRTIs and PIs. This is the first HIV-1 drug resistance threshold survey in the Khuzestan province of Iran and shows a predominance of NNRTI TDR mutations in this area.

Swift Intrahepatic Accumulation of Granulocytic Myeloid-Derived Suppressor Cells in a Humanized Mouse Model of Toxic Shock Syndrome.

Toxic shock syndrome (TSS) and other superantigen-mediated illnesses are associated with 'systemic' immunosuppression that jeopardizes the host's ability to fight pathogens. Here, we define a novel mechanism of 'local' immunosuppression that may benefit the host. Systemic exposure to staphylococcal enterotoxin B (SEB) rapidly and selectively recruited CD11b(+)Gr-1(high)Ly-6C(+) granulocytic myeloid-derived suppressor cells (MDSCs) to the liver of HLA-DR4 transgenic mice. Hepatic MDSCs inhibited SEB-triggered T cell proliferation in a reactive oxygen species-dependent manner, and ex vivo-generated human MDSCs also similarly attenuated the proliferative response of autologous T cells to SEB. We propose a role for MDSCs in mitigating excessive tissue injury during TSS.

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

CONTEXT: Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. OBJECTIVE: We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. MATERIALS AND METHODS: A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector. RESULTS: Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. DISCUSSION AND CONCLUSION: The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Suppressive Effects of Chronic Stress on Influenza Virus Protection after Vaccination with Plasmid DNA-Encoded Nucleoprotein.

BACKGROUND: Influenza is a highly infectious and acute respiratory disease caused by an infection of the host respiratory tract mucosa by the influenza virus. The use of DNA vaccines that express conserved genes such as nucleoprotein (NP) represents a new method of vaccination against influenza. In this study, the effect of chronic stress on the efficiency of this type of vaccine has been evaluated in a mouse model. METHODS: The NP DNA vaccine was administered intradermally 3 times on days 0, 3 and 6 to stressed and nonstressed male BALB/c mice. Two weeks after the last immunization, half of these mice were challenged with A/Puerto Rico/8/34 (PR8) influenza virus and were weighed for 12 days, and their mortality rate was assessed during this period. The cellular immune response of the other half of the mice was evaluated by cytotoxicity assay. RESULTS: The results indicate a significant reduction in the cytotoxic T-lymphocyte response of stressed mice in comparison with unstressed mice. Also, the percentage of weight loss and mortality after the challenge in stressed mice was significantly increased compared to the other group. CONCLUSION: These results indicate that the NP DNA vaccine is not able to induce any effective cytotoxic T-lymphocyte response against influenza virus in stressed mice and cannot induce protective immunity against influenza infection in this group of mice.

Enhancement of HCV polytope DNA vaccine efficacy by fusion to an N-terminal fragment of heat shock protein gp96.

Induction of a strong hepatitis C virus (HCV)-specific immune response plays a key role in control and clearance of the virus. A polytope (PT) DNA vaccine containing B- and T-cell epitopes could be a promising vaccination strategy against HCV, but its efficacy needs to be improved. The N-terminal domain of heat shock protein gp96 (NT(gp96)) has been shown to be a potent adjuvant for enhancing immunity. We constructed a PT DNA vaccine encoding four HCV immunodominant cytotoxic T lymphocyte epitopes (two HLA-A2- and two H2-D(d)-specific motifs) from the Core, E2, NS3 and NS5B antigens in addition to a T-helper CD4+ epitope from NS3 and a B-cell epitope from E2. The NT(gp96) was fused to the C- or N-terminal end of the PT DNA (PT-NT(gp96) or NT(gp96)-PT), and their potency was compared. Cellular and humoral immune responses against the expressed peptides were evaluated in CB6F1 mice. Our results showed that immunization of mice with PT DNA vaccine fused to NT(gp96) induced significantly stronger T-cell and antibody responses than PT DNA alone. Furthermore, the adjuvant activity of NT(gp96) was more efficient in the induction of immune responses when fused to the C-terminal end of the HCV DNA polytope. In conclusion, the NT(gp96) improved the efficacy of the DNA vaccine, and this immunomodulatory effect was dependent on the position of the fusion.

Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice.

Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine.

Molecular characterization of HAO3, the homologue of the Bm86 tick vaccine antigen, from the Iranian isolate of Hyalomma anatolicum anatolicum.

Hyalomma anatolicum anatolicum tick is widely distributed in many parts of Iran and while the commercial vaccines based on the application of midgut-derived recombinant Bm86 antigen are used for its control, limited information about the efficiency of this vaccination in Iran is available. Herein, with the final aim of evaluation of Bm86-based heterologous vaccination, as the primary step the Bm86 homologue of the H. a. anatolicum (Hao3) from an Iranian isolate was characterized and compared with the commercialized Bm86 and other Bm86 homologoue sequences available in GenBank. Our in silico predictions resulted in the identification of seven epidermal growth factor (EGF)-like domains, one hydrophobic transmembrane region, one leader sequence and several glycosylation sites within the structure of both Hao3 and Bm86 proteins, which suggested the pattern of extracellular membrane-bound glycoproteins with the role of regulation in cell growth for both proteins. Moreover, while the nucleotide and amino acid sequences corresponding to Bm86 homologue showed a high level of conservation among the Iranian isolates (Hao3, Hao3-1 and Hao3-2, more than 99%), the Hao3 amino acid sequence had a homology of around 89%, 64% and 65% with that of Indian, Australian and Argentinean isolates, respectively. This indicated a considerable variation between commercial Bm86 antigen and H. a. anatolicum Bm86-like protein of Iranian and Indian isolates. Taking together, these results imply that the efficiency of commercial Bm86-based vaccine against the Iranian H. a. anatolicum may be under the question and indicates the value of the development of Hao3-based recombinant vaccines and further planning for their in vivo evaluation.

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK.

Streptokinase (SK), the heterogeneous protein family secreted by some groups of ß-hemolytic streptococci (ßHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 ßHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI-NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10(4) IU mg(-1)). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.

Novel insights in CAR-NK cells beyond CAR-T cell technology; promising advantages.

CAR-T (chimeric antigen receptor T cell) technology, which has recently showed successful results in the treatment of hematological tumors, has been the focus of attention as one of the most potent approaches in tumor immunotherapy. However, side effects and limitations of this application, such as the risk of graft versus host disease (GvHD), make it challenging to be as accessible as other treatments. Natural killer cells (NK) could be transplanted without alloreactivity, making them as an off-the-shelf product. CAR-NK (chimeric antigen receptor NK cell) therapy can circumvent some serious limitations of CAR-T cell therapy. Application of CAR-NK cells have some considerable advantages over CAR-T cells. These include lack of cytokine release syndrome (CRS), neurotoxicity, and GvHD when using allogenic CAR-T cell. These features lessen the risk of tumor antigen loss and disease relapse. Moreover, NK cells which were derived from different sources, can make the CAR therapy more feasible. In this narrative review, we outlined the key features of CAR-NK cells as an alternative to CAR-T cell therapy in cancer immunotherapy and highlighted the main advantages.

PSC associated inflammatory bowel disease: a distinct entity.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a rare, chronic, and progressive cholestatic disease involving intra- and/or extrahepatic bile ducts. PSC in many patients results in end-stage liver diseases. Nearly 60% of the PSC patients suffer from concomitant inflammatory bowel diseases (IBDs). Classically, IBDs are divided into two principle types: Crohn's disease (CD) and ulcerative colitis (UC). However, with growing knowledge, PSC-associated IBD (PSC-IBD) seems to be a rather distinct entity with specific genetics, clinical, and microbiota characteristics. AREAS COVERED: In this article, we aim to review the unique characteristics of PSC-IBD from clinical, genetic, and microbiota point of view. EXPERT OPINION: PSC-IBD's unique characteristics contribute to the notion that it could be a distinct entity. Acknowledgment of PSC-IBD as a novel entity necessitates designing new clinical guidelines for diagnosis and developing novel therapies.

Subcutaneous transplantation of human thyroid tissue into a pre-vascularized Cell Pouch™ device in a Mus musculus model: Evidence of viability and function for thyroid transplantation.

This study aimed to investigate the survival and efficacy indicators of human thyroid tissue transplantation into a retrievable, prevascularized implanted Sernova Corp Cell Pouch™ (CP) device. Thyroid tissue from human donors was transplanted subcutaneously into the pre-implanted CP device or into the subcutaneous (SC) space alone as a control in a nude Mus musculus model. Transplanted M. musculus were monitored for human serum thyroglobulin (TG) levels for 3 months until the transplants were removed for histological assessment. Human thyroid tissue survived and continued to produce TG in transplanted nude M. musculus in the CP, with no adverse events. CP transplants exhibited more persistent and robust production of human TG than tissue placed in the SC space alone from 3 to 13 weeks post transplantation. Fresh thyroid transplants had better survival and function compared to cryopreserved transplants. Thyroid transplant viability correlated with TG levels at 3 months post-transplant (p = 0.03). Immunofluorescence staining of transplants for TG and TPO localized in thyroid follicles. Human thyroid tissue transplanted into the subcutaneously implanted pre-vascularized CP in nude M. musculus survived and continued to produce robust and persistent human TG and warrants further investigation as a treatment for postoperative hypothyroidism.