Glioblastoma (GBM) is the most malignant brain tumor in adults, with a dismal prognosis despite aggressive multi-modal therapy. Immunotherapy is currently being evaluated as an alternate treatment modality for recurrent GBMs in clinical trials. These immunotherapeutic approaches harness the patient’s immune response to fight and eliminate tumor cells. Standard MR imaging is not adequate for response assessment to immunotherapy in GBM patients even after using refined response assessment criteria secondary to amplified immune response. Thus, there is an urgent need for the development of effective and alternative neuroimaging techniques for accurate response assessment. To this end, some groups have reported the potential of diffusion and perfusion MR imaging and amino acid-based positron emission tomography techniques in evaluating treatment response to different immunotherapeutic regimens in GBMs. The main goal of these techniques is to provide definitive metrics of treatment response at earlier time points for making informed decisions on future therapeutic interventions. This review provides an overview of available immunotherapeutic approaches used to treat GBMs. It discusses the limitations of conventional imaging and potential utilities of physiologic imaging techniques in the response assessment to immunotherapies. It also describes challenges associated with these imaging methods and potential solutions to avoid them. Glioblastoma (GBM) is a devastating and universally fatal brain cancer . The curIn the quest for an effective treatment, several immunotherapeutic approaches have been introduced in recent years that have been designed to harness patient’s immune response to fight and eliminate tumor cells. Broadly, these novel strategies can be divided into four major classes: immunomodulators, active immunotherapy, adoptive immunotherapy, and oncolytic viral therapy ,9,10,11.It has been reported that patients treated with immunotherapy demonstrate profound inflammation at the tumor sites, often referred to as treatment-induced pseudoprogression (PsP), which can suggest a favorable treatment outcome . UnfortuThis review is structured into three sections to cover the role of imaging in immunotherapy of GBMs. The first section covers commonly used immunotherapeutic approaches used to treat GBM patients. The second section discusses the limitations of conventional imaging methods to emphasize the need for alternative imaging techniques in the evaluation of treatment response to immunotherapies. Finally, in the third section, potential applications of physiologic MR and PET imaging methods are described for the assessment of immunotherapies in GBMs. The challenges associated with these imaging methods and possible solutions to avoid those pitfalls have also been described.For several years, it was assumed that the brain lacks a lymphatic system since it was considered an immuno-privileged organ (devoid of any immune cells). However, a strong body of evidence ,33,34 haSeveral promising novel immunotherapeutic approaches are being actively investigated in clinical trials for GBM treatment  7,8,9,1,19,10,11Essentially, these studies have been conducted to test the feasibility, to establish safety profile and to evaluate the therapeutic efficacy of novel immunotherapies in a diverse population of GBM patients. However, the food and drug administration (FDA) has not yet approved any immunotherapeutic approach for the treatment of GBM despite the completion of several clinical trials, probably due to the variability in the success rate achieved with these therapies.In the following sections, a brief overview of commonly used immunotherapeutic approaches currently used in treating GBM patients is described. In particular, the action mechanism, safety profile and therapeutic efficacies of major classes of immunotherapies are discussed. Readers are referred to excellent articles available for a detailed overview on immunotherapies in GBMs ,38,39.Immune checkpoint inhibitor (ICI)-based immunotherapy has revolutionized the treatment of several cancers, including metastatic melanoma , non-smaUnder physiological conditions, checkpoint pathways play a critical role in maintaining immune homeostasis by inhibiting the proliferation, activity and responsiveness of cytotoxic T-lymphocytes A. While Some ICIs such as pembrolizumab (anti-PD1 antibody), nivolumab (anti-PD1 antibody), and ipilimumab (anti-CTLA-4 antibody) have been used for the treatment of GBMs in several large multicentric early- and late-phase clinical trials ,47,48,49Some investigators have also suggested the use of bevacizumab (an antivascular agent) as a steroid substitute to control peritumoral edema and to avoid immunosuppressive actions of steroids. While preliminary results from these studies support the safety of adding bevacizumab to immunotherapies, clinical benefits have not yet been established ,52.Active immunotherapy exploits the use of neoantigens that are overexpressed (tumor-associated antigens) or exclusively expressed (tumor-specific antigens) by tumor cells to train the host’s immune system to target tumor cells and in turn inhibit tumor growth. This type of immunotherapy elicits an immune response by presenting tumor antigens towards major histocompatibility complexes (MHCs), which allows T cells to bind, multiply and target tumor cells to be destroyed . VariousDendritic cells (DCs) are potent immune stimulators that can prime and activate T cells in several organs. These primed T cells have additional potential to mount secondary memory responses. Autologous DCs are differentiated from harvested monocytes, cultured and loaded with antigens (whole tumor cell lysate) from the patient’s tumor specimens obtained at surgical resection. These modified DCs are then treated with a differentiation factor such as granulocyte-macrophage colony-stimulating factor and finally administered back to the patients intradermally. Rindopepimut is a vaccine that has been widely used in the treatment of GBMs ,39. This+ GBM patients -2-Fluoro-2-deoxy-D-glucose ([18F]-FDG) has been the most commonly used PET tracer to study brain tumors [18F]-FDG uptake in neoplastic cells reflects increased expression of glucose transporters and/or enzymatic activity of hexokinase [18F]-FDG in studying metabolic activity in different types of cancers. However, normal brain parenchyma has naturally high uptake of [18F]-FDG, which undermines the diagnostic accuracy of FDG tracer for accurate delineation of brain tumor margins, especially from adjacent gray-matter regions. On the other hand, several amino acid-based PET imaging tracers have emerged as alternative candidates for metabolic imaging of brain tumors [18F]fluoroethyl)-L-tyrosine ([18F]-FET), [11C]methyl-L-methionine ([11C]-MET), and 3,4-dihydroxy-6-[18F]fluoro-L-phenylalanine ([18F]-FDOPA) targeting energy independent amino acid transporters of L-type (LAT) that are known to be upregulated in brain tumors [18F-fluorothymidine ([18F]-FLT), a pyrimidine analog, has been studied as a surrogate marker for cellular proliferation in neuro-oncology because of its preferential uptake by rapidly dividing neoplastic cells where it indicates the activity of thymidine kinase-1 (a key enzyme involved in DNA synthesis) . Su. Su95]. Taken together, the results of these two studies signify ADC as a potential imaging to differentiate TP from immunotherapy-induced inflammatory response. In general, highly malignant regions of GBMs harboring closely packed tumor cells and reduced extracellular volume demonstrate low ADC values. It is hypothesized that successful immunotherapy would result in the accumulation of vasogenic edema and would reduce tumor cellularity, leading to increased ADC values. On the other hand, diffusivity of water molecules decreases in TP with high cellularity and reduced extracellular space. Anti-PD1 therapy is known to activate effector and cytotoxic T cells, leading to secretion of cytokines, which in turn causes tumor regression and also decreases the proliferation and infiltration of regulatory T cells within the tumor ,98. It i+ T cells count was high (indicating low tumor cell density) -FET as a PET imaging tracer, Kebir S et al. [18F]-FET-PET has been that FET tracer exhibits high uptake by neoplastic cells and less uptake by inflammatory cells -2-fluoro-d-(arabinofuranosyl) cytosine ([18F]-FAC) PET imaging and post-contrast MR imaging. [18F]-FAC is a deoxycytidine analog that is a specific substrate for dCK and is specially taken up in activated cytotoxic T cells indicating regions of immune cell activity. The authors reported significantly increased [18F]-FAC uptake in DC vaccinated + anti-PD1-treated mice compared to DC-vaccinated or untreated control mice. The imaging finding was in agreement with immunohistochemical analysis showing the maximum population of infiltrating T-lymphocytes in the DC-vaccinated + anti-PD-1 therapy treated mice followed by DC vaccinated than control animals. Not surprisingly, post-contrast MR images demonstrated comparable tumor growths in glioma-bearing animals, suggesting that standard MR imaging is not effective in distinguishing tumor growth from antitumor response induced by immunotherapies. When the preclinical work was extended to evaluate treatment response in three GBM patients receiving tumor lysate-pulsed DC vaccine with or without pembrolizumab, post-treatment PET images demonstrated enhanced tumor uptake of [18F]-CFA. However, tumor volumes remained constant before and after treatment. Due to the preliminary nature of these findings, additional studies are required to understand the significance of [18F]-CFA-PET findings in GBM patients receiving immunotherapeutic treatments.In a study by Antonios et al. , PET proTreatment response to CAR-T cell therapy from 10 recurrent GBM patients was evaluated at 1, 2 and 3 month follow-up periods using MR methods  115]. I. I115]. max from the contrast-enhancing regions were able to differentiate TP from PsP with an accuracy of 90%. While evaluating treatment response to EGFRvIII CAR-T cell therapy in GBM patients in another study  guanine ([18F]-FHBG) to track IL13 Rα2 CAR T cells expressing a wild-type HSV1-tk reporter gene in a population of seven GBM patients. Each patient underwent [18F] FHBG PET imaging before and after infusion of CAR T cells. Moreover, this method demonstrated the presence of a variable degree of infiltrated cytotoxic T-lymphocytes expressing CARs targeting IL13Rα2 antigens in the tumor regions. Further optimization of this imaging approach for monitoring in vivo cell trafficking may greatly benefit cell-based immunotherapies for GBMs.To optimize the therapeutic potential of CAR T cell immunotherapy, it is also important to monitor the trafficking, biodistribution and viability of immune cells within the tumor site. In this effort, reporter gene-based PET imaging is being explored in monitoring trafficking, targeting and activation of therapeutic cells in tumors ,125. Keu124I-labelled 2′-fluoro-2′-deoxy-1-D-arabino-furanosyl-5-iodo-uracil, ([124I]-FIAU) was used as a specific marker for gene expression of HSV-1-tk to identify the location, magnitude and extent of vector-mediated HSV-1-tk gene expression in a phase I/II clinical trial of gene therapy in recurrent GBM patients -2-methylpropanoic acid (18F-FIMP) [89Zr-IAB22M2C. Preliminary data indicated that CD8 T cells in tumors were detected in tumors by 24 hours post-infusion, suggesting the usefulness of this new PET tracer in tracking immune cells in tumors.PET imaging also provides important functional information about the tumor microenvironment and has been useful in evaluating treatment response to immunotherapies in GBMs. An important component of immunotherapies is the presence of profound inflammation at the tumor bed that presents a significant challenge in the accurate assessment of treatment response. PET imaging probes that selectively accumulate in immune cells rather than in neoplastic cells may be valuable in the therapeutic assessment. In this direction, potential utility of PET imaging probes such as translocator protein (TSPO)  and (S)-8F-FIMP)  targetin8F-FIMP) , patientWith the advent of integrated PET-MR imaging scanners, we believe that simultaneous acquisition and analysis of co-registered PET and MR imaging data providing complementary physiologic information will allow more accurate assessment of treatment response to immunotherapies in GBMs than a single imaging modality alone. Besides treatment evaluation, there is an urgent need for the development of objective, reliable and quantitative prognostic imaging biomarkers to facilitate treatment stratification and selection of GBM patients for enrollment in various immunotherapeutic trials. In this direction, a newer generation of molecular PET imaging tracer (zirconium-89 labeled atezolizumab) is currently under investigation to predict response to anti-PD-LI immunotherapy and upfront patient selection in extracranial cancers . We beli


This ecological momentary assessment study examined the effect of naturally occurring stressors and perceived stress reactivity on alpha-amylase, a proxy of sympathetic nervous system arousal. There are age-related changes in physiological systems sensitive to stress, so the sample included 174 adults ages 20-78 . At the beginning of the study, participants completed the Perceived Stress Reactivity Scale . For 10 consecutive days, participants were prompted five times per day to report exposure to stressors. During the same 10-day period, participants provided seven saliva samples per day, assayed for alpha-amylase. Multilevel modeling was used to examine daily and momentary associations between stressors, the PSRS, and alpha-amylase activity. On a daily basis, stressors did not predict changes in the diurnal alpha-amylase pattern, but higher perceived stress reactivity predicted steeper diurnal slopes and lower total daily output. A significant cross-level interaction emerged showing people higher in perceived stress reactivity had steeper awakening responses on days they experienced more stressors than usual. On a momentary basis, alpha-amylase levels were higher on occasions when participants reported stressors. In addition, higher levels of perceived stress reactivity predicted lower overall alpha-amylase levels. Findings suggest that 1) stressors are associated with elevations in momentary but not daily aggregate levels of alpha-amylase, and 2) the PSRS has prospective validity as a predictor of stress-related fluctuations in diurnal alpha-amylase patterns. Age was not a significant moderator of the relationship between stressors and alpha-amylase, potentially suggesting the effect of stressors on alpha-amylase activity is age invariant.


Chronic hepatitis B virus (HBV) infection is a global healthcare burden in the form of chronic liver disease, cirrhosis, liver failure and liver cancer. There is no definite cure for the virus and even though extensive vaccination programs have reduced the burden of liver disease in the future population, treatment options to eradicate the virus from the host are still lacking. In this review, we discuss in detail current updates on the structure and applied biology of the virus in the host, examine updates to current treatment and explore novel and state-of-the-art therapeutics in the pipeline for management of chronic HBV. Furthermore, we also specifically review clinical updates on HBV-related acute on chronic liver failure (ACLF). Current treatments for chronic HBV infection have seen important updates in the form of considerations for treating patients in the immune tolerant phase and some clarity on end points for treatment and decisions on finite therapy with nucleos(t)ide inhibitors. Ongoing cutting-edge research on HBV biology has helped us identify novel target areas in the life cycle of the virus for application of new therapeutics. Due to improvements in the area of genomics, the hope for therapeutic vaccines, vector-based treatments and focused management aimed at targeting host integration of the virus and thereby a total cure could become a reality in the near future. Newer clinical prognostic tools have improved our understanding of timing of specific treatment options for the catastrophic syndrome of ACLF secondary to reactivation of HBV. In this review, we discuss in detail pertinent updates regarding virus biology and novel therapeutic targets with special focus on the appraisal of prognostic scores and treatment options in HBV-related ACLF. The prevalence of hepatitis B virus (HBV) surface antigen positivity among the general population differs according to geographical region, which also dictates possible routes of transmission. In low prevalence (<2%) regions such as North America and Western Europe, age at infection in early adulthood and route of transmission is mostly sexual and percutaneous. In regions of moderate prevalence (2-8%), age at infection in childhood and perinatal transmission is the most common mode of spread. Previously, age-dependent phases of HBV were described as immunotolerant phase , immunoactive phase, inactive carrier state  and reactivation. Nonetheless, these have been renamed recently as HBV envelope antigen (HBeAg)-positive infection, HBeAg-positive hepatitis and HBeAg-negative infection and HBeAg-negative hepatitis. Progression to cirrhosis in HBeAg-positive patients occurs at a rate of 2 to 5.5% per year increasing from 8 to 20% in five years. Inactive carriers who have normal aminotransferase levels and HBV DNA levels <2,000 IU/ml experience disease regression at the rate of 0.5 to 2% per year. HBeAg-negative hepatitis, or the reactivation phase, represents a progressive stage of chronic HBV. Anti-HBe (antibody to e-antigen)-positive patients experience rapid progression to cirrhosis at an annual rate of 8 to 20%. Patients with cirrhosis progress to advanced liver disease and hepatic failure at a rate of 16% over five years . ChronicUpdates on HBV-related applied biologyThe HBV StructureHBV infection is a dynamic disease that encompasses biochemical, histological and clinical changes that occur over time, depending on the mode of acquisition, host and environmental factors. Within the host, HBV can exist in three forms, the infectious virion (Dane particle) and non-infectious particles that include enveloped nucleocapsids containing immature DNA/RNA, subviral particles  and naked nucleocapsids ,6. AccorThe viral genome encodes four overlapping open reading frames (ORFs): C (pre-core and core regions), P, S , and X . The core antigen (nucleocapsid) protein, HBcAg; and the ‘e’ antigen (HBeAg) and 22-kDa pre-core protein (p22cr) are produced from ORF-C core and pre-core regions respectively. The polymerase protein (Pol) encoded from ORF-P is made of terminal domain with functions of encapsidation and initiation of minus strand synthesis; the reverse transcriptase domian (RT) which catalyzes genome synthesis; and the ribonuclease (H) domain which degrades pregenomic RNA and facilitates replication. HBV X antigen protein (HBxAg) is encoded by ORF-X and has multiple functions that support various stages of viral replication including signal transduction, DNA repair, activation of transcription pathways and inhibition of protein degradation along with participation in the oncogenic potential of HBV -11. In tUpdates on Viral EntryThe mode of entry and HBV replication steps within the hepatocyte has been extensively updated in the last decade. The virus attaches to the host cell surface  through initial low-affinity binding on highly sulfated-heparan sulfate proteoglycans (HSPG) such as hepatotropic glypican-5 followed by high-affinity binding on target receptor. This binding to the HSPGs is mediated by electrostatic interactions between the negatively charged HSPG and two positively charged residues of the S-domain . InitialThere are three different types of endosomes: early endosomes, late endosomes, and recycling endosomes, differentiated by their morphology, the time taken for the endocytosed material to reach them, and by markers such as Ras superfamily of G proteins called Rabs. Once endocytic vesicles uncoat, they fuse with early endosomes (via Rab5A) which then mature into late endosomes before fusing with lysosomes (via Rab7A) . In the Updates on Nuclear Transport, Assembly and ReleaseAfter escape from the late endosome, the viral particles traverse the cytoplasm towards the host cell nucleus. As previously stated, the membrane fusion leads to direct release of nucleocapsids into the cytoplasm. A conserved membrane-permeable peptide within the surface protein of HBV was recently identified, of the pre-S2 domain, called the translocation motif (TLM). The TLM promotes delivery of proteins and nucleic acids into cells and tissues. Surface exposure of TLM peptides on the HBV surface protein due to proteolytic processing leads to fusion of peptides to HBc protein enabling formation of fully assembled capsids -32. ThesUpdates on Viral Transcription and cccDNAWithin the cytoplasm, along with viral polymerase, pre-genomic RNA is encapsidated by HBV core protein to form a viral capsid. Inside the viral capsid, pre-genomic RNA undergoes reverse transcription to generate single-stranded negative-strand DNA , further followed by generation of partially double-stranded DNA  yielding viral rcDNA. These capsids containing rcDNA are either transported back into the nucleus to increase the cccDNA pool or enveloped and released as progeny virions. HBsAg production is predominantly from cccDNA in younger HBeAg-positive patients. Hypo-phosphorylation of capsid proteins produces regular virions while hyper-phosphorylation produces empty virions -44. The With respect to cccDNA clearance, two pathobiological processes are pertinent. One, hepatocyte proliferation itself contributes to reduction in load of cccDNA within infected hepatocytes. In patients with advanced fibrosis or cirrhosis, the hepatocyte replicative senescence adds to the burden of cccDNA formation. Destruction of infected hepatocytes in the presence of potent replication suppression will help reduce cccDNA formation - a therapeutic approach that would require combination of multi-targeted treatment strategies. The cccDNA removal also occurs via non-cytolytic clearance of infected hepatocytes in the presence of antiviral cytokines, specifically interferon-α. It was shown that higher levels of interferon-α were associated with improved cccDNA clearance through triggering of non-cytolytic degradation of cccDNA from infected hepatocytes through induction of the nuclear deaminase A3A or A3B. However, such high doses in a clinical scenario can lead to adverse events and hence are impractical. Recently, the PASylation  of interferon-α was found to improve antiviral effect without additional toxicity -59. FiguUpdates on immunopathogenesis, genetic variants and applied biologyHost immune response against HBV infection includes innate immunity and adaptive immunity. The former includes downstream responses that are activated by pattern recognition receptor (PRR), natural killer (NK) cells, NK-T cells, and monocytes and macrophages; while the latter includes cluster of differentiation (CD)4 + T lymphocytes, CD8 + T lymphocytes, and B lymphocytes. In chronic HBV infection, the virus limits and evades antiviral effects of the innate immune and adaptive immune system through various mechanisms, resulting in continuous replication associated with dysfunction of various immune cells. HBeAg is immunomodulatory and is involved in antigen presentation and recognition by CD4+T cells. In HBeAg negative HBV infection (associated with pre-core stop codon mutation), there is rapid progression, cirrhosis and liver cancer development due to amelioration of host innate immune functions. Similarly, the HBV core promoter mutation in enhancer II which results in enhanced viral replication is accompanied by a reduction or loss of HBeAg leading to fulminant or progressive chronic hepatitis. The HBsAg mutant - defect in S region to arginine at amino acid position 145 and loss of group-specific antigenic determinant a (target of vaccine response) - escapes immune surveillance and infection even in the presence of antibodies to surface antigen and also development of occult HBsAg negative HBV infection. Vaccine-escape mutations occurred in particular when lamivudine (currently not utilized) was used in the long term -64. The Some preclinical studies have shown that, in early HBV infection, PRR-mediated innate immune responses are not activated - the stealth virus phenomenon where the virus interferes with innate signaling pathways to attenuate intrinsic antiviral immune responses -67. HBsACellular Level Immune Activity in HBV InfectionNK cell dysfunction is also central to viral persistence in HBV infection. The ability of myeloid DCs to activate NK cells is impaired due to weak action in decreasing activating cytokines  resulting in reduced secretion of interferon-γ and lowered activity of interferon-α. IL-10 secretion from Kupffer cells (liver resident macrophages) promotes cytokine blunting and hence lowers NK activation. IL-10 is an immune-suppressive cytokine  that maintains the immune tolerance during persistent HBV infection. Programmed death-ligand 1 (PD-L1) on suppressive monocytes also inhibit autologous NK cell activation. Reduction in the NK-cell mediated cytotoxic prowess and IFN-γ production contribute to HBV persistence. The expression of activating receptors on NK cells such as the NKG2D and 2B4 are also reduced in chronic HBV infection.The myeloid-derived suppressor cell (MDSC) with predominant granulocytic subset (gMDSC) and monocytic MDSC (mMDCS) has an inverse relation with T cell function and hepatitis in chronic HBV infection. MDSCs potentiate CD4+ and CD8+ T cell responses via arginase-dependent pathways. High arginase levels reduce amount of arginine required for lymphocyte physiology and growth and resulting in lymphocyte dysfunction. Disruption of MDSC differentiation and T-regulatory cells (Tregs) resulted in immunosuppressive cytokine reduction which inhibited HBV replication. NKT cells of a special subset of T lymphocytes that express surface markers of T lymphocytes and NK cells - the invariant NKT cells (iNKT) lose functionality in the presence of HBV infection through an increase in T-cell immunoglobulin and mucin domain-3 (Tim-3) and programmed cell death protein 1 (PD-1) - antiviral treatment or Tim-3 blocking restores immune function of iNKT cells and improves viral clearance -74.Interferon-γ secreted by lymphocytes in HBV infection induces Kupffer cells to produce chemokine (C-X-C motif) ligand 9 (CXCL9) and recruits HBV-specific CD4 + T lymphocytes to enter the liver for apoptosis, leading to chronic HBV. Defects in CD8 + T-lymphocyte functions through multiple pathways  result in reduced HBV clearance from hepatocytes. PD-1 blockade can partially restore B cell function and CD 8+ T cell functions for viral clearance. Higher level of T helper cell 17 (Th17) lymphocytes  in the liver and peripheral blood was associated with acute and acute on chronic liver failure due to HBV. In chronic HBV infection, a follicular helper T (Tfh) cells response to HBsAg was required for HBV clearance which was blocked by Treg cells -82.Endoplasmic reticulum (ER) stress also plays an important role in viral persistence. In HBV-infected cells, a large number of viral surface proteins are folded in ER during the replicative phase, resulting in disruption of ER homeostasis and ER stress. This is identified as ground-glass hepatocytes that accumulate ER mutant surface proteins (pre-s1 and pre-s2 mutants) which represent ER hypertrophy. ER stress leads to activation of ER degradation enhancers and hence reduction in the immune responses for viral clearance. The intracellular imbalance in favor of L-HBs compared with M- and S-HBs leads to ER stress, which can trigger cellular signals for apoptosis or uncontrolled cellular growth . HBV RNAUpdates on HBV genotypes and their clinical importanceCurrently, 10 genotypes of HBV exist, with additional subtypes (mutations or recombinant strains), which are identified by the letters A to J and numbered respectively, which, through genetic mutations and the lack of proofreading in reverse transcriptase, have evolved over the long term, creating challenges to their elimination. An example is the HBV genotype B2 which is a recombinant, with majority of the genetic framework from HBV genotype B, and the precore/core region from genotype C. Coinfection with different HBV genotypes and intergenotypic recombination of HBV strains are extensively documented. Most commonly associated recombinants include genotypes B/C or A/D. Each genotype is classified by an 8% or more divergence in the nucleotide sequence of the genome. Genotypes A to D are the four predominant genotypes; B and C are most common in eastern and southeastern Asia, A and D are found in North America, Africa and Europe and genotype E is found in West Africa. Genotypes A and B have a greater response to interferon therapy than C and D, but none of the genotypes have differential responses to oral antivirals -93. DelaEvaluation and treatment of HBV related liver diseaseCurrent Approaches to Diagnosis and EvaluationPresence of HBsAg indicates acute or chronic infection and is the first serologic marker to appear. HBV infection is considered chronic if HBsAg persists beyond six months. The HBeAg indicates active replication while its absence can also indicate mutations in the pre-core region of the e-antigen that prevent production of HBeAg. Antibody response to HBeAg (anti-HBe) indicates that the virus is non-replicative, but is also seen among HBV patients with HBeAg mutation with active disease. Antibody to HBc antigen can be present in acute infection and reactivation (IgM) and with past exposure to HBV (IgG). It can be seen in solitude when antibody response to HBsAg is waning. Patients who are HBsAg-positive and anti-HBc-positive need further evaluation for initiation of treatment. Those who are anti-HBs-positive and anti-HBc-positive are considered to have infection in the past and currently resolved. Nonetheless, in these patients infection may remain latent, only to reactivate under special circumstances  along with re-emergence of HBsAg. Patients who are HBsAg, anti-HBs, and anti-HBc negative do not possess immunity needs vaccination. Patients who are only anti-HBs-positive are immune or have undergone vaccination -100.In patients who are HBsAg and antibody  to HBcAg positive, further differentiation is made on the presence or absence of HBeAg after which classification into chronic infection and chronic hepatitis is made for treatment decisions. Patients with chronic infection have normal alanine aminotransferase (ALT) and no or minimal liver damage (fibrosis grade <2) while those with chronic hepatitis have elevated HBV DNA and ALT with ongoing necro-inflammatory liver damage or significant fibrosis (grade ≥2), for whom treatments are to be directed ,102. TheThere are three types of treatment end points or cure in HBV infection. In functional cure, there is resolution of clinical infection which is sustained off drug treatment - no inflammation, normal ALT level and normal liver biopsy, HBsAg seroclearance  with or without the emergence of anti-HBs. Protective immunity is when the anti-HBs level is greater than 10 IU/ml. Complete cure is virologic cure, consisting of all the elements of functional cure plus loss of cccDNA within the liver. In clinical practice, most of the treated patients fall into an interim cure period in which there is disease inactivity - absence of inflammation , low or undetectable HBV DNA level in the presence of HBsAg positivity. In this situation, a patient with chronic hepatitis is effectively down staged to one with chronic infection. An HBsAg level of 100 IU/ml in Asian HBeAg-negative patients is predictive of spontaneous HBsAg seroclearance within six to eight years. Inactive - low replicative chronic HBV patients have high rates of spontaneous HBsAg seroclearance, - 8.1% and 44.7% after 10 and 25 years of follow-up, respectively -110. In 7 IU/mL and normal ALT) if aged above 30-40 years benefit from antiviral therapy irrespective of other standard inclusions for treatment initiation. Nonetheless, one must be aware that spontaneous HBeAg and HBsAg clearance with remission of liver disease can occur in 70 - 80% of patients at median follow up of approximately 10 to 20 years [It is important to note that among HBsAg-positive cases who additionally suffer from obesity or metabolic syndrome, the risk of development of cirrhosis is higher than those with HBV infection alone ,112. In 20 years -115.New updates on diagnosis and monitoringThe HBV core-related antigen (HBcrAg) is a new indicator that encompasses amino acid sequence common to HBeAg and HBcAg as well as the 22-kDa precore protein. HBcrAg positivity correlates with intrahepatic HBV DNA and pregenomic RNA levels among patients on antiviral treatment. This makes HBcrAg measurement a good serum marker of the active transcriptional activity of liver cccDNA and higher levels correlate with increased risk of hepatocellular carcinoma -118. AnoStreamlining the HBV diagnostic process to identify those who would benefit from screening, surveillance or therapy through artificial intelligence-related machine learning (ML) is a novel technique. With the help of ML, a predictive model for inflammation grades of chronic HBV was proposed utilizing a combination of gene expression data and three clinical parameters  over which a user-friendly web tool (LiveBoost™) was applied for the clinical prediction of hepatic fibrosis. It was demonstrated that the ML system outperformed FIB-4 scoring in predicting advanced hepatic fibrosis. Additionally, an artificial neural network (ANN) model was found to be effective in diagnosing liver fibrosis regression in HBV patients on therapy. ML-based models were also found to accurately identify persons at risk for HBsAg positivity, predict HBsAg seroclearance, predict treatment decisions in HBV carriers; 28- and 90-day mortality of HBV related ACLF, determine viral variants; and interactions between viral and host proteins to map pathways in hepatocellular carcinoma -128.Current measures and updates on prevention and treatmentThe immunogenic first-generation active HBV vaccines were made from materials extracted directly from plasma of chronically HBV-infected patients. It was not the virions that were utilized, but the large amounts of non-infectious spherical viral particles in carrier plasma, which were easily separated by biophysical methods. After cloning the HBV genome, large-scale production of spherical viral particles within recombinant yeast cells (yeast-derived second-generation vaccines) showed comparable protection with plasma-derived vaccines. Vaccine response rates with yeast-derived vaccines are>99% among infants and adolescents, but insufficient in 5% of healthy adults. The majority of yeast-derived vaccines consist only of the S-HBs of the globally underrepresented HBV genotype A2, dominant only in Northern Europe and North America. Very high anti-HBs titers  are protective, but low or waning anti-HBs-titers over time increase risk of breakthrough infection with antigenically distant HBV genotypes . The minimal infectious dose of HBV is as low as 16 virions (or 3 IU) when transmission occurs through HBV-contaminated blood transfusions -132.Current therapies for the management of HBV include interferon-α (standard or pegylated) and orally administered NAs. First-line therapy should be with an oral antiviral with a strong genetic barrier to viral resistance such as either entecavir, tenofovir disoproxil (TDF) or tenofovir alfenamide . Short-term treatment with NA is feasible in HBeAg-positive patients experiencing seroconversion to anti-HBe during treatment. A randomized controlled study (FINITE) analysed outcome when TDF therapy was withdrawn in a set of HBeAg negative patients who had achieved suppression of HBV DNA. Interestingly, 43% of patients achieved either HBsAg loss or suppressed DNA without any significant safety concerns -136.8 IU/mL), genotype A, and elevated serum ALT (> 2-5 times ULN) along with necroinflammation on liver biopsy, peginterferon-α could be used as first-line antiviral agent. HBeAg negative, genotype D patients who do not experience decrease in HBsAg levels and 2 log10 IU/ml reduction of HBV DNA at 12 weeks peginterferon-α treatment are considered non-responders. The HBsAg level is useful for prediction and motoring of response to therapy with peginterferon. HBeAg-positive patients, with HBsAg level of 20,000 IU/ml at week 24 are considered non-responders and treatment can be stopped early. Peginterferon leads to higher rates of HBeAg and HBsAg loss at one year mainly in patients with genotype A infection. Overall rates of sustained response (HBeAg seroconversion and undetectable HBV DNA in HBeAg positive patients and DNA <2000 IU/mL in HBeAg negative patients) after a one-year course of treatment is 27-36% and 28% respectively. Combination of NA and peginterferon can be performed via two protocols - de novo combination or the simultaneous administration of the two agents in treatment-naïve HBV patients; and the sequential combination, which features “add-on” or “switch-to” strategy in those who are already on treatment with either drug. This strategy improves HBsAg loss. Nonetheless, the benefits are mainly limited to specific group of patients - those with low baseline HBsAg level and on-treatment HBsAg response, high baseline ALT and viral load and genotype A. Peginterferon should not be used in decompensated cirrhosis, but can be used with caution in patients with compensated cirrhosis [Peginterferon for chronic HBV-related hepatitis is not widely used, even though the treatment period is finite (48 wks therapy). For HBeAg positive patients with low HBV DNA  score between 20-30, while the mortality rate in those with MELD >30 was >90% even in the presence of early antiviral treatment - these patients need early referral for liver transplantation ,142. AftFuture directions for HBV treatmentHBV entry inhibitors targeting NTCP receptors include myrcludex-B  and cyclosporine A (CsA). The former, a synthetic lipopeptide derived from pre-S1 domain blocks infection of new hepatocytes and hinders amplification of intrahepatic cccDNA of infected hepatocytes. The latter, a cyclic non-ribosomal peptide inhibit NTCP transporter activity blocking viral entry into hepatocytes - but can impair sodium dependent bile acid uptake resulting in various adverse events. Nonetheless, recently discovered SCY450 and SCY995 derivatives of CsA do not impair bile acid uptake ,146.Streptococcus pyogenes and S. thermophilus targeting conserved regions of the HBV genome resulted in degradation of > 90% HBV cccDNA by six days. Nonetheless, even though deep sequencing revealed that Streptococcus-CRISPR/Cas9 had no effect on the host genome, it induced intrinsic off-target adverse effects such as mutagenesis [APOBEC3 cytidine deaminase activators  via engineered non-lytic T cells with HBV-specific T-cell receptors inhibited HBV replication in small animal models. LTBR agonists were found to degrade cccDNA and exhaust intrahepatic pool. Genome-editing using transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) or locked nucleic acid technology (LNA) can be used to target specific DNA sequences for cleaving. TALENs comprise a nonspecific Fok1 nuclease domain fused to a customizable (can be engineered to target and disrupt any specific DNA sequence) sequence-specific DNA-binding domain. However, the safety of such DNA sequence cleavage on ‘HBV integrated host genome’ and its consequences remain to be studied. HBV-specific CRISPR/Cas system-mediated removal of the full-length integrated HBV DNA and the disruption of HBV cccDNA in a stable HBV cell line was demonstrated recently. CRISPR/Cas9 system from agenesis -150.RNA interference (RNAi) by which siRNA produces gene silencing at the post-transcriptional level to downregulate the expression of targeted genes is another novel therapeutic area. The siRNA therapeutic, ARC-520 that targets cccDNA-derived pre-genomic RNA was found to reduce HBsAg levels in HBeAg-positive patients, but not HBeAg-negative patients as in the latter, HBsAg arises not from cccDNA pool, but from HBV DNA integrated with the host genome. The novel ARO-HBV (JNJ-3839), targeting two sources of HBsAg, pre-genomic and integrated DNA, is currently under evaluation. Another siRNA molecule called AB-729 using novel conjugated N-acetyl galactosamine delivery technology with strong anti-HBV activity which acts on all HBV RNA transcriptions is under evaluation -153.Virus nucleocapsid assembly inhibitors/modulators  limit HBV replication by causing capsid destabilization is under multiple trials. A novel acyclic nucleotide phosphonate called besifovir is recently approved for trial studies. The main adverse event noted was L-carnitine depletion  in treated patients requiring supplementation. Another new lipid conjugated nucleoside analogue under clinical development is tenofovir exalidex (TLX) which shows enhanced hepatic targeting that maximizes liver activity while reducing systemic drug exposure and was found to enhance HBsAg loss and reduce the cccDNA amount. Another novel nucleoside analogue under phase II clinical study is CMX157 -157. InhBased on our advances in understanding immunopathogenesis of HBV, multiple immune modulating therapeutic agents are under development which would help promote functional cure of HBV. TLR agonists  induce endogenous interferon production, activate innate responses, leading to induction of interferon-stimulated genes  and other signaling cascades that inhibit HBV replication. Nonetheless, phase II studies have shown that even though T cell increase, NK cell responses and interferon signaling were improved with TLR agonists, reduction in HBsAg levels were not identified. This means that monotherapy with these agents is probably of low clinical relevance and hence combination strategies are warranted. Pattern recognition receptor agonists such as RIG-I and NOD-2 agonists activate interferon signaling pathways and proinflammatory cytokines that improve viral clearance. The RIG-I agonist, inarigivir soproxil, a novel oral modulator of innate immunity when used along with TDF significantly increased reduction of HBV replication, HBV RNA and HBsAg levels in a dose-dependent manner in both HBeAg-positive and HBeAg-negative patients -165.Saccharomyces cerevisiae yeast-based vaccine expressing HBsAg, HBcAg, and HBx, did not provide significant reductions in serum HBsAg levels when used alone, but induced strong immunomodulatory effects when used along with TDF. The non-replicative adenovirus 5 vector vaccine TG1050 encodes a large fusion protein made of modified HBV core, HBV polymerase, and selected envelope protein domains. TG1050 was found to have a good safety profile and induced appreciable HBV-specific cellular immune response in early trials [PD-1, a highly expressed inhibitory receptor on HBV-specific T cells, along with increased expression of PD-L1 (PD-1 ligand), contributes to T cell exhaustion and high HBV replication in chronic HBV. Thus, PD-1/PD-L1 pathway blockers induce proliferation of HBV-specific T cells, thus restoring functioning T cells and helping control HBV. A pilot study showed that the PD-1 blocker nivolumab along with HBV therapeutic vaccine GS-4774 achieved significant and sustained HBsAg loss -168. Finy trials -171.In another study, 12 chemical compound candidates for alpha-glucosidase inhibitors were identified from a library of chemical compounds and used to treat fresh human hepatocytes infected with HBV and monitored for their anti-viral effects. It was found that HBV replication was inhibited by one candidate, a tetramethylpiperidinol derivative in a dose-dependent manner, through interaction with HBV nuclear transcription factor Sp1 which was also associated with significant reduction of cccDNA production, compared to entecavir . To summUpdates on HBV-related ACLFAcute on chronic liver failure is a recently described entity in the natural history of cirrhosis, defined by acute insult leading to rapid hepatic decompensation, multiple organ failure and a high risk of short-term mortality, usually less than four weeks. Acute alcoholic hepatitis, drug-induced liver injury, infections and surgical stress are the most frequent precipitants for ACLF. Central to the pathophysiology of ACLF is the state of unchecked persistent inflammation and immune dysfunction with increased propensity to sepsis and organ failure. Reactivation of chronic HBV infection is an important and modifiable cause for ACLF ,178. StuIn HBV-related ACLF, viral factors were found to have strong association with the development of the catastrophic syndrome. The HBV basal core promoter/precore mutations such as T1753V, A1762T, G1764A, A1846T, G1896A, and G899A correlated with an increased risk of HBV-related ACLF which was supported by the fact that ACLF patients had distinct quasi-species characteristics and higher complexities and diversification within the precore/core gene .A genome-wide association study identified HLA-DR and rs3129859*C allele as the major locus for susceptibility to HBV-related ACLF. This allele was associated with prolonged prothrombin time, faster progression to ascites development and higher 28-day mortality in HBV-ACLF. The authors concluded that the HLA class II restricted CD4+ T-cell pathway on the immunopathogenesis of HBV-related ACLF . Some stPrognosis of HBV-ACLF can be ascertained by a variety of scoring systems which include the standard MELD and MELD-sodium (MELD-Na) scores, EASL chronic liver failure (CLIF)-Consortium-ACLF score , integrated MELD, which includes hepatic encephalopathy and age, with an improved sensitivity of approximately 70-80% and the recently proposed AARC score, which integrates bilirubin, creatinine, prothrombin time, lactate, and hepatic encephalopathy which was found to be superior to the MELD in predicting outcomes ,179,184.The NLR was also found to be an independent predictor of mortality in patients with HBV-ACLF undergoing treatment with artificial liver support systems  suggesting that liver function in most patients with baseline NLR ≤3 recovered with ALSS treatment, and those with NLRs >6 require emergency liver transplantation . The ChiThe age-bilirubin-international normalized ratio-creatinine (ABIC) score >9.44 was superior to the MELD score in predicting short-term survival (one and three month) in HBV-ACLF patients . AnotherOur understanding of the structure, biology, viral and immunopathogenesis in chronic HBV-related hepatitis has come a long way. Nonetheless, knowledge gaps still persist that currently limit our therapies toward a complete cure from this globally burdening disease. With the advent of new technologies and better tools such as next-generation sequencing, genome-wide association studies, single-cell RNA sequencing, gene editing and rigorous and well-coordinated collaborative clinical trials, we now understand viral and host-related factors in disease development and progression better than before. Novel modalities of treatments, such as viral RNA interference molecules, capsid assembly blockers, immune checkpoint inhibitors, HBsAg and cccDNA generation blocking molecules and innate immune system modulators, are in the pipeline and will eventually help us improve HBV-related patient outcomes.


Myasthenia gravis (MG) is an autoimmune disorder characterized by abnormal neuromuscular transmission. The thymus is believed to play a key role in the pathogenesis of MG, and thymectomy has been an optional treatment for the disease. Relapse of MG after thymectomy has been reported. Exacerbations and new onset of MG following COVID-19 vaccination have also been documented. This report presents a case of a stable MG patient with recent COVID-19 vaccination experiencing flare-ups of symptoms shortly after video-assisted thoracoscopic (VATS) thymectomy. A 31-year-old female received the second dose of the BNT162b2 mRNA COVID-19 vaccine eight days before thymectomy and developed flare-ups of symptoms four days after the surgery. Although the substantial link between MG exacerbations post-thymectomy and pre-thymectomy COVID-19 vaccination cannot be concluded, this observation warrants further research. Myasthenia gravis (MG) is an autoimmune neurologic disease characterized by abnormal neuromuscular transmission. Antibodies against acetylcholine receptors (AChRs), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-related protein 4 (LRP4) are the predominant autoantibodies in MG . The disSurgical removal of the thymus has been an alternative treatment for MG. A prospective, randomized, controlled clinical trial has shown conclusive evidence that thymectomy benefits patients with non-thymomatous MG and reduces the requirement for immunosuppressive therapy . RelapseNew onset of MG has been reported in two individuals after receiving the second dose of the BNT162b2 mRNA COVID-19 vaccine . It is nThe rapid advancement of COVID-19 vaccines has prompted concerns regarding vaccine safety and efficacy in immunocompromised individuals, such as those with MG. Whether vaccination increases the risk of MG symptoms worsening remains unknown. It's challenging to estimate the safety of mRNA, DNA, and viral vector COVID-19 vaccines in MG because of their novelty. This paper reports a temporal relationship between the BNT162b2 COVID-19 vaccination and symptom exacerbations shortly after thymectomy in a young female MG patient.A 31-year-old Thai female first presented with fatigable bilateral ptosis and proximal muscle weakness 10 months ago and was diagnosed with MG. The symptoms were partially improved with rest. Her blood test was negative for anti-AChR antibodies. Thymic mass screening with chest multidetector computed tomography (MDCT) disclosed prominent soft tissue at mid-superior mediastinum without abnormal enhancement, measured about 0.98 cm x 2.29 cm in its greatest transverse dimension  class IIa. She received the first and second doses of the BNT162b2 COVID-19 vaccine approximately four weeks and eight days prior to the scheduled thymectomy. The preoperative nasopharyngeal and throat swab RT-PCR test for SARS-CoV-2 RNA was negative. Thymectomy was performed via the lateral approach using right-sided video-assisted thoracoscopic surgery (VATS) under general anesthesia with endotracheal intubation. The pathological result revealed lobulated yellow tissue containing islands of involuting thymic tissue and foci of hemorrhage , the patient reported more weakness on her hips, thighs, and shoulders. Fatigable ptosis was more frequent with occasional diplopia. Her voice was faded away when talking longer, and she felt tired more easily. Laboratory investigation ruled out possible infections, and serum potassium was normal. Prednisolone was gradually increased to 20 mg daily and pyridostigmine was increased to two tablets four times a day. At a two-week follow-up, the patient reported improvement of muscle strength, the disappearance of diplopia, a normal voice, and having regular daily activities.The flare-ups of MG symptoms after thymectomy in this patient suggested the temporal relationship with the second dose of the mRNA-1273 COVID-19 vaccine. It is well known that infections are closely associated with the onset of MG and are the most common contributor to MG progression . In ordeIt is uncertain whether thymectomy performed soon after COVID-19 vaccination contributed to MG aggravation. Vaccines or adjuvants could theoretically cause or aggravate autoimmune illnesses by triggering a generalized autoimmune response, resulting in multiple autoantibodies, and reproducing human autoimmune diseases . Thymic Due to mild flares, doses of prednisolone and pyridostigmine were increased to control the patient’s symptoms, with considerable improvement. A previous study showed that prednisolone dose-dependently inhibited the release of pro-inflammatory mediators such as tumor necrosis factor α (TNF-α), IL-6, and IL-8 while enhancing the release of the anti-inflammatory cytokine IL-10 . In critThis case report provides clinical observation on a temporal relationship between the BNT162b2 COVID-19 vaccination and MG flare-ups shortly after thymectomy in a young female patient. Guidance on the care of patients with MG during the COVID-19 pandemic has been issued by a variety of professional groups. Unless there is a specific contraindication, COVID-19 vaccination is suggested for patients with MG. Although there is no strong evidence that COVID-19 vaccines induce cytokine storm, decisions on timing for thymectomy or other major scheduled surgery should be made on a case-by-case basis as inflammatory cytokines released during thymic injury and possibly as a result of COVID-19 vaccines or adjuvants may aggravate the symptoms of MG. Several types of COVID-19 vaccines, including mRNA, DNA, viral vector, protein-based, and inactivated vaccines, have been developed in the SARS-CoV-2 virus pandemic situation. The safety profile and efficacy of these COVID-19 vaccines in MG patients should be further investigated.


Solid tumors adopt multiple mechanisms to grow, evade immune responses, and to withstand therapeutic approaches. A major breakthrough in the armamentarium of anti-cancer agents has been the introduction of monoclonal antibodies (mAbs), able to inhibit aberrantly activated pathways and/or to unleash antigen (Ag)-specific immune responses. Nonetheless, mAb-mediated targeted pressure often fails due to escape mechanisms, mainly Ag loss/downregulation, ultimately providing therapy resistance. Hence, in order to target multiple Ag at the same time, and to facilitate cancer-immune cells interactions, bispecific antibodies (bsAbs) have been developed and are being tested in clinical trials, yielding variable safety/efficacy results based on target selection and their structure. While in hematologic cancers the bsAb blinatumomab recently reached the Food and Drug Administration (FDA)-approval for B Cell Acute Lymphoblastic Leukemia, bsAbs use in solid tumors faces considerable challenges, such as target Ag selection, biodistribution, and the presence of an immune-suppressive tumor microenvironment (TME). This review will focus on the state-of-the art, the design, and the exploitation of bsAbs against solid malignancies, delineating their mechanisms of action, major pitfalls, and future directions. Immunotherapy has recently changed both the treatment modalities as well as the prognosis across several malignancies. Such immune-stimulating therapies entail either passive  or active  approaches ,2. CliniMonoclonal antibodies (mAb) represent another way to induce Ag-specific immune responses. Differently from CAR-T cell therapies, mAbs have the advantage of being ready, off-the-shelf, drug products with a potentially safer therapeutic index, also considering chemotherapy conditioning for CAR-T cell therapies. Clinical results with mAb, especially against hematologic malignancies, further sped up development of the entire field ,6. NowadThe advent of mAbs for cancer treatment has drastically changed clinical practice, leading to improved survival outcomes in several malignancies . They arSubsequently, bsAbs soon became an attractive area for drug development purposes. A key challenge is represented by incorrect protein heterodimerization in the production pipeline: this may involve both heavy and light chains mispairing, which may account for up to 90% of the total mass . To overBsAbs are categorized based on their structure and mechanisms of action (MoA), which are dictated by their specificity , valency , and affinity (interaction strength). For instance, it has been shown that reducing CD3 affinity allows for better biodistribution in tumor sites (versus lymph nodes) and lower rates of cytokine release, while preserving T-cell engaging/activation properties ,9,17,18.A key structural feature of bsAbs is represented by the presence/absence of the Fc region.Fc-free bsAbs display better biodistribution into tumor tissues, higher potency, and less common incidence of immune-related adverse effects (irAEs) ,22. HoweFc-containing bsAbs, instead, are characterized by longer half-lives  and by tIgG-like Fc-based bsAbs are characteristically limited by design to a fixed Ag specificity and a valency of two. IgG-like bsAbs comprise CrossMab 1:1, Triomabs and Fab-arm exchange (FAE) platforms. CrossMabs  have two asymmetric Fab arms, because the desired light chain pairing is forced by swapping either the constant or the variable domains between heavy and light chains, creating two asymmetric Fab arms . TriomabIgG-modified bsAbs, instead, have a fixed specificity of two but can have a valency of two or more. Examples of IgG-modified bsAbs are CrossMab 2:1 or 2:2 , Half-Life Extended BiTEs (HLE-BiTEs) , Dual Affinity Re-targeting (DART)-Fc , Dual variable domain (DVD)-IgG , and Fabs-in-tandem (FIT)-Ig . Of note, FIT-Ig are composed by two fused Fabs in a crisscross orientation, resulting in a tetravalent IgG-like molecule . InsteadRegardless of structure and design, bsAbs mechanisms of action can be summarized in immune cell engagement (ICEs), tumor associated antigen (TAA)-targeting, immune checkpoint blockade (ICBs), or Ab-drug conjugation (bsADCs)  16]..16].TAA-targeting bsAbs may present the advantage of targeting more efficiently single or multiple signaling cascades. For example, ZW25 (zanidatamab) targets two different epitopes in the extracellular domains II and IV of human epidermal growth factor receptor (HER)-2. These epitopes are the same target of mAbs pertuzumab and trastuzumab, respectively . JNJ-611Antibody drug conjugates (ADCs) are dramatically changing the landscape of cytotoxic treatments of different tumors. Compared to mAb-based ADC, bsADCs may theoretically have the advantage of improved specificity and drug internalization, with potentially enhanced tumor cell killing and potentially improved therapeutic indices . The mosICEs are characterized by one single chain variable fragment (scFv) recognizing a surface TAA and a second scFv specific for an effector immune cells’ membrane receptor . They woLastly, bsAbs with specificities for immune checkpoint inhibitor/blocking  receptors exert their activity by enhancing and re-directing host’s immune responses against cancer cells . While tOverall, key determinants of the future positive applications of bsAbs in solid tumors involve the discovery of novel TAAs, ICB receptors or drug conjugates, the correct matching of bsAb design with its biological purpose and the clinical scenario.Although many bsAbs showed remarkable activity especially in the context of B-ALL , most clAmivantamab (JNJ-61186372) is a fully-human anti-EGFR/cMET bsAb with immune cell directing activity ,41. WhilAmivantamab was recently granted accelerated approval by the FDA for patients affected by non-small cell lung cancer (NSCLC) harboring exon 20 insertion mutation in EGFR gene, with disease progression on/after platinum-based chemotherapy . RemarkaThe results of CHRYSALIS study led to the design of other clinical trials. For instance, in the phase III MARIPOSA trial NCT04487080), patients with locally-advanced or metastatic NSCLC harboring EGFR mutations (exon 19 deletion or L858R mutation) are randomized to receive either the combination of amivantamab and lazertinib, lazertinib alone or the standard of care (SoC) osimertinib alone as first-line therapy 7080, pat.Zanidatamab (ZW25) is a humanized biparatopic (targeting two different epitopes of the same Ag) IgG1 antibody: the HER2 extracellular domains (ECDs) ECD2 (pertuzumab binding domain) and ECD4 (trastuzumab binding domain) . In NoveZanidatamab has also been tested against HER2-high  breast (17 patients), gastric/esophageal (11 patients), or other (5 patients) cancers with progressive disease after SoC. HER2-high breast cancer (BC) patients had received prior trastuzumab and T-DM1 (100%), pertuzumab (82%) and lapatinib (53%), with a median of six HER2-targeted regimens for metastatic disease, whereas all gastrointestinal patients had received prior trastuzumab, with a median of four lines of treatments. The DCR was 54% in the BC cohort, 57% in the gastroesophageal cohort, and 33% in other cancers . In HER2Moreover, the combination of zanidatamab plus immunotherapy is under evaluation in a phase IB/II clinical trial in first line treatment (NCT04276493): in cohort 1, patients affected by HER2+ metastatic BC will receive the drug in combination with docetaxel; in cohort 2, patients affected by HER2-positive advanced gastric/gastroesophageal junction adenocarcinoma will receive zanidatamab plus chemotherapy and tislelizumab (anti-PD1) . Lastly,KN026 is a Fc-based, biparatropic, bsAb exploiting the same specificities as trastuzumab (ECD4) and pertuzumab (ECD2). In the first in-human phase I trial (NCT03619681), no dose-limiting toxicity was observed, with an ORR of 32.1%  and a DCR of 76.8%  . KN026 wZenocutuzumab (MCLA-128) is a humanized IgG1 bsAb targeting both HER2 (on a different epitope than trastuzumab) and HER3. Zenocutuzumab was designed with the purpose of overcoming the HER3-mediated resistance to HER2 target therapy by inhibiting HER2–HER3 interaction, downstream phosphoinositide 3-kinase (PI3K)-AKT signaling cascade, and by mediating ADCC activity .Zenocutuzumab showed a favorable toxicity profile and promising single agent activity in heavily pre-treated patients with gastric/gastroesophageal cancers progressing to anti-HER2 inhibition . MoreoveAnother application of zenocutuzumab is represented by metastatic solid tumors harboring Neuregulin 1 (NGR1) gene fusions . Indeed,ZW49 is a zanidatamab-based bsAb conjugated, via a protease-cleavable linker, with a novel N-acyl sulfonamide auristatin payload, which blocks tubulin polymerization and cell division. Auristatin conjugation has been shown not to negatively influence HER-binding in vitro . Phase ITF2 is a tri-Fab bsAb, targeting Carcinoembryonic Antigen (CEA) twice and histamine-succinyl-glycine (HSG). HSG is a unique synthetic hapten, which can be labelled with a wide range of radionuclides, suitable for therapeutic or imaging purposes. Pre-targeted radioimmunotherapy (pRAIT) with TF2 exploits the radio-labelled IMP-288 binding to TF2 molecules on the cancer cells. A vast array of radionuclides can be utilized with IMP288 , as depicted in Cinrebafusp alfa (PRS-343) is a bispecific fusion protein targeting both HER2/4-1BB on tumour cells and T cells, respectively. Seventy patients with HER2-positive tumors received cinrebafusp alfa in a phase I clinical trial (NCT03330561) . This drp < 0.0001). Overall survival (OS) demonstrated a positive trend in the intervention group and was prolonged in patients with gastric cancer . However, intraperitoneal catumaxomab followed by chemotherapy failed to improve PFS and OS compared to chemotherapy alone in patients with peritoneal carcinomatosis by metastatic gastric cancer in a phase II trial [Catumaxomab (Removab) was the first bsAb approved by the European Medicine Agency (EMA) for the treatment of solid tumors. In 2009 it was approved for the intraperitoneal treatment of malignant ascites in adults with Epithelial cell adhesion molecule (EpCAM) positive carcinomas. However, it was subsequently withdrawn for commercial reasons in 2017 . StructuII trial . NotwithConcerning other routes of administration, systemic intravenous administration was evaluated in pre-treated patients affected by NSCLC, observing dose limiting toxicities such as G3–4 elevation of liver enzymes . In addiKN046 is a IgG1 bsAb targeting both PD-L1 and CTLA-4 and preventing their binding to PD1 and CD80/CD86, respectively. It is under evaluation in a Phase I clinical trial (NCT03529526) , where pInterestingly, KN046 has been granted orphan drug designation for the treatment of patients with metastatic thymic epithelial tumors progressing on a platinum-based chemotherapy and is under evaluation in a phase II clinical trial (NCT04469725) . MoreoveCadonilimab (AK104) is a humanized tetravalent IgG1 bsAb targeting PD-1 and CTLA4, being granted the FDA orphan drug designation for the treatment of metastatic cervical cancer. This was due to preliminary, unpublished results obtained in a phase II for second- and third-line therapy, reporting an ORR of 47.6% and a DCR of 66.7% (NCT04380805). Cadonilimab is also under evaluation in patients affected by relapsed/refractory malignant mesothelioma (NCT03261011). Early results demonstrated an ORR of 15.4% (2/13 patients) and a DCR at eight weeks of 84.6% (11/13). A percentage of 16.7% patients experienced a grade 3–4 AEs . Grade 1–2 AEs were mainly represented by rash and IRR.Cadonilimab has also demonstrated a manageable safety profile with early anti-tumour activities upon combination with mXELOX chemotherapy protocol as first-line regimen for metastatic gastric/gastroesophageal junction adenocarcinoma, regardless of the PD-L1 status in the NCT03852251 phase I trial . Most frIn recent years, clinical testing of bsAb for the treatment of solid cancers has steadily grown. Some bsAbs have shown encouraging clinical response rates with favorable toxicity profiles across different patient populations. However, several factors challenge the implementation of bsAbs in solid tumors, such as limited biodistribution, microenvironmental resistance mechanisms, the emergence of anti-drug antibodies, or Ag-escape. Several tools are currently being investigated to improve bsAbs application and their efficacy in solid tumors: simultaneous multiple interaction BiTEs (SMITEs), T Cell Receptor (TCR)-armed bispecific molecules targeting tumor-specific antigens, BiTE-armed oncolytic viruses (OVs), bsAb-armed CAR-T cells, endogenous secretion of T cell redirecting Abs (STAb), or combinatorial drugs acting on the Tumor Microenvironment (TME).SMITEs exploit the combination of two distinct BiTEs to increase specificity, augment T cell responses or convert inhibitory into stimulatory signals . Interesp < 0.0001) compared to investigator’s choice of either dacarbazine, ipilimumab, or pembrolizumab [Bispecific targeting of tumor cells can also be exploited by utilizing fusion proteins composed by anti-CD3 scFv coupled with a soluble, tumor-specific, TCR. Despite being burdened by MHC-restriction, this approach may allow for therapeutic targeting of large numbers of tumor- or patient-specific epitopes. A notable example is tebentafusp (IMCgp100), constituted by TAA-targeting bispecific molecules specific for CD3 and gp100 . Tebentaolizumab . Full pholizumab ,81. Aparolizumab . Since molizumab ,84. Receolizumab .Another tool to improve bsAb activity and reduce toxicities is to promote local expression of bsAbs within the TME. To this end, oncolytic viruses’ ability to selectively replicate within cancer cells has been exploited in preclinical models to deliver BiTEs at the tumor site. For example, the OV ICOVIR-15K has been engineered to express an EGFRxCD3 BiTE, increasing the accumulation of tumor-infiltrating T cells compared to a parental in a pre-clinical model of human lung or colorectal carcinoma . MoreoveIn addition, another combinatorial approach envisions the use of CART.BiTEs, in which CAR-T cells also co-express and secrete functional BiTE molecules. This platform has been tested in a pre-clinical model of human glioblastoma, with T cells encoding for an anti-EGFRvIII CAR together with an anti-EGFR-CD3 BiTE, resulting in elimination of tumors displaying heterogeneous cells (EGFRvIII positive and negative), with considerable potential advantages to EGFRvIII-specific CAR-T cell therapy alone .Alternatively, ex vivo gene-modified cells can be engineered to produce bsAbs endogenously, as for STAb immunotherapies ,41,89,90Lastly, the presence of an inflamed TME, heralded by interferon-γ signaling, is linked with clinical responses to immunotherapies . HoweverOverall, however promising, these approaches need to be clinically tested to further assess the potential role of achieving bispecific targeting for the treatment of solid cancers.The deployment of bsAbs for the treatment of solid malignancies is recently experiencing a fast-paced evolution. Indeed, while increased knowledge both on cancer biology allowed for the identification of microenvironmental- or tumor-specific targets, improved drug engineering processes led to the design of bsAbs with better therapeutic indexes and/or with therapeutic cargos. Remarkable advancements in the field have been witnessed by regulatory approval of amivantamab, as well as by breakthrough designation of zanidatamab, fast-track designation for zenocutuzumab and orphan drug designations for KN046 and cadonilimab, between 2020 and 2021. Although encouraging, most preliminary results still need proper validation in large, phase II–III randomized clinical trials, when possible. Most notably, amivantamab has already changed the clinical approach to NSCLC patients harboring EGFR exon20 mutation, a population of patient devoid of any efficacious treatment regimen so far. In general, to attain positive efficacy results in solid malignancies being investigated, the identification of the most suitable bsAb molecule must match with different disease indications and with the most appropriate combination therapies. In addition, novel approaches may also promote bsAb entry in the anti-cancer armamentarium of solid tumors, such as combinations with TME-altering agents, or bsAbs delivery at the tumor site. Overall, the entire field is moving fast to finally achieve robust safety and efficacy data, potentially envisioning bsAbs as a novel treatment in the upcoming years.


Current procurement of specimens for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection requires trained personnel and dedicated equipment. We compared standard nasopharyngeal swabs with self-collected gargle lavage fluid obtained from 80 mostly symptomatic outpatients. After RNA extraction, RT-PCR to detect SARS-CoV-2 was performed. Qualitative results obtained with the paired samples from individual outpatients were 100% congruent. Therefore, self-collected gargle lavage fluid can serve as a suitable specimen for coronavirus disease 2019 (COVID-19) testing in outpatients.IMPORTANCE The SARS-CoV-2 pandemic still strains health care systems worldwide. While COVID-19 testing is considered an essential pillar in combating this infectious disease, shortages in supplies and trained health care personnel often limit the procurement of patient samples, in particular in outpatient settings. Here, we compared the simple self-collection of gargle lavage fluid with the gold standard nasopharyngeal swab as a specimen for COVID-19 testing. By finding complete congruence of results obtained with paired samples of a sizeable patient cohort, our results strongly support the idea that the painless self-collection of gargle lavage fluid provides a suitable and uncomplicated sample for reliable SARS-CoV-2 detection. Fast and reliable testing of persons with suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a key element in combating the coronavirus disease 2019 (COVID-19) pandemic. Early in infection, high viral loads can be found in the nasal and oral cavities. Hence, nasopharyngeal swab specimens (NPS) are considered the gold standard material for SARS-CoV-2 testing in this period. However, obtaining NPS by medical staff has several drawbacks: the shortage of trained professionals, significant discomfort for patients, and an increased risk of infection for the medical staff during the procedure. In outpatient settings, these factors can compromise sampling and can delay diagnosis of patients with COVID-19. To achieve maximum testing throughput, alternative sampling strategies, such as self-collection of saliva or gargle lavage (GL) fluid, have been suggested 16. Howeve1–7–10–We compared the suitability of self-collected GL versus NPS taken by health care professionals as testing materials for reverse-transcription PCR (RT-PCR)-based SARS-CoV-2 detection in outpatients.First, we asked if a GL specimen is suitable for procuring cellular material to an extent comparable to that of the standard NPS, which samples mucosal material in a locally defined area. To this end, we obtained paired GL specimens and NPS from 11 healthy volunteers, isolated RNA, and performed RT-PCR with primers directed against the human RNase P gene as a housekeeping gene. Interestingly, GL fluid yielded similar but slightly larger amounts of host material . This fiNext, we recruited patients with possible/probable SARS-CoV-2 infection between October and December 2020. Participants had been traced by health authorities as close contacts of SARS-CoV-2-positive persons and had attended different doctors’ offices. The sampling took place when patients first visited doctors’ offices because of possible COVID-19. The study protocol (DRKS number DRKS00023904) was approved by the Institutional Review Board of the University of Konstanz, Germany, and was carried out according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants. From each patient, a nasopharyngeal swab   was takeOf 80 patients enrolled in the study, 26 (32.5%) tested positive for SARS-CoV-2 using the professionally acquired NPS (referred to here as positive patients). The gender and age distribution and the reported symptoms of all patients are summarized in Cq) values of the 26 positive patients, a substantial difference was observed between the self-collected samples  and the NPS  (n = 4) of the positive samples showed a low viral titer with Cq values of >30, in line with the idea that participants were identified early during the course of infection around symptom onset, when viral titers are highest  and a higher half , we observed that the differences in Cq values between NPS and GL samples were substantially more pronounced for samples which showed lower Cq values in NPS   had a reduced difference (mean difference of 2.9) in Cq values between NPS and GL fluid .Upon direct comparison of the RT-PCR cycle threshold (< 0.001) , corresp highest . Interes n = 13) . In contGL fluid . MoreoveCq values compared to GL specimens. However, also at this early phase there is enough virus contained in GL to reliably detect SARS-CoV-2. At later stages, when viral concentrations are generally lower, the virus has moved further down the airways and might be as efficiently collected by GL as by NPS. Consequently, the testing from GL fluid may be sufficiently sensitive to detect SARS-CoV-2 infection at both early and later stages of the infection. This idea is in line with the findings by Mittal et al. (These findings demonstrate that the detection of SARS-CoV-2 from the (mostly symptomatic) participants by self-collected specimens yields congruent qualitative results. Accordingly, self-collected GL specimens may be suitable for detection of SARS-CoV-2 in symptomatic outpatients by RT-PCR. Furthermore, we showed that viral RNA levels were significantly higher in NPS than in GL and this difference was particularly pronounced at high virus concentrations, whereas the difference between NPS and GL was small at lower virus concentrations. One possible explanation for this might be the altered tissue distribution of the virus during the course of the COVID-19 infection. The early phase of the disease is characterized by high viral loads in the upper airways . Here, tl et al. , who fouWe believe that these results are important, since the numbers of outpatients substantially exceed those of inpatients, and this self-collection technique saves human and material resources and helps to protect medical professionals from infection. Though self-collection might in some cases lead to contamination of the outer surface of the sample containers, the safe handling of such potentially contaminated containers by trained personnel in appropriately equipped central diagnostic laboratories poses only a minor infection risk. Moreover, since this simplified procedure has a better acceptability than NPS , it may


Outbreak and rapid spread of coronavirus disease (COVID-19) caused by coronavirus acute respiratory syndrome (SARS-CoV-2) caused severe acute respiratory syndrome (SARS-CoV-2) that started in Wuhan, and has become a global problem because of the high rate of human-to-human transmission and severe respiratory infections. Because of high prevalence of SARS-CoV-2, which threatens many people worldwide, rapid diagnosis and simple treatment are needed. Genome editing is a nucleic acid-based approach to altering the genome by artificially changes in genetic information and induce irreversible changes in the function of target gene. Clustered, regularly interspaced short palindromic repeats (CRISPR/Cas) could be a practical and straightforward approach to this disease. CRISPR/Cas system contains Cas protein, which is controlled by a small RNA molecule to create a double-stranded DNA gap. Evidence suggested that CRISPR/Cas was also usable for diagnosis and treatment of SARS-CoV-2 infection. In this review study, we discoursed on application of CRISPR technology in detection and treatment of SARS-CoV-2 infection. Another aspect of this study was to introduce potential future problems in use of CRISPR/Cas technology. Coronavirus disease (COVID-19) was spread in December 2019 and was recognized as a zoonotic disease . Severe Some model systems, including mammalian cells, can efficiently cleave any complementary sequence to the gRNA and target and cleave the RNA. Targeted genome editing, often called CRISPR/Cas9, is increasingly recognized as an effective tool in medicine . It can The coronavirus family (CoVs) contains many virus species, and these viruses can cause various diseases in birds, livestock, and humans . MembersHost-virus conflict is a dynamic process. Viruses use host factors to complete their life cycle, and the host uses the body’s immune system to fight off the viral infection, so it does not waste energy. The virus must enter the host cell to replicate its genome and complete its life cycle  Figure . TheoretHost cell factors such as the Apo-Lipoprotein B Editing Complex (APOBEC3) and Tripartite Motif Containing 5 (TRIM5) have been identified as viral limiters for HIV infection . APOBEC3Some host factors are essential for virus replication, assembly, and budding. Therefore, knocking out their genes could be an alternative to preventing HIV-1 infection ; such geThe worldwide pandemic of SARS and CoV-2 poses a significant threat to global public health and societal stability and has become a significant global public health problem. Regrettably, current diagnostic and therapeutic methods to prevent and control SARS-CoV-2 have many limitations. CRISPR/Cas technology has emerged as a potential complement to conventional methods in recent years. Biomedicine has extensively used biological tools based on the CRISPR/Cas systems. They are helpful in pathogen detection, clinical antiviral treatment, and drug and vaccine discovery. Therefore, CRISPR/Cas technology could be promising in preventing and treating SARS-CoV-2 and other emerging infectious diseases.CRISPR-Cas systems could be used for molecular diagnosis of nucleic acids  Jia et . CRISPR-2 site) and 2) integration with low-cost portable reporting sheets and side streptometers. 3) isothermal signal amplification for rapid target detection in the absence of termocycling. CRISPR-based assays are more accessible and convenient than RT-PCR viral RNA identification assays because the CRISPR system does not require bulky instrumentation or complicated processes , influenza A virus (IAV), and vesicular stomatitis virus (VSV) in human cells . ConversTo reprogram CRISPR-Cas13b against the genomic and subgenomic RNAs of SARS-CoV-2, we performed genome-wide computational predictions and screens at single-nucleotide resolution. Cas13b effectors reprogrammed to target accessible segments of spike and nucleocapsid transcripts had silencing efficiencies greater than 98 percent in virus-free animals. Tailored and multiplexed Cas13b CRISPR RNAs (crRNAs) inhibit viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including novel dominant variants. CRISPR-Cas13-based viral suppression strategy is readily adaptable and can be extended to harmful viruses other than SARS-CoV-2 and, therefore, could provide an effective platform for antiviral treatments . HoweverHowever, it is critical to identify and investigate the deleterious effects of using single-guide RNAs (sgRNAs), the CRISPR/Cas system, or PAC-MAN on host physiology. Although the Cas method seems to have a high chance of successfully identifying therapies, it needs further investigation.CRISPR technology is advancing rapidly. Although recently discovered and new, CRISPR/Cas is a tool with multiple genome engineering capabilities. Because of its ability to edit genomes in such a user-friendly way, it has attracted the attention of biomedical researchers. CRISPR can appropriately solve various viral diseases. In cell-based and animal studies, successful results have been achieved in several human viral infections . The theAlthough few studies have shown off-target editing and most studies support CRISPR/Cas, one of the major concerns associated with the CRISPR/Cas system is the possibility of off-target activity and mutant viruses. Viral escape mutations are caused by deletions (indels) at the Cas9 segregation site . Using NTransferring CRISPR/Cas9 to virus-infected cells is another limitation of this new method. The success of this technology in the clinical setting is necessary to control the most severe viral diseases, including SARS-CoV-2 . As CRISEmerging viruses such as SARS-CoV-2 are responsible for hundreds of thousands of illnesses and deaths worldwide each year. The disease is spreading everywhere and destroying the economies of affected populations. Genome editing strategies to deactivate the viral genome could be a suitable way to treat such diseases. The lack of effective drugs and vaccines may be contributing to so many SARS-CoV-2 samples being collected for rapid diagnosis. Containing and preventing further spread of the virus appears to be critical, and rapid detection of infection in organisms may prevent further spread of the disease. CRISPR/Cas is a solution for treating virus-related diseases with many future applications. Therefore, the CRISPR/Cas9 system could be helpful, especially if some valuable and specific sgRNAs are developed. If the CRISPR/Cas system leads to therapeutic and diagnostic solutions, the financial burden will be reduced because CRISPR/Cas-based therapies will eliminate the need for drugs. In addition, the treated individual will not suffer repeated disease relapses. Therefore, advances in the CRISPR/Cas system and the success of clinical trials in animal models are critical


Concurrent kidney and ureteral stones are always complicated and a clinical challenge. Improvements in endoscopic equipment have led to the widespread adoption of retrograde intrarenal surgery, which has a good stone clearance rate. On the other hand, laparoscopic ureterolithotomy (LUL) has been reported to be non-inferior to retrograde flexible ureteroscopy in stone-free rate and the need for axillary procedures, and to have a significantly lower rate of post-operative sepsis compared to retrograde flexible ureteroscopy. We describe a case managed with LUL followed by laparoscope-assisted retrograde intrarenal surgery (LA-RIRS) in a single operation for a large upper ureteral stone and small renal stones, which is usually challenging and requires axillary procedures.The patient was a 66-year-old male with underlying hypertension and diabetes mellitus. He reported severe flank pain after receiving endoscopic management of concurrent right ureteropelvic junction stone and multiple renal stones about 1 month previously.The residual stones were reassessed using non-contrast computed tomography before surgery. A 2.8-cm residual ureteropelvic junction stone and multiple renal stones with a maximum length of 1 cm were found. A second operation was considered to be necessary due to the deterioration of his renal function and refractory flank pain.We performed LUL followed by LA-RIRS. Two surgeries were completed in a single operation. The Jackson–Pratt drain was removed 3 days after the operation.After the surgery, no high-grade complications were recorded according to the Clavien Dindo classification. A follow-up kidney, ureter, and bladder radiograph performed 2 months after the operation revealed no residual stones. Renal echo revealed no obstructive nephropathy 1 month after double-J catheter removal.LUL with LA-RIRS with a stone basket for renal stone extraction is a safe and feasible technique, and no step surgery or axillary procedures were needed in our case. If clinical cases with a huge stone burden over the ureter are indicated for LUL with concurrent small renal stones, LUL with LA-RIRS can be an alternative option. In addition, the flexibility of a flexible ureteroscope is limited in a renal pelvis with high-grade hydronephrosis, which then affects the operation field and stone-free rate.,9 On the other hand, laparoscopic ureterolithotomy (LUL) has been shown to be non-inferior to retrograde flexible ureteroscopy (fURS) in stone-free rate and the need for axillary procedures, and to have a significantly lower rate of post-operative sepsis with huge ureteral stones.–13 In this article, we report the case of a 66-year-old male patient who had previously undergone endoscopic management with double-J catheter (D-J) stenting but still had a heavy stone burden in the ureteropelvic junction (UPJ) and lower pole renal calyx. We conducted LUL with laparoscope-assisted retrograde intrarenal surgery (LA-RIRS) for residual stones and achieved excellent results.Concurrent kidney and ureteral stones have increasingly been managed with endoscopic methods in recent years due to the development of newer generation lithotripters and semi-rigid ureteroscopy along with holmium laser lithotripsy.2A 66-year-old male patient presented with a history of hypertension, diabetes mellitus, and concurrent renal and ureteral stones after receiving endoscopic management at another hospital about 1 month previously. Residual stones in the UPJ and lower calyx were noted in a kidney, ureter, and bladder radiograph  was required.The authors would like to thank China Medical University Hospital for providing the opportunity to conduct this study.Conceptualization: Sheng-Feng Chou, Po-Fan Hsieh.Formal analysis: Sheng-Feng Chou.Funding acquisition: Chi-Ping Huang.Investigation: Sheng-Feng Chou.Resources: Po-Fan Hsieh.Software: Wei-Ching Lin.Supervision: Po-Fan Hsieh, Chi-Ping Huang.Validation: Wei-Ching Lin.Visualization: Wei-Ching Lin.Writing – original draft: Sheng-Feng Chou.Writing – review & editing: Po-Fan Hsieh.


Most patients undergo multimodal treatment approaches and post-treatment follow-up with conventional MRI (CE-T1-weighted and FLAIR/T2-weighted) of the brain is performed to monitor for disease recurrence. However, owing to the similar appearance of treatment-related changes like radiation necrosis with that of true recurrence, conventional MRI alone suffers from low specificity. Given the high mortality of patients with brain metastases and the considerable treatment-associated morbidity, a need remains for an imaging modality that accurately differentiates recurrence from treatment-related changes. Accurate imaging is key to preventing unnecessary surgery or changes in effective therapy in patients mistaken for disease progression as well as prevent continuation of ineffective therapy if radiation necrosis is incorrectly diagnosed. To this end, 18F-fluciclovine PET studies of recurrent brain metastases. Forty subjects with solid tumor brain metastases who have undergone radiation therapy will be enrolled across ~8 US sites if they have a reference lesion considered equivocal on MRI for recurrent disease and are planned for craniotomy. Subjects will undergo 18F-fluciclovine PET <42 days after the MRI and 1–21 days before planned craniotomy. Outcome measures comprise the diagnostic performance of 18F-fluciclovine PET at different thresholds of 18F-fluciclovine uptake compared with histopathology, subject- and lesion-level diagnostic performance based on established image interpretation criteria, and safety evaluations. Enrolment began in August 2020 and the trial is open at the time of submission.NCT04410367 is a prospective, open-label, single-arm, single-dose (185 MBq ± 20%) study with a primary objective to establish visual image interpretation criteria for