Aedes species mosquitoes; however, ZIKV can also be sexually transmitted. During the initial epidemic and in places where ZIKV is now considered endemic, it is difficult to disentangle the risks and contributions of sexual versus vector-borne transmission to adverse pregnancy outcomes. To examine the potential impact of sexual transmission of ZIKV on pregnancy outcome, we challenged three rhesus macaques (Macaca mulatta) three times intravaginally with 1 x 107 PFU of a low passage, African lineage ZIKV isolate (ZIKV-DAK) in the first trimester . Samples were collected from all animals initially on days 3 through 10 post challenge, followed by twice, and then once weekly sample collection; ultrasound examinations were performed every 3-4 days then weekly as pregnancies progressed. All three dams had ZIKV RNA detectable in plasma on day 3 post-ZIKV challenge. At approximately 45 days gestation , two of the three dams were found with nonviable embryos by ultrasound. Viral RNA was detected in recovered tissues and at the maternal-fetal interface (MFI) in both cases. The remaining viable pregnancy proceeded to near term  and ZIKV RNA was detected at the MFI but not in fetal tissues. These results suggest that sexual transmission of ZIKV may represent an underappreciated risk of pregnancy loss during early gestation.Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) and is primarily transmitted by  Aedes species mosquitoes is thought to be the most common route of infection in endemic areas, the contribution of sexual transmission in epidemics remains poorly understood, in part because during an outbreak, both transmission routes occur simultaneously and can be challenging to disentangle (Zika virus (ZIKV) emerged from relative obscurity five years ago to sweep through tropical and subtropical regions of the Western hemisphere. More than a million cases between 2015 and 2018 were reported in Pan American Health Organization (PAHO) regions alone . Human i8 copies/ml reported  models have been utilized to examine aspects of sexual transmission of ZIKV , 30, 31. in mice , 30–39.  in mice .Macaca mulatta) intravaginally with ZIKV. Our goal was to investigate the potential impact of intravaginal ZIKV challenge during the first trimester on fetal and pregnancy outcomes and to develop a model for sexual transmission during early pregnancy.Because infection during the first trimester is associated with the highest risk for adverse pregnancy outcomes and, since women may be unaware of a pregnancy and may potentially be less likely to utilize barrier methods to prevent sexually-transmitted ZIKV infections during the early first trimester , 24, we All animal procedures conformed to the requirements of the Animal Welfare Act and protocols were approved prior to implementation by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Davis. Activities related to animal care, housing, and diet were performed according to California National Primate Research Center (CNPRC) standard operating procedures (SOPs). SOPs for colony related procedures are reviewed and approved by the UC Davis IACUC.Macaca mulatta, N=3) were time-mated and identified as pregnant by ultrasound according to established methods . Challenge stocks were prepared from this master stock by inoculation onto a confluent monolayer of C6/36 mosquito cells as described previously  was originally isolated from  syringe . Animals syringe .Plasma and PBMCs were isolated from blood placed in EDTA vacutainers and processed at 1500 RPM for 15 minutes according to standard protocols. Serum was isolated from whole blood collected into glass vacutainers without additives. Viral RNA was extracted from 300 µl plasma as previously described with a Maxwell 16 MDx instrument  and evaluated using qRT-PCR . RNA conin situ hybridization (ISH).Dams 049-102 and 049-103 were scheduled for hysterotomies in the late first trimester (nonviable embryos detected 3 days prior to hysterotomy). The hysterotomy for dam 049-101 was performed at approximately 155 days gestational age according to the original study design Figure 1For dams 049-102 and 049-103 a modified collection was performed, consistent with the early developmental stage of the conceptus Table 2.Fresh samples collected from the 049-101 fetus  were shipped with cold packs for additional assessments as noted above; the eye was analyzed by the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). Placental tissues from all dams and tissues for the fetus from dam 049-101 were assessed as described previously in Koenig et al. .Maternal-fetal interface (MFI) and fetal tissue vRNA loads were determined from approximately 20 mg of each specimen. ZIKV RNA was isolated from tissues using the Qiagen AllPrep DNA/RNA Mini Kit  using the QIAcube following the manufacturer’s protocol. Viral RNA was isolated from 140 µl maternal urine or amniotic fluid using the QIAmp Viral RNA minikit  following the manufacturer’s protocol. Following isolation, cDNA synthesis was performed using the Qiagen Sensiscript RT kit  according to the manufacturer’s protocol. Quantification of vRNA load was performed by real-time PCR using the Taqman amplification system and the QuantStudio 12 K Flex Real-Time PCR System  as described previously . The est® Red 2.5 kit  according to the manufacturer’s protocol. After deparaffinization with xylene, a series of ethanol washes, and peroxidase blocking, sections were heated with the antigen retrieval buffer and then digested by proteinase. Sections were then exposed to the ISH target probe and incubated at 40°C in a hybridization oven for two-hours. After rinsing, ISH signal was amplified using the provided pre-amplifier followed by the amplifier-containing labelled probe binding sites, and developed with a Fast Red chromogenic substrate for 10 minutes at room temperature. Sections were then stained with hematoxylin, air-dried, and mounted.ISH probes against the ZIKV genome were commercially purchased . ISH was performed using the RNAscope90)  on Vero cells (ATCC #CCL-81) with a cutoff value of 90% (PRNT90) . Neutral90) , 46.4 copies/ml for 049-101 to 1.30x105 copies/ml for 049-103 (2 copies/ml). Dam 049-103 had a detectable plasma vRNA load until day 14 (2.46x103 copies/ml) but was negative on day 17 (the next time point samples were collected). Dam 049-102 was consistently positive for ZIKV vRNA until day 14, was negative on day 17, and then positive again on days 21 and 24 post challenge. Dam 049-102 was positive for ZIKV RNA in blood plasma collected at hysterotomy, the last time point sampled for the study. Overall, maternal plasma vRNA loads for dams 049-101, 049-102, and 049-103 were somewhat delayed compared to animals subcutaneously inoculated with French Polynesian or Puerto Rican ZIKV isolates in our previous studies, but were consistent in magnitude with previous observations . In addition, ZIKV RNA was detected in the brain and liver of both non-viable embryos, as well as in MFI tissues including the primary and secondary placental disks and membranes (amnion and chorion) (5 copies/mg). ZIKV RNA was not detected in amniotic fluid collected from the fetus of dam 049-101 at hysterotomy. Although a large number of fetal and MFI tissues were assessed following hysterotomy, the presence of ZIKV RNA was only detected in a subset of sections of MFI tissues from 049-101 . ZIKV genomic RNA was detected in both the primary and secondary placental disks from dams 049-102 and 049-103  were assessed by ZIKV ISH using RNAscope . Serum samples from 0, 14, 27, and 122 days post-challenge collected from dam 049-101 were similarly assessed. Samples collected on day 0  from all animals were negative for ZIKV nAbs. Neutralizing Ab titers above 1:10 are indicative of immunity against ZIKV. Serum collected on day 14 post-challenge from all animals neutralized ZIKV-DAK at levels considered protective by PRNT90 . Serum collected on day 24 post-challenge from dams 049-102 and 049-103, and on day 27 post-challenge from dam 049-101 showed an increased neutralization response relative to baseline (day 0) and day 14 for each individual animal (90 titer approximately 1:300)  against ZIKV were evaluated for dams 049-102 and 049-103 on days 0, 14, and 24 post-challenge by 90% plaque reduction neutralization tests  , 48, 49.Here we describe a proof-of-concept study that indicates intravaginal challenge with ZIKV during early pregnancy results in productive maternal infection and suggests that infection by this route can result in embryonic demise. ZIKV RNA was detected at the MFI and in embryonic tissues, as well as in the amniotic fluid from the pregnancies of dams 049-102 and 049-103, supporting a role for ZIKV in the adverse pregnancy outcomes for these animals. Although ZIKV was detected by qRT-PCR in the MFI tissues from dam 049-101, no vRNA was detected in fetal tissues directly. Interestingly, although vRNA was detectable in the placenta of dam 049-101 by qRT-PCR, it was not detected by ISH. Given the focal nature of ZIKV RNA detected in the placental tissue samples collected from dam 049-101, it is likely that the samples evaluated by ISH were simply from areas without vRNA present Table 2.Our results indicating early demise as a result of ZIKV infection are consistent with those described previously in a cross-center, cross-NHP species study . InteresSome limitations of this study include the use of a relatively high dose of ZIKV to inoculate the dams, the inclusion of multiple challenges over a short timeframe, and the small number of animals included in the study. The dose of inoculum chosen for this study is representative of the high end of the ZIKV vRNA range reportedly detected in human semen, which can be up to 100,000 times higher than that in blood –18. In pWe chose to utilize a low passage African ZIKV isolate (ZIKV-DAK) rather than a more contemporary isolate such as the commonly utilized PRVABC59 because, although it is also low passage, recent studies have suggested that this virus may have an attenuated phenotype and is not as pathogenic as ZIKV-DAK in mice , 60. In via an intramuscular route during the first or second trimesters has also been shown to result in spontaneous pregnancy loss  has the potential to result in embryonic demise in rhesus macaques when infection occurs intravaginally and in early pregnancy. To our knowledge, this is the first NHP study to show clear evidence of vertical transmission of ZIKV following intravaginal infection, which has only previously been observed in mice , 30, 36.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by University of California, Davis Institutional Animal Care and Use Committee.CN, AT, CM, and DHO designed the study. AT provided animal oversight and monitoring, ultrasound imaging, and performed all sample collections. AT and CM performed animal infections. AT, MM, MB, XZ, HS, TM, MA, EB analyzed samples. CN, DD, and JR curated data. CN and JR prepared the figures. CN prepared the initial manuscript draft. All authors contributed to the article and approved the submitted version.DHHS/PHS/NIH R01 A|1116382-01A1, NIH P51 OD011107 and NIH S10 OD016261.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The uncertainty of the pandemic of Coronavirus Disease 2019 (COVID-19) brought about tremendous psychological harm for pregnant women, causing their high rates of prenatal anxiety. The impacts of COVID-19 pandemic and symptoms of pregnant status are highly linked with prenatal anxiety. Whereas, self-efficacy and support from family and friends could attenuate the development of prenatal anxiety. Thus, the purpose of the study is to evaluate the prevalence of prenatal anxiety and its influence factors among pregnant women during the pandemic of COVID-19 in Shenyang, China.A cross-sectional study with face-to-face interview between April 24, 2020 and May 3, 2020 during the COVID-19 pandemic was applied among pregnant women in Shenyang Women’s and Children’s Hospital. Chi-square tests were calculated to determine the differences in prenatal anxiety among categorical variables. Multivariable logistic regression was employed to investigate the risk factors of prenatal anxiety.The percentage of prenatal anxiety (GAD-7 score ≥ 7) among pregnant women during the pandemic of COVID-19 was 34/304 (11.18%). Logistic regression indicated that vomiting  and feeling susceptible to SARS-CoV-2 infection  increased the odds of prenatal anxiety. Satisfaction with medical care  and self-efficacy  decreased the odds of prenatal anxiety. High monthly income  reduced the chances of suffering from prenatal anxiety.The pregnant women in China exerted a higher prevalence of prenatal anxiety during the COVID-19 pandemic than that without COVID-19 pandemic. Effective management on symptoms of pregnant status should be delivered to relieve prenatal anxiety for the pregnant women. Furthermore, interventions on self-efficacy enhancement and high-quality medical prenatal care should be provided to prevent from the susceptibility of SARS-CoV-2 infection and reduce prenatal anxiety. Pregnant women experience considerable physical and psychological changes in the process of pregnancy, which may elevate their likelihood of suffering from mental disorders , 2. SincPregnant women may cancel or delay obstetric examinations because of worries about the risk of SARS-CoV-2 infection at health facilities, routine inspection and the process of delivery , 8. The Factors that influence pregnant women’s psychological state are complicated and multifaceted. Demographic characteristics and characteristics of pregnancy such as monthly income and vomiting were found to be associated with prenatal anxiety , 17. PreSelf-efficacy, as a positive resource, is defined as the perceptual ability to execute and perform required actions in order to achieve a specified type of performance, which plays a vital role in the transition into motherhood and has a strong relationship with reduced anxiety , 24. As There is an urgent need to explore the mental health among pregnant women during COVID-19 pandemic. The target of this study was to assess the levels of prenatal anxiety and its associated protective and risk factors during the COVIID-19 pandemic, including demographic characteristics, symptoms of pregnancy status, and positive resource, as well as the pandemic of COVID-19. This study provides evidence that can be used to improve psychological counseling and psychological crisis interventions for pregnant women.A cross-sectional study with face-to-face interview between April 24, 2020 and May 3, 2020 during the COVID-19 pandemic was measured among pregnant women in Shenyang Women’s and Children’s Hospital, Liaoning province, China. Pregnant women who came for routine obstetric examinations or hospitalizations, met the inclusion criteria, were continuous selected in this study. This study was implemented and approved by the Standards of Ethics Committee at China Medical University. Questionnaires were set up in advance before the survey and the questionnaire was preset, only after all questions have been answered by the respondents that could be submitted. The questionnaire included the Generalized Anxiety Disorder 7-item Scale (GAD-7), General Self-Efficacy Scale (GSES), questions on demographic characteristics, questions on symptoms of pregnant status, and questions on impacts of COVID-19 response, taking about 25 min to complete.The inclusion criteria for the participants was: I. Eighteen years and older; II. Visited the hospital and had maternal health records available between April and May 2020; III. Able to use a mobile phone and complete the questionnaire independently or with the help of the interviewer; IV. Willing to participate and provide signed web-based informed consent. Study exclusion criteria included: I. Reported a hearing or vision impairment, mental illness, or clinical diagnosed psychiatric disorder including anxiety, depression, mania, schizophrenia.; II. Reported fetal malformations, stillbirths and other fetal abnormalities requiring the induction of labor.A comprehensive explanation of this survey purpose, content, was provided by experienced surveyors in advance of this questionnaire, and then pregnant women who prefer to participate in this study were required to sign an online informed consent forms, data were attained through trained surveyors with face-to-face interview through a link to an electronic version of questionnaire on a WeChat platform via Wenjuanxing Platform, a widely used social media and professional online survey platform in China. Among totally 355 pregnant women identified, 304 consecutive pregnant women validly completed questionnaires, obtaining a valid response rate of 83.3%.Demographic characteristics of the participants including age , education , monthly income , trimester of gestation , history of abortion (yes/no), were collected in this study.Symptoms of pregnant status included gravidity (first pregnancy/two or more previous pregnancies), pregnancy complications (yes/no), vomiting (ever/never), mood fluctuations (yes/no).Self-efficacy was conducted with the assessment using the Schwarzer General Self-Efficacy Scale (GSES) . The ChiMeasures of COVID-19 worries regarding the COVID-19 pandemic include the questions on whether or not they felt large impacts on daily life during the COVID-19 pandemic (yes/no), whether or not they felt susceptible to SARS-CoV-2 infection (yes/no), whether or not communities where they lived were under lockdown during the COVID-19 pandemic (yes/no), whether or not they were satisfied with their medical care (yes/no),and timely support from family and friends (yes/no).Prenatal anxiety during pregnancy was measured by the Generalized Anxiety Disorder 7-item Scale (GAD-7) . The ChiP values < 0.05 were thought as statistically significant for the study. Odds ratios (OR) and 95% confidence levels (95% CI) were calculated to analyze risk factors associated with anxiety. All analyses were employed with SPSS 24.0 statistical software version 3 for Windows version 17.0 .Chi-square tests were employed for the categorical variables to compare the differences in the prevalence of prenatal anxiety. Multivariable logistic regression was employed to investigate the associated factors of prenatal anxiety. The demographic characteristics and characteristics of pregnancy, symptoms of pregnant status as well as impact of COVID-19 were selected as the independent variable. Anxiety were converted into categorical variables as a dependent variable in this study based on the research purpose. A total of 304 women with pregnancy completed this online survey. The demographic characteristics and the prevalence rate of prenatal anxiety in pregnant women are exhibited in Table P < 0.05). Primigravid women reported a higher rate of anxiety compared with those who had two or more previous pregnancies (P < 0.05). Pregnant women with vomiting symptoms experienced a higher prevalence of anxiety than those without vomiting symptoms (P < 0.05). Pregnant women who felt susceptible to SARS-CoV-2 infection suffered a higher percentage of anxiety than those who felt they could not possibly be infected with SARS-CoV-2 (P < 0.01). Participants who were dissatisfied with medical care had higher pregnancy-related anxiety compared with participates who were satisfied with their medical care (P < 0.01). Pregnant women enjoying with more self-efficacy had a higher prevalence of anxiety compared with other groups (P < 0.01).The factors associated with prenatal anxiety during the COVID-19 pandemic are represented in Table The associated factors of prenatal anxiety was analyzed with multivariable logistic regression analyses. High monthly income  and , satisfaction with medical care , and self-efficacy (OR 0.253 95% CI 0.100 ~ 0.639) decreased the chances of suffering from prenatal anxiety. Conversely, vomiting  and felt perceived susceptible to SARS-CoV-2 infection  increased the odds of prenatal anxiety.The results of the final multivariable logistic regression model and the forest plot are listed in Fig. The study demonstrated the impacts of the COVID-19 pandemic on the prevalence of prenatal anxiety among 304 pregnant women in Shenyang, the capital city of Liaoning province, China. The results revealed that 11.18% pregnant women experienced prenatal anxiety during the COVID-19 pandemic (GAD score ≥ 7), which was slightly higher than the prevalence before the pandemic according to the previous studies (4% VS 7%) , 34. PreThis study revealed that high monthly income was a protective factor of pregnant women’s prenatal anxiety, which was consistent with previous studies . Having Notably, vomiting was found to be a risk factor for prenatal anxiety. Women with a history of severe vomiting were susceptible to have psychological disturbances, such as anxiety and depression . VomitinThe results of this study indicated that pregnant women who perceived susceptible to SARS-CoV-2 infection were prone to have higher levels of prenatal anxiety. Among our participants, 13.5% of women reported feeling very possible about SARS-CoV-2 infection. Given the common modalities of routine examination, pregnant women were afraid of being exposed to hospitals where there were relatively high cases of COVID-19 patients. Besides, with the uncertain features of the COVID-19 virus and symptoms, the excessive concerns and worries about their own health and vertical mother-to-fetus transmission infection might have increased the prevalence of anxiety . In addiAnother finding of our survey was that satisfaction with medical services serves as a protective factor against prenatal anxiety. The majority of pregnant women  reported they were satisfied with medical services, which implied that the quality of prenatal care improved and the expectations of pregnant women’s health were met during the COVID-19 pandemic. Chinese pregnant women were satisfied with local health facilities due to the effective measures to facilitate prenatal care, such as prompted interpreted the guidelines through new media to popularized professional knowledge, launched online education to answer questions, opened online appointments and green channels . PregnanFinally, our findings illustrated that self-efficacy played a positive role in relieving anxiety, which is consistent with previous studies , 27. SelThere are several limitations for the present study. First, this study’s cross-sectional design indicating that it is impossible to draw conclusions about the causal relationships between anxiety and its associated factors. Therefore, prospective longitudinal studies are needed to confirm the results presented in this study. Second, participants in this study were only from a single hospital in an urban area in northeastern China, which does not allow for the generalizability of this results to other populations with selection bias and information bias. Therefore, expanding hospitals and regions to improve the results of generalizability should be prominent in the future study. Third, this survey did not take account of the influence of individual factors , obstetric characteristics  on pregnant women’s anxiety, which could be confounding factors –52. TherOur study revealed that there is a slightly high rate of prenatal anxiety among Chinese pregnant women in COVID-19 pandemic (11.18%). Pregnant women who suffered from vomiting and perceived susceptible to SARS-CoV-2 infection experienced the elevated levels of prenatal anxiety. Moreover, high monthly income, satisfaction with medical care, and self-efficacy were found to be protective factors against prenatal anxiety for pregnant women. This study indicated that interventions aiming to target reactions including improvement of self-efficacy, provision of high quality prenatal care and management of pregnancy-related complications are of significant importance to alleviate anxiety among pregnant women while they are encountering public health emergencies.


Immune checkpoint inhibitor (ICI)-related myositis is a rare, potentially fatal condition that warrants further studies. Its incidence, clinical features, and prognosis remain poorly understood. To address these gaps, we conducted a systematic review and meta-analysis to evaluate the risk of myositis associated with ICI for solid tumors by analyzing phase III randomized controlled trials of anti-programmed death-1/ligand-1 (PD-1/PD-L1) and anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4). To complement this analysis with clinical data, we evaluated published ICI case reports along with cases from our institutional registry. This registry comprised 422 patients treated with ICIs alone or in combination from September 2014 to June 2021. The analysis revealed an incidence of ICI-related myositis in 6,838 patients in 18 randomized controlled trials of 0.38%  for patients receiving ICIs compared with controls. Detailed analysis of 88 cases from the literature search and our registry showed that myositis induced by PD-1 inhibitors was more frequent than that induced by anti-CTLA-4 agents, revealing a clinically diverse trend including myasthenia gravis and myocarditis. Importantly, having ptosis at the time of onset was significantly associated with the development of concomitant myocarditis , which is associated with poor prognosis. Regarding treatment, most patients received glucocorticoids, and some received immunosuppressants. Our study revealed the incidence of ICI-mediated myositis and the clinical features of myocarditis, highlighting the need for recognition and early intervention. Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment by exerting anti-tumor effects on various types and stages of cancer that cannot be achieved with existing drugs, and numerous clinical trials are underway to expand indications . HoweverA review by Kadota et al.  reportedA systematic review on myocarditis has been published , but, toThe systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines , 10 and via discussion between the two investigators.In the electronic search, we systematically searched PubMed, EMBASE, the Cochrane Central Register of Controlled Trials, and Web of Science Core Collection (up to August 16 2021) for randomized controlled trials (RCTs) reporting the risk of myositis associated with ICIs for the treatment of patients with solid tumors. Search formulae are presented in Supplementary Text 1. Two investigators  independently screened candidate articles by checking the title and abstract after uploading the citation list into Endnote X9 software . Inclusion criteria were as follows: (1) phase III RCT study design; (2) the experimental group was treated with at least one type of ICI with or without other systemic chemotherapy and the control group with non-ICIs; (3) three-arm studies where an ICI was included in at least one arm; (4) patients clinically diagnosed with any solid tumor; and (5) the study included the incidence of myositis. Exclusion criteria were as follows: (1) systematic review or meta-analyses; (2) retrospective analyses; (3) single prospective cohort studies with no control group; (4) ICI two-drug combination therapy; (5) republished research literature; (6) studies with no or insufficient safety results at the time of the literature search; and (7) studies published in languages other than English. Only full-text papers were used for analysis. Disagreements in assessing cases or data were resolved 2 statistics and P-values. Heterogeneity was indicated by I2 wherein 0% indicated no heterogeneity and 100% indicated the strongest heterogeneity. Statistical analyses were performed using Review Manager 5.3 . The Cochrane Risk of Bias Tool was used to evaluate the risk of bias for each RCT (Odds ratios (ORs) of any-grade myositis between the ICI treatment arm and non-ICI arm were calculated using a random-effect model meta-analysis. Heterogeneity was assessed using Ieach RCT . The quaConsecutive cancer patients  who received ICIs  for approved cancer types at the collaborative research department of Yokohama City University Hospital between December 2014 and May 2021 were included. The study was approved by the ethics committee of Yokohama City University (A200500004). In this study, we included patients treated with ICI monotherapy or combination therapy. Demographic, clinical, and treatment data were obtained from clinical charts. The study followed the Ethical Guidelines for Epidemiology Research, published by the Japanese Ministry of Health, Labour and Welfare, and had an opt-out strategy.We searched the literature for cases of ICI-related myositis using PubMed on September 7, 2021. ‘ICI-related myositis’ was defined according to the previously reported literature review .Of 5,471 candidate articles, 120 were potentially eligible after abstract and title screening. From these 120, 19 studies were excluded, and 101 articles were reviewed in detail. Studies of 93 RCTs were insufficient to assess the incidence of myositis. Finally, 18 RCTs –30 were 2 = 0%)  from 18 RCTs. Twenty-six events were observed in the ICI arm and seven events were observed in the non-ICI arm. Among the 26 ICI-related myositis, 11 events were caused by anti-PD-1, 13 by anti-programmed death ligand 1 (PD-L1), and two by anti-CTLA-4, suggesting that any ICI can elicit this condition. We calculated OR and 95% CI to compare PD-1/PD-L1 and CTLA-4 blockade therapies. There was no significant difference in frequencies of myositis between two groups . The overall incidence of ICI-related myositis was 0.38% , and did not seem to be affected by the cancer type or ICI regimen. We found that myositis was significantly increased with ICI treatment compared with non-ICI treatment  Figure 2Meta-analysis provides epidemiological data such as the incidence of ICI-related myositis, but it is difficult to analyze detailed clinical characteristics. To understand the prognosis of ICI-related myositis, we first examined our institutional database. A total of 422 patients were identified. Among these, 365 cases were treated with a PD-1 or PD-L1 inhibitor alone, nine were treated with a CTLA-4 inhibitor alone, and 47 cases were treated with a PD-1 inhibitor and CTLA-4 inhibitor combination. There were three cases of ICI-related myositis (incidence 0.71%). Baseline characteristics are shown in To clarify the clinical features of ICI-related myositis in a larger patient population, we evaluated the clinical features of published case reports of ICI-related myositis. Of 75 candidate articles, 63 were identified as eligible –93. A fuAll cases except one were treated with glucocorticoids. Thirty-three patients died, of which 19 deaths were associated with myositis. Because myocarditis is known to be a frequent and fatal complication of ICI-related myositis , we compThere were some significant differences in clinical characteristics in the group that developed myocarditis. First, we analyzed overall ICI use and found that the occurrence of myocarditis was significantly affected by the type of ICI regimen (p=0.015), but was underpowered to detect an association with a specific regimen. Second, in terms of clinical features at the time of myositis onset, ptosis and respiratory muscle paralysis (type II respiratory failure) were significantly more frequent in the myocarditis group . The ORs were calculated from multivariate logistic regression analysis, which also revealed that ptosis and respiratory muscle paralysis were independently associated with the development of myocarditis .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


HIV-1 infection and its progression to AIDS remains a significant global health challenge, particularly for low-income countries. Developing a vaccine to prevent HIV-1 infections has proven to be immensely challenging with complex biological acquisition and infection, unforeseen clinical trial disappointments, and funding issues. This paper discusses important landmarks of progress in HIV-1 vaccine development, various vaccine strategies, and clinical trials. In 2020, 37.6 million people were living with HIV infections, and 1.5 million people acquired HIV infections within the year. Since the HIV epidemic began, 34.7 million people have died due to an AIDS-related illness. In 2020, 690,000 people died due to an AIDS-related illness [Treatment of HIV-1 infections drastically changed as antiretroviral drugs evolved. This started with azidothymidine, an inhibitor of viral reverse transcriptase, in 1987, and it decreased the amount of HIV RNA in the bloodstream. This treatment plan was altered from a single-drug regimen for a more effective two-drug regimen. Then, clinicians tested a three-drug regimen, including the newly developed protease inhibitors, and this was referred to as the highly effective combination antiretroviral therapy (ART) in 1996. Provided that patients with HIV-1 infections consistently adhere to ART, they have close to normal life expectancies and do not transmit the virus to an uninfected sexual partner. The virus is not transmitted in this circumstance because ART suppresses the level of the virus to incredibly low levels .Fourteen years later, in 2012, clinicians started using ART to prevent HIV-1 infections and referred to this as preexposure prophylaxis or PrEP. A single pill taken once daily has been shown to be 99% effective in preventing HIV-1 infection via sexual acquisition . CurrentWhile the development of effective antiretroviral drugs for patients with HIV-1 infections and their application as PrEP to help prevent infection to at-risk people is an important landmark in scientific history, it does not replace the need for an effective vaccine ,4,5,6. GTo end this epidemic, two possible immunization strategies must be considered as possible solutions, therapeutic and prophylactic vaccines. The aim of prophylactic vaccines is to prevent the infection or disease while therapeutic vaccines are aimed at treating the individual already infected with HIV. This review is focused on the prophylactic vaccines, and as such, it considers clinical trials that assess the risk of contracting HIV-1 infections after receiving a prophylactic vaccine instead of reduction in viral load.The biological challenges of HIV vaccine development include a high rate of mutation and recombination during viral replication, four main groups of HIV with nine subtypes/clades across the world, no appropriate animal models, and limited information regarding the correlates of immune protection.The high rate of mutation of HIV is due to the error-prone viral reverse transcriptase and has been estimated to lead to 1–10 mutations per genome per replication cycle. This mutation rate mostly leads to changes within the Env glycoprotein, allowing glycan shielding so the virus can evade the immune system. Though there is considerable genetic diversity present in the Env glycoprotein, this structure is the main target of neutralizing antibodies .HIV-1 infections are classified into a group  and subtype or clade . Within a clade, genetic variation can be as high as 30%. Additionally, 10–20% of people infected with HIV in certain regions of Africa have two or more viral subtypes . These vThe lack of appropriate animal models posed a unique challenge to researchers before the early 1990s. Chimpanzees, an endangered species classified as a nonhuman primate (NHP), contracted HIV, but it did not follow the course of the disease in humans. This led to the US and Japan separately developing SHIV, a chimeric virus with gag and pol genes from SIV and env gene from HIV, because it is a more pathogenically relevant model for the generation of an HIV vaccine. The standard animal model widely accepted today is macaque monkeys infected with SHIV administered with low doses and intravaginally . It is wResearchers are also challenged by the lack of information regarding the correlates of immune protection. This is due to the complex progression of HIV-1 infection since the infection is never able to be cleared by the immune system because of the reservoir of latently infected memory CD4+ T-cells .An interesting characteristic that may provide insight into immune correlates is the three unique subsets of the population with HIV-1 infections. There are viremic controllers who suppress the viral load from 50 to 2000 RNA copies per mL, elite controllers who suppress the viral load to <50 RNA copies per mL, and long-term nonprogressors. Long-term nonprogressors maintain stable CD4+ T-cells above 500 cells per microliter for a decade without ART. The mechanisms behind these subsets’ response to HIV-1 are not fully understood. Some genetic studies suggest the difference in amino acids in proteins that code for HLA class I alleles are correlated to controllers or progressors. HLA-B*57, HLA-B*27, HLA-B*52, and HLA-B*14 are more frequently found in controllers, while HLA-B*07, HLA-B*08, and HLA-B*25 are linked to an increased risk of progression. Though genetic variation is an important aspect of understanding the biology behind viremic and elite controllers, no HLA Class I allele is able to suppress the virus. Researchers believe that the mechanism likely involves HLA association with CD8+ T-cells and other nongenetic factors . Another characteristic that may provide insight into immune correlates is the neutralizing antibodies present in approximately 10–30% of people with HIV-1 infections. These antibodies are developed from heavy chain mutation and are not produced until years into a chronic natural infection [There is a gap between the resources and funding needed to develop an effective vaccine and the countries that have large populations with HIV-1 infections. Unfortunately, the financial backing behind HIV vaccine research and development has decreased since 2010. Eighty-five percent of the funding has been contributed by the US government and the Bill & Melinda Gates Foundation. In 2018, the combined contribution from these two groups amounted to approximately 680 million US dollars .From 1987 to 2021, there have been three major approaches driving HIV-1 vaccine development. Each of these approaches involved one or more clinical trials and is summarized in In the late 1980s, the first approach focused on generating a vaccine that would induce neutralizing antibodies because neutralizing antibodies and their associated subsequent cytotoxic T lymphocyte responses were believed to provide enough protection against HIV-1. Vaccines designed and tested targeted gp120 or gp160 HIV-1 envelope proteins . This waThe second approach was based on administering a viral vector to induce a CD8+ T cell response . In the Briefly, the STEP trial starting in 2004 and its associated counterpart, “Phambili”, starting in 2007 were the first T-cell-based vaccine candidates. Both tested recombinant Ad5 vector with HIV-1 clade B gag/pol/nef inserts. It is worth noting that no envelope genes were present  because The third and current approach is to utilize a heterologous prime-boost to elicit humoral and cell-mediated immune responses . The priThis approach was employed in the only modestly successful trial to date, RV 144, and its results were made public in 2009, only two years after STEP’s disappointing results . This trThough the prime-boost approach has proven to be somewhat successful, there has yet to be an HIV vaccine, so more research into the understanding of HIV’s mechanism and its immune correlates, development of an effective and safe vaccine, and clinical trials are necessary.Since researchers have been developing an HIV vaccine for two and a half decades, the fields of molecular biology, bioinformatics, and “omics” based technology have all developed from inception or significantly expanded ,12.Molecular biology and bioinformatics techniques rapidly evolved and led to the HIV genome sequencing and cloning and identification of structural proteins of the virus . “Omics”Since the beginning of HIV vaccine development, it is worth noting the significant changes from the initial recombinant vectors to more effective and safer vectors. This transition to improve recombinant vectors was specifically between the first and second approaches discussed in the history of HIV development section. Initially, a recombinant vaccinia virus was utilized in 1987, and it posed serious potential concerns . IndividIn 1975, researchers developed MVA, a nonreplicating highly attenuated vaccinia virus with over 200 poxvirus proteins . It was Given the numerous challenges in HIV-1 vaccine development, scientists doubted whether a vaccine could be generated to provide immunity against this virus. One significant breakthrough that illustrated that a preventative HIV-1 vaccine is possible was the result of the RV144 trial, obtained in 2009 [Briefly, a summary of the RV144 trial protocol is as follows. This phase 3 efficacy trial conducted in Thailand tested ALVAC-HIV, a recombinant canarypox vector. This vector included Env (clade E), group-specific antigen (gag) (clade B), and protease (pro) (clade B) and is classified as a prime . FollowiIt is worth noting that both of these components had previously been tested in other trials. ALVAC-HIV, the vector prime, was not as immunogenic as some of the other vectors. AIDSVAX with a bivalent clade B gp120, the protein boost, had also been tested separately with no vector prime, and this was unsuccessful in preventing HIV-1 infection .RV144 trial had a vaccine efficacy of 60% at 12 months and 31% at 3.5 years . Though Given the unexpected results of RV144, there is renewed interest in the role of antibodies outside the classic role of neutralization, and this is particular focused on antibody-dependent cell-mediated cytotoxicity (ADCC) .Based on the RV144 trial’s modest efficacy, a similar trial in South Africa, HVTN 702, was launched. The vaccine regimen was designed to increase the efficacy and immune response duration of RV144. These modifications from RV144 included changing the clade present in the vaccine due to regional differences, changing the adjuvant in the protein boost from alum to MF49, and changing the timing of the vaccinations from four injections in six months to five injections over twelve months .HVTN 702 was terminated prematurely because the independent data and safety monitoring board found that the vaccine was not effective, with approximately the same number of HIV infections in the participants who received the vaccine as the participants who received the placebo .The rationale behind the study design of HVTN 702 may have led to this product failure in clinical trial. The HVTN 702 clinical trial differed from the RV144 clinical trial in terms of vaccination schedule, clades, subtypes of proteins, lack of tagging of proteins, genes in immunogens, and/or adjuvants. Based on nonhuman primate studies, the alum to MF49 adjuvant change may have contributed to low efficacy in HVTN 702 . MF49 adResearchers have attempted to produce immunogens that induce the immune system to synthesize broadly neutralizing antibodies (bNAbs) for several years. BNAbs inhibit the virions from entering the host cells, preventing HIV integration into the genome. Their role is particularly important because bNAbs are able to protect against the strain that the patient has been infected with as well as multiple different immunological strains . Though The breakthrough that synthesized bNAbs was high throughput single-cell BCR-amplification assays . This waTo investigate whether these bNAbs could induce a protective immune response in human subjects with HIV-1 infections, two early phase clinical trials were completed. Caskey et al. studied how a passive infusion of 3BNC117 affected the viral load in participants with HIV-1 infections and without HAART treatment . Lynch eResearchers assessed VRC01 further and found that it protected against HIV-1 clades B and C in vitro. Subsequently, they tested VRC01 in two simultaneous proof-of-concept trials, HVTN 703 and 704, starting in 2016. These trials were completed to learn whether this bnAb could prevent HIV-1 acquisition .The results of the antibody-mediated protection (AMP) trials published in 2021, shown in BNAb as passive immunotherapy would be challenging to administer to a small population with the health infrastructure in place and the cost of biological production. Given the obstacles to administering this therapy, it is clear that an active vaccination eliciting bNAbs would be more effective as a prophylactic vaccine. A concept to circumvent this issue is vectored immunoprophylaxis, where an adeno-associated vector and the bnAb genes are injected into the muscle. Researchers used this gene therapy concept and conducted a phase 1 trial (IAVI A003/CHOP HVDDT 001) with rAAV vector coding for PG9 antibody, shown in Given HIV-1’s vast genetic diversity and several strains, some researchers reconstructed global HIV-1 sequences in silico to generate mosaic immunogens . These iAPPROACH, a phase I/II clinical trial, investigated the safety and effects of two different mosaic vaccines in Ad26 or MVA vectors followed by a protein boost of Env gp140 . At the Building on the positive phase I/II results of the Ad26 mosaic vaccine, a phase II clinical trial, HVTN 705/Imbokodo, evaluated the efficacy and was expected to be completed in 2022 . UnfortuSome research groups believe that a rationally designed HIVconsv immunogen is the key to an effective HIV vaccine. In theory, this immunogen causes T-cells to bind conserved regions of HIV-1 proteins and is designed with alternating clade consensus sequences between each conserved sequence. In humans, a vaccine of HIVconsv inserted into DNA, simian adenovirus, and poxvirus MVA vectors induce broadly specific T cell responses .A phase I clinical trial, known as HIV-CORE 004, evaluated pSG2.HIVcons plasmid DNA, MVA.HIVconsv and Ad35-GRIN. Ad35-GRIN contained HIV-1 clade A Gag, reverse transcriptase, integrase, and Nef fusion protein (GRIN). All participants had HIVconsv-specific T cell responses. In vitro, vaccine-induced T-cells inhibited replication of the majority of viruses tested from HIV-1 clades A, B, C, and D .A second generation tHIVconsvX, based on this concept, will be studied in HIV-CORE 0052, a phase I clinical trial. This is designed to test the safety and immunogenicity of the conserved mosaic HIV-1 vaccine .In an attempt to produce an effective HIV-1 vaccine, researchers are developing messenger ribonucleic-acid (mRNA)-based vaccines. mRNA vaccines have been developed for Zika, influenza , and 2 vGiven the biological challenges of HIV-1, as discussed previously, this strategy to vaccination will likely be more complicated than other strategies. Mu et al. hypothesized that this scenario for a possible effective HIV-1 mRNA vaccine involves a sequential immunization of mRNAs encoding Env-based immunogens that will produce germline precursors that develop into bNAb in the germinal centers .In phase 1 clinical trial of an mRNA-based HIV vaccine, IAVI G001, researchers administered participants with two doses of eOD-GT8 60mer vaccine or placebo . The eODThough there have been significant achievements in preventing HIV-1 infections in at-risk populations, it does not replace the necessity for the efficacious prophylactic vaccine. This has become apparent now more than ever because of the pandemic of COVID-19. This pandemic illustrated the significant gaps in healthcare and its accessibility to populations at risk for developing HIV-1 infections. People at risk for developing HIV-1 infections and people with HIV-1 infections have been negatively impacted in terms of HIV testing, treatment access, availability of preexposure prophylaxis, and treatment of opportunistic infections . The sucMore research, funding, and clinical trials are necessary to eradicate the HIV-1 epidemic. Though researchers have been developing a vaccine for thirty years, the mosaic antigens, broadly neutralizing antibodies, and gene therapy application to induce broadly neutralizing antibodies show significant advancement and may potentially provide us with an HIV-1 vaccine in the future.


Mycobacterium tuberculosis antigens as subunit vaccines. Such vaccines may be used either alone or in the prime-boost model in BCG-vaccinated people. However, the antigens for subunit vaccines require adjuvants and/or delivery systems to induce appropriate and protective immune responses against tuberculosis and other diseases. Articles published in this Special Issue have studied the pathogenesis of BCG in children and the use of BCG and recombinant BCG as potential vaccines against asthma. Furthermore, the use of different adjuvants and delivery systems in inducing the protective immune responses after immunization with subunit vaccines has been described.The only licensed vaccine against tuberculosis is BCG. However, BCG has failed to provide consistent protection against tuberculosis, especially pulmonary disease in adults. Furthermore, the use of BCG is contraindicated in immunocompromised subjects. The research towards the development of new vaccines against TB includes the use of  Mycobacterium bovis after subculturing on artificial medium for 13 years (1908–1921) and 231 passages [Vaccines, invited experts have contributed articles related to the pathogenesis of BCG in neonates, the protective potential of BCG against asthma, and the use of mycobacterial antigens as subunit vaccines in inducing protective immune responses by using different adjuvants and delivery systems.Tuberculosis (TB) is a global infectious disease that has been known to humans since ancient times. Although, various steps to control TB have been undertaken by different governments, as well as the World Health Organization, TB remains among the top 10 diseases in the world . The onlpassages . Currentpassages . Howeverpassages . In thisThe article by Al Busaidi et al. reports the spectrum of BCG-induced disease among children in Oman . They inThe BCG vaccine has also been reported to protect against other mycobacterial diseases   and non-Mycobacterium tuberculosis antigens as subunit vaccines [BCG has been used as a vaccine to protect against TB since 1921, but it has failed to provide consistent protection in different parts of the world, especially against pulmonary TB in adults [vaccines . Such vavaccines .M. tuberculosis subunit vaccine Mtb10.4-HspX (MH) plus ESAT6-Ag85B-MPT64<190–198>-Mtb8.4-Rv2626c (LT70), together with adeno-associated virus-mediated IL-7 or lentivirus-mediated transcriptional factor Id3, Bcl6, Bach2, and Blimp1. The vaccine-induced immune responses were evaluated at 25 weeks after the last immunization. The results showed that IL-7 plus the TB subunit vaccine induced the formation of long-lived memory T cells and increased the expression of Id3, Bcl6, and bach2, which are the three main transcription factors involved in the generation of long-lived memory T cells. The results of immunization with the transcriptional factors along with the subunit vaccines showed that Bcl6 and Id3 resulted in the increased production of antigen-specific antibodies and long-lived memory T cells. These results showed that IL-7 and transcriptional factors Id3 and Bcl6 acted as adjuvants in helping the TB subunit vaccine to induce long-term immune memory, which plays a role in providing protection against M. tuberculosis infection.In this Special Issue, the results of the adjuvant effects of the cytokine IL-7and transcriptional factors  in increasing the duration of the immune memory induced by tuberculosis subunit vaccines was studied in mice by Han et al. . Groups Many other adjuvants and delivery systems have been used to improve the protective immune responses induced by subunit vaccines against TB, which include bacterial and viral vectors, liposomes, nanoparticles, plasmid DNA, and edible-based strategies, etc. ,25,26. AThe subunit vaccines listed in In conclusion, the use of appropriate adjuvants and delivery systems for tuberculosis subunit vaccines has gone beyond the experiments in animals and several clinical trials in humans have been conducted with satisfactory outcomes. These results provide hope for new TB vaccines that may have a safety profile better than the currently used BCG vaccine. Furthermore, such subunit vaccines may be able to replace BCG or can be used as boosters in BCG-vaccinated subjects in prime-boost strategies.


Extracorporeal photopheresis (ECP), an immunomodulatory therapy for the treatment of chronic graft-versus-host disease (cGvHD), exposes isolated white blood cells to photoactivatable 8-methoxypsoralen (8-MOP) and UVA light to induce the apoptosis of T-cells and, hence, to modulate immune responses. However, 8-MOP-ECP kills diseased and healthy cells with no selectivity and has limited efficacy in many cases. The use of 5-aminolevulinic acid (ALA) and light (ALA-based photodynamic therapy) may be an alternative, as ex vivo investigations show that ALA-ECP kills T-cells from cGvHD patients more selectively and efficiently than those treated with 8-MOP-ECP. The purpose of this phase I-(II) study was to evaluate the safety and tolerability of ALA-ECP in cGvHD patients. The study included 82 treatments in five patients. One patient was discharged due to the progression of the haematological disease. No significant persistent changes in vital signs or laboratory values were detected. In total, 62 adverse events were reported. Two events were severe, 17 were moderate, and 43 were mild symptoms. None of the adverse events evaluated by the internal safety review committee were considered to be likely related to the study medication. The results indicate that ALA-ECP is safe and is mainly tolerated well by cGvHD patients. The modification of today’s standard extracorporeal photopheresis (ECP) with the introduction of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) may improve treatment efficacy. Since the treatment of cutaneous T-cell lymphoma was approved more than 30 years ago, ECP has been used to treat patients with other T-cell-mediated disorders, including chronic graft-versus-host disease (cGvHD) ,2,3. GraECP is a leukapheresis-based therapeutic procedure. The mechanism of its immunomodulatory effect is not fully understood but involves altered T-cell function and the modulation of dendritic cell maturation, with the modification of the cytokine profile and stimulation of regulatory T-cells ,7,8. DurThe idea of using 5-aminolevulinic acid (ALA) as a photosensitiser precursor for ECP has emerged from decades of research and experience with photodiagnosis and PDT. ALA-PDT is well established for topical use in the treatment of patients with non-melanoma skin cancer, and the oral intake of ALA is approved for clinical use in the photodetection of glioma and for the treatment of Barrett’s oesophagus ,10,11. AThe aim of this study was to evaluate the safety and tolerability of multiple treatments with ALA-ECP in patients with cGvHD. In addition, data are presented from assessments of selected organs, performance, and quality of life in patients during the study period.9/L, platelet count < 20 × 109/L, aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, or an international normalised ratio (INR) value ≥ 3× upper limit of normal or significant ECG findings. Patients considered unlikely to comply with the study procedures were also excludedPatients with cGvHD who responded inadequately to 8-MOP-ECP after a minimum of 3 months of treatment at St. Olavs Hospital, Trondheim University Hospital, Norway, were considered for study inclusion. Inadequate response was defined as the progression of cutaneous cGvHD (>25% worsening of skin score from baseline or insufficient response with a <15% improvement in skin score compared with baseline or a ≤25% reduction in corticosteroid dose). Excluded were patients under 18 years of age; with body weight below 40 kg; and those with photosensitive comorbidities such as porphyria or known hypersensitivity to ALA or porphyrins, aphakia, pregnancy or breastfeeding, polyneuropathy, ongoing cardiac and pulmonary diseases, uncontrolled infection or fever, history of heparin-induced thrombocytopenia, an absolute neutrophil count < 1 × 10www.clinicaltrials.gov (accessed on 20 September 2017) as NCT03109353. The study was performed in accordance with the Helsinki Declaration, monitored by the Clinical Research Unit Central Norway, Norwegian University of Science and Technology (NTNU), and patients provided written informed consent.This prospective, open, single-centre, phase I–(II) study was approved by the regional committee for medical research ethics (REK-Nord 2014/2316), the Norwegian Medicines Agency  and was registered at The CELLEX Photopheresis machine  was used. This is a closed system in which leukapheresis, photoactivation, and the reinfusion of white blood cells are achieved sequentially. The patients were connected to the machine through single or double venous access to collect whole blood. Then, through centrifugation, the fraction of white cells (buffy coat) was separated and collected into a treatment bag (buffy bag), while the other blood components, mainly red cells and plasma, were returned to the patient. A commercially available ALA-hydrochloride powder for systemic oral use  was used at a dose of 0.168% w/v (10mM). The pharmacy at St. Olavs Hospital reconstituted ALA-hydrochloride in 0.9% sodium chloride (NaCl) physiological saline under sterile condition to a stock solution of 30 mg/mL. Since the amount of buffy coat collected varied on different days for the same patients and between patients, the volume of the ALA solution added to the buffy coat was adjusted to the volume collected on each treatment to reach a dose of 10 mM. After the ALA-solution was added, a 1-h incubation period in the dark was allowed for intracellular PpIX production before UVA light exposure of the cells. Although the most effective light wavelength for PpIX activation is at 400 nm, the use of the UVA light source in the Therakos photopheresis machine can also effectively kill ALA-incubated human T-cell lines [Finally, the treated buffy coat was reinfused into the patient. One treatment-cycle represents two treatments: one treatment given on two consecutive days. Each patient could receive up to 20 ALA-ECP treatments corresponding to 10 treatment-cycles within a year. The duration between two treatment-cycles varied depending on each patient’s symptoms and treatment response.Representative buffy coat samples for the investigation of ALA-induced PpIX were taken after a 1-h ALA incubation at room temperature. The plasma samples for PpIX determination were taken immediately after the reinfusion of the ex vivo ALA-ECP-treated buffy coat and 24-h after treatment. Measurements of ALA-induced PpIX in the buffy coat and plasma samples were performed in an extraction solution ,18. The Safety and tolerability were regularly monitored through clinical and laboratory examinations and patient reports . Safety Additional blood tests to analyse AST, ALT, bilirubin, and INR were performed to evaluate the effect of ALA-ECP on the liver. Blood analyses were performed at the Department of Clinical Chemistry, St. Olavs Hospital, and reference values were used. Urine was examined using a dipstick test.Tolerability was observed through the patients’ reported adverse events (AEs). Any event during the study period in which the patients felt unwell or different from usual was recorded as an AE. Conceivable AEs of systemic ALA treatment included nausea, vomiting, headache, photosensitivity, and chills. Patients received a diary in which AEs were noted between visits. The seriousness of all events was graded  according to Common Terminology Criteria for Adverse Events (CTCAE) v4.03. The assessment of study continuation was based on the stated protocol criteria. An internal safety review committee (ISRC) was appointed to assess safety and tolerability data and to evaluate the reported AEs.Assessments of organ, performance, and quality of life were repeated at approximately 3-month intervals. Investigator-performed evaluations and patient-reported outcome measures were used. The use of immunosuppressive therapy was monitored. Parts of Filipovich and colleagues’ clinical organ scoring system were used in the evaluation of skin manifestations . This scDescriptive data are reported as numbers, percentages (%), and mean (min–max.) and standard deviation (±), together with line graphs and box plots . IBM SPSS software version 27 was used.Five patients (three women and two men) were included, of which four completed the study. The mean age at entry was 48 years (32–60). Four patients were diagnosed with acute myeloid leukaemia, and one was diagnosed with high-risk myelodysplastic syndrome before undergoing allogeneic bone marrow transplantation. Prior to study inclusion, the patients had received a mean of 25 (10–49) treatment-cycles of 8-MOP-ECP. Three patients received systemic steroids.During the study period, the patients received a total of 82 single treatments (42 treatment-cycles) with ALA-ECP. One patient missed one treatment due to clotting in the buffy coat. A second patient missed one treatment because of a gastric flu event. The mean volume of the buffy coat collected at treatments was 146 mL (100–251), and the mean irradiation time was 17 min (2–45). As the patients’ symptoms of the disease varied, the treatment intervals were individualised. Two patients had an ECP treatment interval of 4 weeks, each receiving 20 treatments within 9 months. The remaining three patients had an interval of 6–8 weeks and received 18, 13, and 11 treatments, respectively. One patient met a protocol-defined discontinuation criterion 8 months into the study when the treatment with systemic prednisolone was increased from 10/5 mg/every other day to 25 mg/day due to the worsening of symptoms. After discharge from the study, the patient received two 8-MOP-ECP treatments before being diagnosed with lung cancer, after which ECP was discontinued.We did not observe any clinically significant changes in the patients’ vital signs. The details of the results are presented in One patient had an increased QTc interval value of 467 ms on ECG at the 3-month control compared to 450 ms at baseline . Conseq9/L), and lactate dehydrogenase level (239 U/L). No evidence of liver toxicity was observed  and SR values (31 and 21 mm/h), respectively, compared with the baseline. One patient had transient high values of eosinophil count (15%), absolute eosinophil count  with a history of elevated liver enzymes. This patient regularly used paracetamol but had no established liver disease. Another patient (patient five) with elevated liver enzyme and bilirubin levels four days after treatment had a known bile disorder. No clinically significant long-term liver effects were observed. Albumin and total protein levels were stable in all the patients throughout the study. Dipstick urinalysis revealed no severe kidney or urinary tract disorders. One test yielded a positive urine culture, resulting in antibiotic treatment. ALA-induced PpIX was determined from 75 buffy coat and 104 plasma samples from the five patients and was detected in all the buffy coat samples with a mean concentration of 122 (123) nM. No PpIX was found in the plasma samples taken immediately or at 24 h after re-infusion.In total, there were 62 cases of AEs (two serious adverse events (SAEs), 20 AEs, and 40 conceivable AEs). Of the 62 events, two (3%) were severe, 17 (27%) were moderate, and 43 (69%) were mild symptoms . None ofThe assessment scores from the baseline and the patients’ last control are shown in This paper reports the results of 82 ALA-ECP treatments administered to five patients with cGvHD who were considered to respond inadequately to 8-MOP-ECP. We did not observe any clinically significant changes in vital signs or detect any persistent abnormal laboratory findings. Overall, we registered an improvement in the patients’ skin scores during the study period.The rationale for the clinical use of ALA in ECP is multiple. ALA does not induce carcinogenesis, since ALA-induced PpIX localises to the cellular membrane structures outside the nucleus ,24. TranThis is the first study of ALA administered to patients treated with ECP. We took the precaution of using a dose of ALA corresponding to 11.2 mg/kg if assuming a 75 kg body weight 10mM). When previously used ex vivo for a 24-h incubation, the same dose gave no dark toxicity to leukocytes from cGvHD patients . MoreovemM. When Common side effects observed from the use of oral ALA are nausea, vomiting, and a transient rise in some liver enzymes and bilirubin, which typically resolve after 48 h ,28. SimiConventional ECP with 8-MOP is considered a safe treatment modality with side effects that are commonly sporadic and mild, such as nausea, fever, or headache . Of all Although safety was the primary focus of this study, organ, performance, and quality of life assessments were regularly performed. It should be emphasised that our observations only reflect changes that occurred during the study period and cannot be attributed to ALA-ECP treatment alone. Factors such as the fluctuating course of the diseases, the comedication of the patients, and the lack of investigator-blinded controls may have influenced the results. Overall, the improvement in scores appears promising, especially for skin. In contrast, tear production was measured to be greatest at baseline. The presence of keratoconjunctivitis sicca is well known in cGvHD  and fourThis study met the aim to evaluate safety and tolerability after multiple treatments with ALA-ECP in patients with cGvHD and lays the basis for designing future clinical ALA-ECP studies.In conclusion, the results indicate that ALA-ECP is safe. Treatment appears to be tolerated by patients, with most adverse events reported to be in the mild-to-moderate range of severity. Apart from reduced eye moisture, no findings suggestive of organ toxicity were observed. Further research is needed to assess the safety and for the optimisation of the treatment.


To investigate liver-related and all-cause mortality among amphetamine users with hepatitis C virus (HCV) infection and compare this with opioid users with HCV infection and the uninfected general population.In this national register study of mortality in persons notified with HCV infection 1990–2015 and a substance-related diagnosis in Sweden, amphetamine users  were compared with opioid users  and a matched comparison group without HCV infection/substance use .Amphetamine users were observed for 91,000 years and 30.1% deceased. Crude liver-related mortality was 1.8 times higher in amphetamine users than opioid users , but there was no significant difference when adjusting for age and other defined risk factors. An alcohol-related diagnosis was associated with liver-related death and was more common among amphetamine users. Crude and adjusted liver-related mortality was 39.4 and 5.8 times higher, respectively, compared with the uninfected group. All-cause mortality was lower than in opioid users , but high compared with the uninfected group. External causes of death dominated in younger ages whereas liver-related death was more common among older individuals.This national register study presents a higher crude risk of liver-related death among HCV-infected amphetamine users compared with opioid users or the uninfected general population. The higher risk of liver-related death compared with opioid users may be explained by lower competing death risk and higher alcohol consumption. Treatment of HCV infection and alcohol use disorders are needed to reduce the high liver-related mortality. Chronic hepatitis C virus (HCV) infection cause significant morbidity and mortality globally . Within An important route of HCV infection is injection drug use. Consequently, there is a high prevalence of HCV among people who inject drugs (PWID) with 48.5% estimated prevalence worldwide, or 5.5 million people . StimulaGlobally it is estimated that 585,000 persons died in 2017 as a result of drug use . People There are studies reporting overall mortality of people who use drugs ,12, but st 1987 -Dec 31st 2017 [In this cohort study, with an external comparison cohort, we used prospectively collected data from national registers. The study population consisted of all persons in Sweden notified with an HCV infection to the Public Health Agency year 1990–2015, and with an amphetamine or opioid diagnosis in the National Patient Register (NPR) at any time point from Jan 11st 2017 . The PubAll residents in Sweden have a unique personal identification number (PIN) that is used when in contact with the health care system and for national registers. These PINs were used to link individual information from the national registers.The HCV notification data were transferred to Statistics Sweden where PINs were checked, residence in Sweden was confirmed for the study, and information on emigration and date of death was obtained. A comparison cohort without HCV infection, referred to as controls in this manuscript, was produced by Statistics Sweden, where each HCV-infected person was matched with 10 randomly selected persons without an HCV diagnosis from the general population. The matching criteria were birth year, sex and county of residence, and the matching date was the date of HCV notification.Thereafter, the data were transferred to the National Board of Health and Welfare, where information on substance use and risk factors was retrieved from the NPR. The NPR contains the dates and diagnoses relating to all inpatient hospital admissions since 1987 and all outpatient specialist care since 2001. The diagnoses are classified according to the WHO International Statistical Classification of Diseases and Related Health Problems (ICD)-9, year 1987–1996, and ICD-10, year 1997-. All ICD codes used for this study are provided in the supplementary material .By using the NPR, the HCV-infected study population with substance use-related diagnoses was identified into the following groups: amphetamine diagnosis only, opioid diagnosis only, and both amphetamine and opioid diagnosis (named as amphetamine-opioid users in this study). A person with an amphetamine and/or opioid use diagnosis was considered a PWID from the HCV notification date even when the person received a substance use-related diagnosis after notification, since HCV usually is contracted through injection drug use. Persons with a liver transplantation diagnosis registered in the NPR (ICD-9 or ICD-10) before the start of observation and persons with an HBV infection notified to the Public Health Agency of Sweden 1990–2015 were excluded. Persons with opioid or amphetamine diagnosis in the NPR were excluded from the controls.Using the above-mentioned procedures, 56,977 persons with HCV infection 1990–2015 were identified from the Public Health Agency . After tThe primary outcomes were liver-related and all-cause mortality. In addition, death from mental and behavioural disorders, external causes , drug- and alcohol-related death, and death from infections (including hepatitis and HIV), neoplasms, diabetes mellitus, the circulatory system, the respiratory system, and the digestive tract were assessed. The National Board of Health and Welfare provided information from the Cause of Death Register (DR), which includes causes of death since the year 1961 , classifst 2005 and includes all prescribed drugs dispensed at pharmacies in Sweden, classified according to the Anatomical Therapeutic Chemical (ATC) classification system. Hepatitis C virus treatment uptake was defined as the proportion of persons with dispensed HCV treatment  therapy) from July 1st 2005, when the SPDR started, until end of follow-up, among all persons who were still alive and were living in Sweden on July 1st 2005. Treatment uptake of DAA was defined as the proportion of persons with dispensed DAA therapy Jan 1st 2014-Dec 31st 2017.The National Board of Health and Welfare also provided information from the Swedish Prescribed Drug Register (SPDR). The SPDR started July 1st 1987- Dec 31st 2017. Diabetes mellitus was defined as one ATC code of diabetes mellitus medication (A10.x) in the SPDR, at any point of time from July 1st 2005- Dec 31st 2017, or a diagnosis in the NPR during follow-up. Diabetes mellitus and HIV were analyzed as time-dependent variables, whereas alcohol use was not, as it was considered to be an indicator of a long-standing condition. HIV was defined as at least two diagnoses in the NPR and/or DR.Alcohol-related diagnosis, diabetes mellitus, and HIV were assessed as risk factors of all outcomes. Alcohol-related diagnosis was defined as an alcohol-related diagnosis in the NPR or the DR Jan 1The Public Health Agency of Sweden, Statistics Sweden, and the National Board of Health and Welfare handled all linkage of data. Only anonymized data were sent to the researchers. No written informed consent was collected. This study was approved by the Ethical Review Board of Stockholm (Ref: 2015/1282-31 and 2019–01972).st 2017.Baseline was set to six months (183 days) after HCV notification to the Public Health Agency to avoid overestimation of the mortality risk due to surveillance bias . Patients were followed until death, or censored at emigration, liver transplantation, or Dec 31Statistical differences between frequencies of risk factors between groups were investigated using the Chi-squared test. A p-value below 0.05 was considered statistically significant. Mortality rates in amphetamine users were compared with the rates in opioid users, amphetamine-opioid users, and controls. All-cause and cause-specific mortality were analyzed using population-averaged Poisson regression models, both unadjusted and adjusted for age, sex, diabetes mellitus, HIV, and alcohol-related diagnosis. Age was included as a time-varying categorical variable with six levels . Sex and an alcohol-related diagnosis were included as binary variables. Diabetes mellitus and HIV were also included as binary variables but were allowed to vary across time. The person-time spent in each age group was included as an offset term and the so-called clock-reset approach was applied . MortaliThe study population consisted of 15,317 HCV-infected persons with a substance use-related diagnosis, of which 6,509 (42.5%) had an amphetamine diagnosis, 5,739 (37.4%) an opioid diagnosis, and 3,069 (20.0%) an amphetamine and opioid diagnosis . The comAn alcohol-related diagnosis was significantly more common among HCV-infected persons with amphetamine use than opioid use (57.3% vs 36.9%) or compared to uninfected controls (57.3% vs 3.2%) (p< 0.001 for both) . DiabeteOverall mortality was high; in total 30.1% of amphetamine users, 27.3% of opioid users, and 32.0% of amphetamine-opioid users were deceased by at the end of the study, to compare with 4.4% of controls. The median age at death was higher among amphetamine users than opioid users . The mosThere was no significant difference in crude all-cause mortality between amphetamine users and opioid users, but in the adjusted analysis, the MR was significantly lower among amphetamine users  0.78, 95% CI 0.73–0.84). The adjusted all-cause mortality was also significantly lower among amphetamine users than among amphetamine-opioid users . When coThe crude analysis demonstrated a significantly higher liver-related mortality rate among amphetamine users compared with opioid users . However, in the adjusted analysis, there was no longer a significant difference , mainly due to adjustment for age and alcohol since the amphetamine users were older and had a higher proportion of alcohol-related diagnosis. Similarly, compared to amphetamine-opioid users, there was a significantly higher rate of liver-related mortality in crude analysis, but not in adjusted analysis. Liver-related mortality was associated with an alcohol-related diagnosis , older age , diabetes mellitus , HIV co-infection , and male sex  in HCV-infected PWID.Compared with controls, liver-related cMRRs were very high across all groups of PWID: 39.35 (95% CI 32.81–47.19) for amphetamine users, 22.10 (95% CI 17.65–27.67) for opioid users, and 26.46 (95% CI 20.62–33.95) for amphetamine-opioid users. With adjustment for age, sex, diabetes mellitus, HIV co-infection, and alcohol, the ratios were lower than in crude analysis: aMRR 5.78 (95% CI 4.18–7.98), 6.23 (95% CI 4.40–8.84), and 4.81 (95% CI 3.32–6.97) respectively.Amphetamine users had a lower rate of drug-related death compared with opioid users  and compared with amphetamine-opioid users. Crude MRR and aMRR were high across all groups of PWID compared with controls .Similarly, death from mental and behavioral disorders  was lower among amphetamine users compared with opioid users and with amphetamine-opioid users, and the mortality was higher across all groups of PWID compared with controls.In addition, the rates of external causes of death were also lower among amphetamine users compared with opioid users  and compared with amphetamine-opioid users. The rates were also high across all groups of PWID compared with controls with the highest ratios for opioid users .Alcohol-related mortality was higher among amphetamine users than opioid users , but there was no significant difference between amphetamine users and amphetamine-opioid users. The rates were high across all groups of PWID compared with controls, for amphetamine users cMRR was 17.57 (95% CI 15.59–19.79) and aMRR 16.53 (95% CI 14.65–18.66).Mortality from digestive tract disorders (including liver disease) was high compared with controls, especially in the amphetamine group. However, it was not as high in the adjusted analysis which was mainly due to adjustment for alcohol. Mortality from neoplasms (including HCC), as well as from infection (including hepatitis and HIV), from diabetes mellitus, and from the respiratory system, was also higher across all groups of PWID compared with controls. Death from cardiovascular disease was higher in all groups of PWID compared with controls and amphetamine users had higher mortality rates than opioid users .The proportions of death by age groups among amphetamine users are presented in This national register study of HCV-infected amphetamine users demonstrates a high risk of liver-related mortality, with 1.8- and 39-times higher crude risk compared with opioid users and the general population, respectively. An alcohol-related diagnosis was present in almost 60% of amphetamine users, contributing with HCV infection to an additive risk for liver disease. At younger ages, death from external causes and mental and behavioral disorders dominated, but liver-related death was one of the main causes of death at older ages.When adjusted for age and other defined risk factors, there was no significant difference in liver-related death between amphetamine and opioid users, even though the crude risk was almost twice as high in amphetamine users. Liver-related deaths occurred mostly in those who were older than 50 years, and in this age group, it accounted for one in five deaths among amphetamine users. The cause of this important finding is multifactorial. Probable explanations are that amphetamine users in this study were born almost 10 years earlier than opioid users, and therefore may have lived longer with HCV infection, and that amphetamine users more often than opioid users reach the age when liver complications may occur since opioid users have a higher risk of drug-related death at younger ages. Furthermore, alcohol diagnosis was significantly more common among amphetamine users than opioid users, which may have contributed to the higher liver-related mortality among amphetamine users. Diabetes mellitus was more common in all groups of PWID compared with the general population with the highest prevalence of 10% in amphetamine users. Thus, an additive effect of alcoholic liver disease, metabolic disease, and viral hepatitis seems to be present in an important number of amphetamine users. Hepatitis C treatment uptake was low among PWID in the period we have studied and only one in six diagnosed with HCV infection had received treatment. However, treatment uptake was equally low in amphetamine and opioid users. Thus, different access to treatment probably does not contribute to the observed difference in liver-related mortality between the two groups of PWID.All-cause mortality among amphetamine users with HCV infection compared with the general population was high, in accordance with previous studies of amphetamine users including both HCV-infected and uninfected ,13. The A previous register study of a national HCV-infected cohort in Sweden reported a higher all-cause standardized mortality ratio (SMR) in the whole cohort than in a subgroup with transfusion-associated HCV infection . In our When comparing all-cause MR in amphetamine users with opioid users there was no significant difference in the crude analysis, but in the adjusted analysis, the mortality was lower in amphetamine users. The finding is mostly explained by the higher age of the amphetamine users. Previous studies similarly have presented lower mortality rates among amphetamine users than opioid users ,12. The The risk of death from the circulatory system was high among amphetamine users compared with controls and compared with opioid users. This has been reported in previous studies as a consequence of amphetamine-induced cardiotoxicity . AnotherThe all-cause cMRR for the respective group of PWID compared with controls was highest among younger persons and decreased with age. This was mainly explained by the high cMRR of external causes and mental and behavioral disorders in younger ages. Liver-related cMRR increased with age, which may be due to that liver-related consequences from HCV and alcohol mostly emerge after several decades of exposure.The main causes of death in the amphetamine group were death from external causes and mental and behavioral disorders in younger ages, but in older ages, death from neoplasms (including HCC), the circulatory system, and liver-related death were dominating . The finA low proportion had been treated for HCV infection in our study. With new effective and tolerable DAA treatment, liver-related death caused by HCV infection could be prevented. A scale-up of HCV treatment of PWID is also important to reduce HCV transmission and reach the WHO goal of HCV elimination by 2030. The high prevalence of alcohol-related diagnoses among PWID is a challenge that also needs to be addressed.There are several strengths in this study, especially considering the nationwide coverage of all people registered with an HCV and amphetamine/opioid diagnosis in Sweden, limiting bias from inclusion. The observation time is rather long, which is important when studying chronic HCV infection with complications developing late. Due to the availability of national registers in Sweden, the data have been prospectively collected, eliminating recall bias.However, our study has some limitations. The date of HCV transmission is unknown since it is common with a delay of several years from transmission to diagnosis. Therefore, the notification date (six months thereafter) was used to set the start of follow-up. As amphetamine users were diagnosed at a higher age than opioid users, delayed diagnosis and lead time bias cannot be excluded.As HCV notification can be based on either anti-HCV or HCV RNA and both acute and chronic infections are notified to the Public Health Agency of Sweden, some persons included in the study may have cleared their infection, which could have underestimated the risk of liver-related death among those who are infected.As the SPDR started in July 2005, previous HCV treatment data is missing. However, the treatment uptake in PWID before 2005 has been reported to be very low . Even thAmong 52,967 notified cases with HCV infection, 15,317 had a substance use-related diagnosis. In most cross-sectional studies in high-income countries, injecting drug use is the route of transmission in the majority of incident HCV cases. A selection bias can therefore have been introduced. Milder substance use disorders may be present among those without a substance use-related diagnosis. The consequences of HCV infection in this group could thus be different from what we have demonstrated in those with a substance use-related diagnosis.In addition, individuals with amphetamine diagnosis might have co-used opioids (and vice versa) without it being diagnosed in the registers, resulting in a misclassification. However, existing literature support our results that only a minority of persons with amphetamine/opioid disorders in Sweden co-use the two drugs ,22.In conclusion, this national register study presents a higher crude risk of liver-related death among HCV-infected amphetamine users compared with opioid users with HCV infection or the uninfected general population. One in five deaths after the age of fifty was liver-related in amphetamine users with HCV infection. All-cause mortality was higher than in the uninfected population but lower than in opioid users. The higher risk of liver-related death compared with opioid users may be explained by lower competing death risk and higher alcohol consumption. Efforts to increase HCV treatment among amphetamine users, as well as alcohol prevention and treatment, may reduce liver-related death in this group, and implementation and further development of evidence-based treatment for amphetamine use disorder are of great importance for the reduction of all-cause mortality.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Fluorescent probes are powerful tools for visualizing cellular and subcellular structures, their dynamics and cellular molecules in living cells and enable us to monitor cellular processes in a spatiotemporal manner within complex and crowded systems. In addition to popular fluorescent proteins, a wide variety of small-molecule dyes have been synthesized through close association with the interdisciplinary field of chemistry and biology, ranging from those suitable for labeling cellular compartments such as organelles to those for labeling intracellular biochemical and biophysical processes and signaling. In recent years, self-labeling technologies including the SNAP-tag system have allowed us to attach these dyes to cellular domains or specific proteins and are beginning to be employed in plant studies. In this mini review, we will discuss the current range of synthetic fluorescent probes that have been exploited for live-cell imaging and the recent advances in the application that enable genetical tagging of synthetic probes in plant research. Over the past decades, a wide range of fluorescent dyes have been synthesized and deployed for addressing cellular biological questions in plant sciences. There has also been a continuous effort by chemists to improve the characteristics and applications of dyes. Many of the original dyes can only be used in fixed tissues since they do not permeate into live cells. Still, they are frequently used to mark or highlight organelles in multicolor imaging approaches. With the advent of fluorescent proteins (FPs), many scientists have shifted to using FPs that can be genetically targeted, not just to specific organelles but to even specific subdomains by selecting suitable FPs . These aA wide range of small-molecule synthetic fluorescent dyes have been developed for live imaging . These ftrans-Golgi network/early endosome (TGN/EE). FM dyes, which initially label the PM, are internalized over time and can be utilized to assess endocytic activities (Fei Mao (FM) dyes are styryl dyes with amphiphilic characteristics that are widely used to stain PMs. Among FM dyes, FM4-64 is one the most popular dyes in the realm of plant live-cell imaging, because of its chemical stability and wavelength  which enable it to be used for live imaging with green/yellow FPs . FM1-43,tivities . Collot 18(3) and DiIC12(3)  have been reported to heterogeneously label Arabidopsis protoplast PMs (12-sphingomyelin (BD-SM) have also been used for protoplast membrane lipid phase analysis where LRB-PE  and BD-SM  stained phospholipid-enriched and sphingolipid-enriched compartments, respectively, and both dyes were retained at PM longer than DiI dyes in Arabidopsis protoplasts  partitioning preferentially in the model membrane systems  have beelast PMs . The liptoplasts . Accorditoplasts , which dtoplasts .+ channels present in the ER. ER-Tracker™ probes have been used in dynamics analyses to stain the ER in pollen tubes (The endoplasmic reticulum (ER) is a membrane network that exhibits dynamic movements and shape transitions within the cell. ER-Tracker™, a fluorescent ER probe, consists of a fluorophore and a drug-derived moiety called glibenclamide that binds to adenosine triphosphate (ATP)-sensitive Ken tubes  and rooten tubes . Howeveren tubes , so ER-T5-ceramide  was used to label the Golgi apparatus of Arabidopsis and tobacco pollen tubes -5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM), which accumulates in the vacuolar lumen, allows the 3D reconstruction of the vacuole and the analysis of its volume and morphology  or the vacuolar lumen. In plant cells, PM lipids are transported to the tonoplast via endosomes including TGN/EE and multi-vesicular bodies/late endosome. Therefore, the dyes used to stain PM such as FM4-64 and FM1-43 can be used to image vacuoles . Howeverrphology .6(3)], a cyanine derivative dye, was also used to visualize plant mitochondria , which is less phototoxic to cells. However, unfortunately, the staining of nuclei was only observed in closed stomata and not in opened stomata  derivatives have been developed, and the nuclei of Arabidopsis root and leaf tissue, including epidermal and mesophyll cells, were well stained by PC1  and PC3 (552/600 nm)  or Hoechst 33342 , but the stomata , and no in maize . Still, /600 nm) . So far,d-manno-oct-2-ulosonic acid (Kdo). Pulse labeling experiments of Kdo analog, Kdo-N3 (8-azido 8-deoxy Kdo), have revealed that RG-II synthesis was reduced in Arabidopsis root differentiation zone. A drawback of Kdo labeling is that it labeled only the cells closest to the surface of the tissue, possibly due to the limited penetrance of the fluorescent dye into deeper regions of the tissue. The secondary cell walls are often visualized noninvasively through lignin autofluorescence with standard green FP filter sets  injected into pathogen-infected tobacco leaves, was oxidized by ROS to produce fluorescent 2ʹ,7ʹ-dichlorofluorescein (DCF) (Govrin and Levine 2000). In addition to H2DCFDA, a chloromethyl derivative, CM-H2DCFDA, has also been employed for the visualization of intracellular ROS in plant cells (2HFF) bovine serum albumin (BSA) has been used for extracellular ROS imaging, successfully monitoring extracellular ROS production during root hair apical growth and ROS accumulation in leaves under high light   and dihydroethidium, have been used to observe ROS accumulation in leaves (Reactive oxygen species (ROS) accumulate under stress conditions and function as stress responses in development and in environmental responses. ROS can be produced by enzymatic activities of several peroxidases . In vivont cells . Anothergh light . Compari 1 (PO1)  has photn leaves .). Seminaphthorhodaflours-1 (SNARF-1) shows varying emission intensities at wavelengths of around 580 nm and 640 nm, depending on the pH when excited by a 488-nm laser (BCECF (pKa 7.0), the most popular pH indicator derived from fluorescein, is suitable for near-neutral pH monitoring. For the use as a cytosolic pH indicator in plant cells, BCECF was fused with dextran and microinjected into cells. The dye was used in a ratiometric assay by exciting at two wavelengths, 450 and 490 nm, and collecting emission at 520 nm, and pH changes in the cytoplasm of lily pollen tube have been quantitatively analyzed . 8-HydroDünser et al. 2019nm laser . The SNAnm laser . In othenm laser .bis--trimethine oxonol [DiBAC4(3)] is transported to the cytoplasm in response to depolarization. DiBAC4(3) is utilized to monitor the membrane potential of single cells such as protoplast and pollen tube and mature plant tissue such as root epidermal cells  have been developed, which enable the measurement of microviscosity when combined with the fluorescent lifetime imaging microscopy. This technique provided PM and cell wall microviscosity maps in epidermal cells of Arabidopsis root and leaf .Chemical dyes localize to compartments and molecules based on their chemical properties, but do not bind to specific proteins by themselves as the localization is merely a passive process. On the other hand, genetically encoded probes that make use of FPs can be targeted specifically to target domains of specific proteins. Covalent labeling technology provides a way to combine the advantages of synthetic fluorescent probes with genetical tagging. It complements and combines the benefits of existing labeling methods with synthetic dyes and FPs and offers advantages over conventional FPs, including access to a wide variety of synthesized and designed organic dyes and smaller size relative to standard FPs (27 kDa).2) and a 15-amino-acid small peptide motif containing the sequence CCXXCC  tag labeling has been developed based on the interaction between fluorescein arsenical helix binder-ethanedithiol , which showed plant cell membrane permeability using tobacco BY-2 cells (Kai Johnsson’s group has pioneered the development of a covalent self-labeling technique that efficiently binds small-molecule-based fluorescent probes to POIs in vivo . SNAP-tahia coli . Recentlhia coli . Benzylg-2 cells . One of -2 cells . This al-2 cells .O2-benzylcytosine (Other covalent tags such as CLIP-tag and HaloTag have also been developed to enable the simultaneous multicolor labeling of multiple proteins , which icytosine  (Fig. 1DLive-cell imaging is an important technology for plant cell biology, and the scope and values of the technology are closely tied to the availability of suitable fluorescent probes. A wide range of dyes available for plant cell biology research are shown in Self-labeling with chemical dyes via SNAP-tag can be effectively used in plant cells. In mammalian cells, a BODIPY-based calcium indicator has been successfully localized to specific compartments using SNAP-tagging to measure local calcium concentrations . Covalen