Vaccination has been confirmed to be the safest and, sometimes, the only tool of defense against threats from infectious diseases. The successful history of vaccination is evident in the control of serious viral infections, such as smallpox and polio. Viruses that infect human livers are known as hepatitis viruses and are classified into five major types from A to E, alphabetically. Although infection with hepatitis A virus (HAV) is known to be self-resolving after rest and symptomatic treatment, there were 7134 deaths from HAV worldwide in 2016. In 2019, hepatitis B virus (HBV) and hepatitis C virus (HCV) resulted in an estimated 820,000 and 290,000 deaths, respectively. Hepatitis delta virus (HDV) is a satellite virus that depends on HBV for producing its infectious particles in order to spread. The combination of HDV and HBV infection is considered the most severe form of chronic viral hepatitis. Hepatitis E virus (HEV) is another orally transmitted virus, common in low- and middle-income countries. In 2015, it caused 44,000 deaths worldwide. Safe and effective vaccines are already available to prevent hepatitis A and B. Here, we review the recent advances in protective vaccines against the five major hepatitis viruses. Viral hepatitis remains an important challenge to human health, as it is a primary cause of death worldwide . Among tHepatitis E virus (HEV) is another leading cause of acute hepatitis, causing an estimated 3.3 million cases of acute hepatitis each year . The sprHowever, infections with hepatitis B, C, or D viruses  are important causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Approximately 296 million people were living with chronic HBV infection in 2019 , and chrProtective vaccination for hepatitis viruses remains the cornerstone of public health policy to prevent its related morbidity and mortality. It also represents a vital component of the global viral hepatitis elimination response . HepatitThe aim of this review is to clarify both the clinical impact of protective vaccines in controlling the five major hepatitis viruses, as well as the frontiers of research in hepatitis vaccines.The authors performed a literature search focused on protective vaccines against the five major hepatitis viruses. This research was conducted by searching PubMed and Google Scholar databases for articles published from 1975 to December 2022. The publications focusing on candidate hepatitis virus vaccines, vaccination coverage, vaccine efficacy, vaccine safety, long-term vaccine protection, impact of immunosuppressants on vaccine efficacy, causes of vaccine failure, and recent advances in protective vaccines against hepatitis viruses were reviewed and essential data are summarized in this review.In developing countries, ≥50% of the population has already been exposed to HAV infection by the age of 15 years . The WHOBased on well-conducted, randomized clinical trials on children in the 1990s , an inacThere are no large-scale studies from different geographic regions comparing the safety and immunogenicity of both inactivated and live attenuated HA vaccines. However, similar safety and efficacy of both vaccines was reported, with a higher seroconversion rate after a single dose of the live attenuated vaccine . In factAn HAV incidence of 6.0–30.0 cases per 100,000 persons/month is reported in nonimmune travelers to risky destinations. HA vaccination is recommended for travelers aged ≥1 year visiting areas with intermediate or high risk and particularly for immunosuppressed patients, patients with chronic liver disease (CLD), and MSM ,29. RemaThe number of HAV cases in developed countries has decreased substantially over the past two decades. This is attributed to the vaccination of travelers against HAV, integration of the HA vaccine into childhood immunization programs, and improvement of sanitation .Periodic HAV outbreaks occur among MSM in developed countries. The most effective approach to control these outbreaks is through HA vaccination . PreemptHAV-seronegative patients with autoimmune diseases are at risk of contracting HAV and are recommended for HA vaccination . There iPatients with CLD are not at an increased risk of contracting HAV infection. However, they are at high risk of developing FHF and acute-on chronic liver failure . HA vaccUp to 87% of persons with HIV infection are at risk of contracting HAV infection due to poor response to HA vaccine or lack of previous immunization. In addition, they are at higher risk of contracting severe HAV infection than people with HAV infection alone. In fact, HIV-infected patients have poor responses to HA vaccine and carry a higher long-term seroreversion rate ,40. The HBV infection is a worldwide leading cause of morbidity and mortality . The WHOa” antigenic determinant. DNA recombination technology was implemented in yeast to synthesize and assemble an “a” antigenic determinant-like protein that was approved by the Food and Drug Administration (FDA) in 1986 [The first commercially available HB vaccine was developed in 1982 . The man in 1986 . The rec in 1986 .®. Heplisav-B® uses recombinant HBsAg along with cytidine phospho-guanosine oligonucleotide (CpG 1018) derived from bacterial DNA as an adjuvant [In November 2017, the United States of America (USA) FDA approved the two-dose HB vaccine Heplisav-Badjuvant . This acadjuvant .® against recombinant vaccine have concluded that the two-dose Heplisav-B® induced higher immunity in healthy individuals than the conventional three-dose recombinant vaccine [® vaccine reached almost 100% as opposed to approximately 93% elicited by the recombinant vaccine [The protective impact of HB vaccine is measurable in different manners, including the efficacy of the vaccine in mounting protective HB surface antibodies (anti-HBs) in vaccinated individuals and the effectiveness in preventing vertical and horizontal transmission. Multiple trials exploring the immunogenicity of Heplisav-B vaccine ,66,67. I vaccine .The risk of vertical transmission for HB envelope antigen (HBeAg)-positive and HBeAg-negative mothers is 70–90% and 10–40%, respectively . The admIn Taiwan, 30 years after the implementation of the national HB vaccination program, the HBsAg-positive rate decreased from 9.7% in university students born before June 1974 to <1.0% in students born after 1992 . Since ta determinant) of small HB surface (S-HBs) protein may infect even vaccinated individuals [The pathogenic mechanism behind the failure of immune response to HB vaccine is the reduced T-cell activation or the failure of T cells to recognize HBsAg . Globallividuals ,90,91,92The immune response to HB vaccination can be further augmented in nonresponders by different interventions, including using additional booster doses, increasing the vaccine dose, use of intradermal route, use of tri-antigen HBV (3A-HBV) vaccine, or use of the large HB surface antigen (L-HBsAg) vaccine ,94,95,96The 3A-HBV vaccine contains three HBV surface antigens, viz., pre-S1, pre-S2, and S, unlike currently available HB vaccines that contain only S-HBs. The pivotal phase III study PROTECT demonstrated that this 3A-HBV vaccine is highly immunogenic for adults, including older adults and those with well-controlled chronic diseases . The 3A-An HB vaccine using the L-HBsAg was recently established . This vaThe primary anti-HB protection after HB vaccination lasts for more than two decades in the vast majority of healthy individuals . NeverthBiological therapy involves treatment with natural molecules made by the body or prepared in a laboratory. It includes treatment with monoclonal antibodies, adoptive cell transfer, gene therapy, treatment with cytokines, cancer vaccines, oncolytic viruses, immunoconjugates, and the use of targeted therapy . ProtectThe immune response rate to HB vaccination significantly decreases in patients receiving tumor necrosis factor-alpha (TNF-α) blockers ,104. RavAltogether, these data suggest that HB vaccine should be administered before the initiation of biological therapy. Ideally, those patients can be provided an accelerated regimen of HB vaccination to achieve early seroconversion.Worldwide, there has been a decrease in the incidence of HB-related HCC after the introduction of HB vaccination . This waA significant decrease in the incidence of HB-related HCC was also reported in Taiwanese children . The incThe impact of HB vaccination on the incidence of HB-related HCC has also been demonstrated in low-endemic countries. A study from Alaska on individuals aged <20 years, who received infant HB vaccination, demonstrated a decline in the incidence of childhood HCC to 0 cases per 100,000 population from a peak incidence rate of 3 per 100,000 before vaccination .The expanded program of immunization attendance and the average monthly immunizations were reduced during the COVID-19 pandemic. This reduction was particularly noticeable for the HepB-BD vaccine . In factThe reduced availability and implementation of HB vaccination during the COVID-19 pandemic will have a deleterious impact on the incidence of HBV infection in the new generations. Moreover, it will impede the response of countries to the WHO hepatitis elimination goals by 2030 .The rate of HB vaccination had increased globally from 30% in 2000 to 85% in 2019. However, African nations have recorded a low vaccination coverage of 73%. Moreover, only 43% of African newborns received the HepB-BD vaccine . SimilarThe WHO emphasizes that universal HB vaccination is pivotal in reducing HBV incidence and eliminating viral hepatitis by 2030 . The WHOIncreasing global infant vaccination coverage to 90% would avoid 4.3 million incident chronic HBV infections and prevent 1.1 million HB-related deaths by 2030. In addition, it is estimated that improving the coverage of infantile vaccination to 90% in combination with a HepB-BD vaccination coverage of 80% would prevent 1.5 million deaths due to HCC by 2030 .In 2009, the WHO recommended that all newborns receive the timely HepB-BD vaccine, irrespective of the mother’s serological status. By 2019, only an estimated 43% of newborns had received it, and only 110 countries were administering the universal HepB-BD vaccine as a routine policy .It was estimated that scaling up the timely HepB-BD vaccination coverage to 90% by 2030 will result in immediate reductions in incident chronic HBV cases. In fact, it would avoid 41,000,000 chronic infections between 2020 and 2100 and will result in 710,000 fewer deaths among those born between 2020 and 2030 worldwide .Delays in the scale-up of the timely HepB-BD coverage will result in both delays in the elimination of HBV and an increase in HBV-related deaths in the coming decades. The challenges to the scale-up of the timely HepB-BD coverage were attributed, in part, to the circumstances created by the COVID-19 pandemic . Home biA recent USA-based study reported that HB vaccination was more cost effective than the test and treat strategy. The study compared the incremental cost-effectiveness ratios (ICERs) of vaccination versus the screening and treating strategy and found that vaccination was more cost effective. In fact, vaccination had ICERs of $6000 per quality-adjusted life-year gained versus $21,000 for the screen and treat strategy .Universal vaccination was found to be cost effective in areas of intermediate/high HBV endemicity, whereas it was ineffective in areas with low endemicity. Cost-effectiveness had been reported in Iran , China , and VieHCV was first suspected in 1975 by Alter et al. when they suspected a non-A non-B viral hepatitis . SubsequTrials to develop an HC vaccine started in the early nineties using viral vectors to induce viral proteins (glycoprotein) that can stimulate the immune system to develop T-cell response against HCV . AlthougPreventive vaccines include HCV-like particle vaccines, recombinant protein vaccines, DNA vaccines, peptide vaccines, and viral vector vaccines, or a combination of these ,137. SomEvidence suggests that HCV infection can be spontaneously cleared in 25–40% of infected individuals because of the interaction between the virus and both innate and adaptive immune systems. Patients clearing the virus spontaneously have a better chance (approximately 80%) to clear it again if reinfected. Although the initial infection was not protective against subsequent infections, this anamnestic immune response encouraged the development of vaccines against HCV . HC vaccNucleic acid vaccines, including DNA and RNA vaccines, exhibit promising potential in targeting HCV. Nucleic acid vaccines provide a number of potential advantages over conventional vaccines, including the stimulation of both B- and T-cell responses, improved vaccine stability, absence of any infectious agent, and the relative ease of large-scale manufacture . MoreovePlasmids that comprise sequences encoding a number of proteins or epitopes of HCV are capable of inducing cellular and humoral immune responses in animal models when used intramuscularly or subcutaneously. Studies have reported that the use of plasmids containing structural or nonstructural genes of HCV in animal models can stimulate the response of viral T-specific cells ,146. It Charles M. Rice, in his Nobel lecture, stated that he hoped we can learn from all the efforts that are being put into developing COVID-19 vaccines . NeverthConsidering the small number of tested HC vaccines in human beings and the small numbers of vaccinated volunteers, it is not reasonable to give solid conclusions concerning the safety and immunogenicity of any of them. However, the initial results are assuring in some trials and raising concerns in others. Frey et al. evaluated the safety as well as the immunogenicity of a novel HC vaccine (HCV E1E2/MF59C.1) administered to three groups of volunteers with different doses  via intramuscular injections at weeks 0, 4, 24, and 48. The vaccine was well tolerated with few nonrelated severe adverse events and with accepted mild-to-moderate adverse events. The vaccine induced both antibody responses and lymphoproliferative responses . AnotherThe majority of tested HC vaccines are administered in frequent, separated doses before evaluating the induced immune response ,152,153.HEV is one of the major infectious causes of acute liver dysfunction, representing a global health problem . MoreoveDue to the lack of efficient cell cultures, various technologies were applied to generate recombinant proteins as potential immunogens for vaccine production. In fact, the HEV genome is a positive-stranded RNA consisting of a short 5′ noncoding region capped with three open reading frames (ORFs) that code for nonstructural proteins (ORF1), capsid proteins (ORF2), and a phosphoprotein (ORF3) . The capThe HEV capsid contains three major domains, viz., S, P1, and P2. The outermost P2 domain  appears to be the core antigenic region of pORF2 harboring the majority of key neutralization sites, some of which are essential for virus–host interactions and are responsible for the induction of neutralizing antibodies .Escherichia coli [E. coli system. Then, another, more recent, HEV vaccine candidate, the p179, was developed based on the 439–617 region of ORF2 of a genotype 4 strain expressed in E. coli [The first report of an HE vaccine candidate was published in 1993 by scientists at the Centers for Diseases Control and Prevention. This was based on the pORF2 from a genotype 1 HEV expressed in hia coli . Subsequhia coli . The sechia coli , which w E. coli .Both genotype 1-derived recombinant vaccines, rHEV and HEV 239, have demonstrated efficacy and safety in preclinical trials in monkey models. In clinical trials, the rHEV vaccine was proceeded into an open Phase I trial , which dIn the same context, HEV 239 was the most extensively studied vaccine and was shown to be safe and effective; however, more data are necessary to recommend its use on a global basis. It has been evaluated through Phase I, Phase II, and Phase III clinical trials in China. Data from these clinical trials suggest that the HEV 239 vaccine is well tolerated with no associated serious adverse events. In the Phase I trial , 44 healConsequently, the HEV 239 vaccine was licensed and has been available in China since 2012 . HoweverRegarding the third recent vaccine, the p179 vaccine proceeded to a phase I trial in 2017, compared with the HEV 239 vaccine. Results showed comparable safety and efficacy with a similar seroconversion as the control vaccine . The autWith an aim to enhance the immunogenicity and, in turn, the efficacy of the HEV vaccine, a recent study developed a fusion protein in a nonparticulate form that could improve the delivery and immunogenicity of E2 epitopes to elicit a better immune response. The study proposed fragment A of CRM197 as an intramolecular adjuvant carrier protein that can be fused to nonparticulate antigens that substantially enhanced the immunogenicity of the E2 protein .According to a recent study by Yu et al. in 2019, the majority (96.7%) of HEV 239-vaccinated elderly persons aged >65 years seroconverted at 1 month following the final dosage of this three-dose vaccine. At 7 months, 97.3% of them had satisfactory anti-HEV IgG levels. The vaccine was well tolerated with no documented adverse events in this cohort of elderly people .HEV infection in pregnant women may cause severe or fatal adverse events . During Solid organ transplant patients and persons with other immunosuppressive disorders, such HIV and rheumatological diseases, are more vulnerable to developing chronic HEV infection . To datePatients with CLD have a higher risk of developing severe hepatitis E if infected . This emIn general, young individuals are at increased risk for infection in HEV-endemic developing countries ,157. In In the extended efficacy study of the HEV 239 vaccine in the Phase III clinical trial, long-term follow-up of up to 4.5 years demonstrated continuous efficacies of 93% (95% CI: 78.6–97.9%) among participants who received all three doses and 85% (95% CI: 67.1–93.3%) among those who received at least one dose. Furthermore, the trial demonstrated cross-protective efficiency across the common HEV1 and HEV4 genotypes in China . HoweverTo date, the HEV 239 vaccine is not prequalified by the WHO , which mThe positive impact of single-dose HA inactivated vaccine programs on FHF and liver transplantation in children has been increasingly demonstrated in Argentina, Brazil, and Russia ,179,180.Based on the latest WHO recommendations , HA vaccImmunotherapy is a new player in the armamentarium against HBV. The major challenge in this strategy is the HBsAg-specific immune tolerance. There is an ongoing quest to breach this barrier by either breaking the immune tolerance and/or enhancing the immunogenicity of the vaccine. It was hypothesized that long-term suppression of HBsAg levels can induce HBsAg-specific B-cell responses ,183. TheE. coli heat-labile enterotoxin B subunit was also investigated recently by Feng et al., which demonstrated promising results in stimulating the secretion of IFN-γ and IL-4 as well as inducing humoral immune responses that could prevent viral entry [No approved HC vaccine to date, even in the most recent trials that utilized more than one vaccine, has reached its predetermined efficacy endpoint to prevent chronic HCV infection, irrespective of the induced T-cell response and relative safety of the used vaccines . A breakal entry . The proE. coli expression system. A nanomaterial  was used to wrap the HEV ORF2 recombinant protein P206 to prepare nanoparticles (P206@PLGA) with a uniform size and stable quality. Experimental studies revealed its efficacy in animal immunization compared with conventional vaccines, with a potential safety profile both in vivo and in vitro [Nanoparticles have emerged as drug carriers that can improve drug bioavailability and efficacy via drug encapsulation. Recently, a veterinary vaccine based on nanoparticle technology “P206@PLGA” was introduced, targeting the zoonotic transmission of HEV genotype 4. This new vaccine is a swine-derived HEV4 HB-S4 strain. The immunogenic recombinant protein P206 was successfully synthesized using an in vitro . This vaThe positive impact of the universal HA vaccination has been increasingly demonstrated in middle-income countries. Moreover, HA vaccination of travelers has substantially decreased the incidence of imported HAV-infected cases in high-income countries. The rate of HB vaccination increased globally to 85% in 2019. Universal infant HB vaccination had significantly decreased the incidence of HCC in high- and low-endemic areas. Recently, an HB vaccine using L-HBsAg that induces antibodies capable of neutralizing HBV with VEMs was stabilized. The recent discovery of E1 and E2 viral structures may significantly help in discovering more effective HCV vaccines. HEV 239 was the most extensively investigated HE vaccine and has been demonstrated to be safe and effective. The veterinary vaccine “P206@PLGA”, based on the nanoparticle technology, was recently introduced, targeting the zoonotic transmission of HEV genotype 4.The WHO recommended a single-dose strategy for HA vaccines for children aged ≥12 years and for HAV outbreaks. The populations in middle-income countries may benefit the most from universal HA immunization programs . The low


Background: Bladder cancer is the most cost-intensive cancer due to high recurrence rates and long follow-up times. Bladder cancer organoids were considered interesting tools for investigating better methods for the detection and treatment of this cancer. Methods: Organoids were generated from urothelial carcinoma tissue samples, then expanded and characterized; the expression of immune modulatory antigens and tumor stem cells markers CD24 and CD44 was explored in early (P ≤ 3) and later (P ≥ 5) passages (P) by immunofluorescence and by quantitative PCR of cDNA. The expression of these factors was investigated in the corresponding cancer tissue samples by immunohistochemistry. Results: The expression of the PD-L1 was detected on some but not all organoids. CD276 and CD47 were observed on organoids in all passages investigated. Organoids growing beyond passage 8 expressed both CD24 and CD44 at elevated levels in early and late cultures. Organoids proliferating to the eighth passage initially expressed both CD24 and CD44, but lost CD24 expression over time, while CD44 remained. Organoids growing only up to the 6th passage failed to express CD24 but expressed CD44. Conclusions: The data indicate that the expression of CD24 in urothelial cancer cell organoids may serve as an indicator for the prolonged proliferation potential of the cells. Bladder cancer (BC) is among the most common malignancies in many countries. A global incidence of more than 350,000 new cases and a prevalence of close to 3 million cases were estimated about a decade ago . CurrentBesides behavioral and acquired risk factors, genetic disposition also contributes to BC . Genome-Depending on the size and stage of development, tumors contain a blend of cells. A small cohort of cancer stem cells (CSC) is present in a specific tumor niche in most types of primary cancers ,10. ThesposCD24pos phenotype was associated with tumor progression and metastases [pos cells may therefore be associated with CSC and stem cell niches in corresponding samples. At the same time, organ-specific CSC markers eventually open new avenues for diagnosis and therapy. In addition, analyses of the expression of these markers may facilitate the identification of CSCs in in vitro cultures as well.Cancer stem cells are recognized by the expression of distinct stem-cell markers. These markers differ in CSCs of tumors of different origins . In solitastases . Howevertastases . CD44 extastases . DetectiIn this study, we therefore investigated the expression patterns of immune checkpoint molecules PD-L1, CD276, and the immune modulatory molecule CD47 on bladder cancer organoids (BCO) in early versus late passages in comparison to the bladder cancer stem-cell markers CD24 and CD44 on protein and transcript levels. The corresponding tumor samples were characterized as well.Organoids BCO#140, #154 and #147 were established from urothelial carcinoma cells and passaged in vitro. Some key clinical data of the patients are listed in In early passage organoids in chamber slides, CD276 was detected in more than 70% of the cells, while PD-L1 was not detected or expressed only on a few cells. The majority of cells expressed CD47 ; Table 2+ and CD47+ cells were distributed randomly in the organoids  cells, visualized by yellow fluorescence, were found. Overall, the red fluorescent CD44+ cells were enriched in an outer ring while the green fluorescent CD24+ cells were found in an inner ring, separated partly by a layer of CD24−/CD44− double-negative (DN) cells  cells .+ cells were not found frequently in the outer zone of the organoid but enriched in the center  and late-passage (Hblac) normal human urothelal cells served as controls . TranscrRNA was extracted from organoids of BCO#140, #154, and #147 in early (plain bars) and late (dashed bars) passages as indicated to generate cDNA for quantitative PCR. Human urothelial cells HL19/16 and Hblac served as normal early- and late-passage controls, respectively. The y-axis presents the transcript amounts normalized to two house-keeping genes; the x-axis shows the target gene investigated as indicated.The expression of the immune modulatory antigens and the stem-cell markers were investigated in the corresponding ex vivo tumor samples of patients #140, #154, and #147 as well . The expTo correlate the expression patterns observed by organoids in vitro with the pathological situation in bladder cancer tissue, paraffin sections were stained with antibodies to PD-1, PD-L1, CTLA-4, CD276, CD47, CD24, and CD44 as indicated, and detected by immunohistochemistry. Tissues were counter-stained by HE. Size bars indicate 100 μm.high urothelial carcinoma cells may therefore attach better to this scaffold when compared to CD24low cells [high bladder cancer cells to the extracellular matrix in organoids may depend not only on β1 integrin-mediated binding but possibly also on additional factors.In this study, we investigated the expression of immune modulatory antigens and CSC markers CD24 and CD44 on BCOs in early and late passages. More than 30 years ago, CD24, then termed NALM-6M1, was described as a cell-surface molecule on pre-B-lymphocytes, and transcripts were found to be enriched in tumor cells which carried functional mutations of tumor suppressor gene p53 ,33. CD24ow cells . Integriow cells . Integriow cells ,41. Howeow cells . This suhigh cells in the organoids. Moreover, CD24 was described as a mucin-like adhesion molecule, specifically binding to P-selectin, but not to E- or L-selectins [high bladder cancer cells are protected in organoids from CD24-induced apoptosis as urothelial cells express the CD24-ligand P-selectin at low levels, while at the same time elevated soluble P-selectin in the serum of bladder cancer patients prevents CD24 cross-linking on the tumor cells, and at the same time apoptosis of CD24high cells in tumor tissues.Insufficient cell binding of adherently growing cells such as epithelial cells in spheroids is associated with apoptosis . This maelectins . Therefoelectins , but eleelectins . As seruelectins ,47. Howepos cells [posCD24pos co-expression [posCD24neg phenotype [high cells in our organoids are epithelial/urothelial cells but not mesenchymal cells based on their expression of cytokeratins [We noted that the CD24 transcript levels in late-stage normal urothelial cells Hblac were clearly lower when compared to less expanded normal urothelial cells HL19/16. The same trend was observed in BCO#140 and BCO#154 cells. Although transcripts encoding CD24 were detected in all late stage BCOs investigated, our results suggest that expression of the CD24 depends on culture time or number of passages of BCOs in vitro. This finding is corroborated by studies employing serial cell expansions to investigate the number of CD24os cells . Moreoveos cells . An invapression . In conthenotype . This cokeratins . In addikeratins . A loss keratins . As bothThe role of CD44 in the context of BC stem-cell markers seems less clear ,52 and apos cells were enriched in bladder cancer tissue. In the BCOs, CTLA-4 transcripts were barely above the detection threshold of this technique and two logs below the transcript amounts recorded for PD-L1 (data not shown). This result was unexpected as CTLA-4 is a well-known T lymphocyte antigen. However, recently it was shown that CTLA-4 can be expressed on thymic epithelial cells upon functional loss of the autoimmune regulator AIRE [Other results also merit discussion. By immunohistochemistry, we detected expression of PD-1 and CTLA-4 on paraffin sections of tumor samples. PD-1 and CTLA-4 are considered immune checkpoint molecules on T lymphocytes . Prolifetor AIRE . We therDue to the sensitive technology employed for immunofluorescence, the signal intensities shown in this study should not be interpreted as quantitative data in the sense of describing expression levels or protein amounts in or on individual cell. Comparably, the staining patterns presented in the tables should also not be interpreted as indicators for staining intensities in or on individual cells in organoids, but strictly taken as indicator of the percentage of cells showing in staining. This challenge in data interpretation becomes evident when staining patterns of whole organoids in 3D structures are compared to the patterns obtained from the same organoids and passage by paraffin sections. One has to take into account that staining of whole organoids in 3D may detect primarily proteins on cell surfaces, while on paraffin sections, antibodies bind to both intracellular antigens and membrane-bound targets. Moreover, in organoids, fluorescence from the whole construct may add up to a collective staining. In contrast, in thin paraffin sections, less antigen mass is accessible. These differences may contribute to the different signal frequencies observed between 3D organoid staining in comparison to staining of paraffin sections. However, investigating BCOs by fluorescence microscopy in their 3D structure as well as in paraffine sections provided another advantage as well. The hollow structure of BCO#147 was clearly demonstrated in paraffine sections, but it was not evident from immunofluorescence analyses of organoids in 3D. Some difference between BCO#140 and BCO#154 and BCO#147 were also observed for expression of BC stem-cell marker CD24 in the three BCOs investigated here.While most BCO studies published to date investigated the expression of cytokeratins, proliferation, and tumor markers in vitro in comparison to the tumor in vivo ,59,60, iOne of the limitations of this study is the low number of individual BCOs investigated at the time of exploration. We intended to study the expression of immune checkpoint antigens and bladder cancer stem-cell markers in BCOs in early as well as late passages. Sufficient material was available from many tumor samples. Cells from many BC tissue samples were seeded in Matrigel. However, after initial growth, some samples failed to generate three-dimensional constructs resembling bona fide BCOs but rather grew in cellular clusters with fuzzy rim and without convincing mitotic activity. In addition, several BCOs retarded proliferation in passages 3 to 5. Only a few passed this crisis, including BCO#140, BCO#147, and BCO#154. Other BCOs passed beyond passage 5 but did not provide sufficient material for molecular analyses at later stages. To overcome these limitations in the future, we investigated all possibilities to improve the efficacies of BCO production and long-term culture. Moreover, only surgical samples from male tumor patients were included. In future studies, tumor samples from female patients and from patients representing clinical pTa to pT4 BC tumors should be included.Bladder cancer-derived organoids have been and will be part of intensive research in tumor biology ,59,61,622, humidified atmosphere). All organoid cultures included in this study were downscaled to match 48-well plates. Normal human urothelial cells HL19/16 in early passage and commercial urothelial cells in late passage  served as controls [Pathologist confirmed urothelial carcinoma samples were obtained from patients undergoing surgery after written and informed consent. The study was approved by the local Ethics Committee under file number 804/2020/B02. Tumor cells were isolated from the tissue by mechanical degradation followed by proteolytic digest and seeded in hydrogel domes as described recently . In briecontrols . A detaiTo characterize the BC organoids, two distinct methods of immunofluorescence were applied. For three-dimensional evaluation of the BCOs, organoids were cultured in 8-well chamber slides. Immediately prior to staining, the medium was aspirated and the BCOs were rinsed twice with PBS. BCOs were fixed by 4% formaldehyde , washed well three times with PBS, blocked , and incubated  with primary antibodies to immune checkpoint antigens PD-L1, CD276, to immunoregulatory molecule CD47, as well as to bladder cancer stem-cell markers CD24 and CD44. Unbound primary antibodies were washed away . Primary antibodies were detected by incubation of the samples with complementary fluorescence-labelled secondary antibodies . Unbound secondary antibodies were rinsed away . Cellular nuclei were counterstained by DAPI, and the expression of the marker genes was visualized by microscopy (Leica Stellaris 8 or Zeiss Axiophot). Antibody diluent was 1% BSA in PBS. Samples omitting the primary antibodies and samples stained with mouse or rabbit IgG isotype antibodies served as controls (data not shown). Details on the antibodies employed are listed in For two-dimensional analyses of BCO paraffin, sections were generated following standard procedures. BCOs were expanded in 48-well plates to reach the culture density desired. The medium was aspirated and BCOs were rinsed twice by PBS and fixed , a.t.). The BCOs were dehydrated and embedded in paraffin. Paraffin sections were generated , dewaxed and rehydrated by aid of incubation in xylene, rehydrated by ethanol in decreasing concentrations, and mounted on glass slides as described . The samImmunohistochemistry of paraffin sections was utilized to detect expression of the immune modulatory antigens and bladder cancer stem-cell markers in bladder cancer tissue samples from the patients corresponding to #140, #154, and #147. In addition to the organoid samples, tumor samples were stained with antibodies to PD-1 and CTLA-4 . The priQuantification of gene expression on mRNA transcript levels was performed as described . In brieWe conclude that elevated expression of CD24 on bladder cancer organoids may serve as an indicator for the in vitro culture and expansion capacities of BCOs. This notion is supported by our results on transcript and protein levels. Studies to investigate the diagnostic and therapeutic potential of CD47 and CD276 by aid of BCOs seem feasible, while the expression patterns observed for CTLA-4 on BCOs require additional research. Using three distinct BCOs is the limitation of our experiments. The study will therefore be complemented by additional research.


N = 53). A subset of these children also completed diffusion and quantitative MRI scans at age 2 years (N = 20). Within this subgroup, diffusion tractography was used to identify white matter pathways that are considered critical to language and reading development, namely, the arcuate fasciculus (AF), superior and inferior longitudinal fasciculi, and inferior occipital-frontal fasciculus. Quantitative macromolecular proton fraction (MPF) mapping was used to characterize myelin density within these separately defined regions of interest. The longitudinal data were then used to test correlations between early language input and output, white matter measures at age 2 years, and pre-literacy skills at age 5 years. Parental language input, child speech output, and parent-child conversational turns correlated with pre-literacy skills, as well as myelin density estimates within the left arcuate and superior longitudinal fasciculus. Mediation analyses indicated that the left AF accounted for longitudinal relationships between infant home language measures and 5-year letter identification and letter-sound knowledge, suggesting that the left AF myelination at 2 years may serve as a mechanism by which early language experience supports emergent literacy.Longitudinal studies provide the unique opportunity to test whether early language provides a scaffolding for the acquisition of the ability to read. This study tests the hypothesis that parental language input during the first 2 years of life predicts emergent literacy skills at 5 years of age, and that white matter development observed early in the 3rd year (at 26 months) may help to account for these effects. We collected naturalistic recordings of parent and child language at 6, 10, 14, 18, and 24 months using the Language ENvironment Analysis system (LENA) in a group of typically developing infants. We then examined the relationship between language measures during infancy and follow-up measures of reading related skills at age 5 years, in the same group of participants ( Examining whether early language skills predict later emergent literacy skills in preschoolers can shed light on the relationship between language and literacy development and may contribute to developing effective early instruction and interventions and early identification of children at risk of developing reading difficulties. Early language measures coupled with neuroimaging measures in the developing brain can further illuminate the interplay between language input, early language skills, and the emerging neural circuitry for reading. The current study examined the relationship between early language skills and parental language input in infancy (6–24 months), white matter structure at age 2 years, and emergent literacy skills in 5-year-old preschoolers. Additionally, white matter structure at 2 years was tested as a potential mediator of the relationship between infant and preschool behavioral measures.Phonological awareness and letter-sound knowledge have consistently been found as the two best predictors of reading acquisition during the first 2 years in school . These tLongitudinal and cross-sectional studies have supported the SVR framework by demonstrating that concurrent language comprehension and decoding skills strongly predict reading comprehension skills at school age. These studies also extend the SVR framework by showing that language comprehension and decoding skills in preschool, before the onset of formal literacy instruction, predict reading comprehension skills at school age even several years later . HoweverDespite the clear contribution of language skills to reading comprehension in school-age children, it is not yet clear how early language skills relate to emergent literacy skills. Different studies show inconsistencies related to the specific nature of these relations and suggest that there are additional contributing factors. One comprehensive study explicitly addressed the question regarding the relations between early language skills and emergent and later literacy skills using path analysis . This stOther studies have mostly focused on the relations between early vocabulary and emergent literacy skills. For example, There is evidence of long-term predictive associations between early language skills on later reading skills. Parental language input is a key component of the early learning environment, and child language development is associated with input quantity, as well as several dimensions of input quality [for a recent review, see Parental speaking style is another important element of input quality. Adults tend to adopt a style known as “parentese” when they address infants and young children . ParenteSocial interaction is also thought to play an important role in language acquisition, and is considered another important dimension of input quality . StudiesThe early language environment has also been linked to specific components of child literacy skills. Parent-child book reading interactions at 1–2.5 years have been shown to predict receptive vocabulary, reading comprehension, and internal motivation to read, but not decoding, external motivation to read, or math skills at elementary school (2nd–4th grade) . FurtherBrain imaging studies have shown that reading involves two main brain systems in the left hemisphere. The dorsal sub-lexical pathways maps between print and sounds of spoken language and include temporoparietal and frontal regions and the white matter pathways that connect them, namely the arcuate fasciculus (AF) and superior longitudinal fasciculus (SLF). This system is more involved in the beginning stages of reading development. The dorsal pathways are also associated with speech production and auditory-motor integration , as wellLongitudinal studies measuring brain function in children from the pre-reading stage throughout reading development have shown that activation in the left posterior superior temporal gyrus and functional connectivity between the left dorsal and ventral pathways, which is related to phonological processing in pre-readers, predict reading skills 1–3 years later at school-age . SimilarStudies with infants (3–12 months) and young children (1–5 years) suggest that white matter development coincides with the emergence of language-related skills. For example, estimates of left AF and SLF fractional anisotropy (FA) obtained shortly after birth have been shown to correlate with receptive and expressive language skills at the age of 2 years . FurtherIn the current study, we examined how infants’ spoken language environments and early language skills relate to emergent literacy skills at the age of 5 years. Based the research described above, we hypothesized that parental input and parent-child interactions in the first 2 years of life would predict later pre-reading skills, and that this relationship might be mediated by structural development of the white matter, specifically within pathways related to expressive language skills. Longitudinal data included: (1) Measures of infants’ spoken language, parental input, and interactions with caregivers from 6- to 24-months of age, manually coded from home language recordings; (2) Diffusion and quantitative MRI at 26 months; (3) Emergent literacy and related skills at 5-years of age. Correlation and regression analyses were used to assess the relationship between early spoken language skills and parental input, decoding skills prior to the onset of literacy instruction, and the brain mechanisms that support these relations.Seventy-nine families from the Seattle metro area were recruited through the University of Washington (UW) Communications Studies Participants registry with UW Human Subjects Approval that provides subject contact information directly to researchers. These families had previously participated in intervention studies at the Institute for Learning and Brain Sciences (I-LABS) , 2021 anN = 38 total, and N = 16 in the MRI group) participated in a parental language intervention from child ages 6–18 months. The intervention increased the quantity of parentese speech and parent-child conversational turns observed in home language recordings  completed an initial phone screening interview to determine whether their children met the following criteria: (1) Pre-K child between the age of 5 years and 5 years and 4 months; (2) Native English is primary in the home ; (3) Children had no clinically diagnosed congenital, neurological or other physical abnormality. Exclusion criteria included: (1) Any brain injury and medications that impact cognition; (2) Intellectual disability, Autism Spectrum Disorder, mood disorders, and other disorders that impact cognition; (3) Significant and permanent hearing impairments. After the initial screening process, 53 eligible participants were invited to take part in the follow-up study at I-LABS when they were 5-years-old .All families who participated in the study when their children were infants were invited to return for a follow-up MRI session when children were approximately 26 months old, as well as a follow-up study of pre-reading skills at the age of 5 years. All families who agreed to have their child participate in a follow-up study at the age of 5 years . Participants’ reading and related skills at the age of 5-years were measured with the following standardized and non-standardized tests that were adapted to online administration by uploading the stimuli to PowerPoint presentations and presented to the participants during online sessions via Zoom . The onlThis test is designed to measure Alphabet knowledge and letter sounds. Participants were shown isolated letters on the screen and instructed to name the letters and their corresponding sounds. This test was administered separately for lowercase and uppercase letters. This test resulted in four measures: Uppercase letter names (ULN), uppercase letter sounds (ULS), lowercase letter names (LLN), and lowercase letter sounds (LLS). All 26 letters were presented in random order.Letter sound scoring was carried out by trained research assistants, under the supervision of the first author. Scorers only accepted isolated pronunciation (not adding any vowel), short vowels, and hard “G,” “C,” and “X” as correct responses. Video recordings of each session were first scored by the research assistant who administered the session. Two other research team members then provided independent scoring for the videos. Inter-rater inconsistencies were discussed in weekly meetings and resolved as a group.This standardized test  is desigThis standardized test  is desigThe Language ENvironment Analysis System  , due to All data were acquired using a 3.0 T Philips Ingenia MRI system with a 32-channel head coil while children were in natural sleep. High resolution T1-weighted images were acquired using a multi-echo MPRAGE sequence with FOV = 230 × 230 × 180, acquisition voxel size 1.0 mm × 1.0 mm × 1.0 mm, reconstructed voxel size 0.5 mm × 0.5 mm × 0.5 mm, TR/TI/TE1/TE2 = 13/1200/3.7/9.7 ms, shot interval 2,250 ms, and flip angle (FA) = 8°. T1-weighted images were used as a common reference space for later anatomically guided analysis of macromolecular proton fraction (MPF) maps and of diffusion-weighted images, as described below.3, and actual voxel size of 1.25 mm × 1.25 mm × 1.24 mm interpolated to 0.625 mm × 0.625 mm × 0.620 mm. Additionally, actual flip-angle imaging (AFI) B1 maps  volumes and 64 diffusion-weighted volumes acquired at either a b-value of 2,000 s/mm2 (52 non-collinear gradient directions) or a b-value of 800 s/mm2 . An additional set of 6 non-diffusion-weighted volumes were acquired using the same parameters with a reversed phase encoding direction (posterior–anterior), for use in correcting EPI distortions  for motion and eddy current correction . Specifically, initial segmentations were made using way-point ROIs in subject native space. The Whole brain tractography was carried out using the MRtrix software package (version 3.0) with the iFOD2 algorithm . The resMacromolecular proton fraction values were extracted within each fiber tract and summarized at the tract core as a weighted-mean across fiber nodes, which penalizes locations farthest from the spatial center of each tract and thereby minimizes the influence of minor variation in the exact outer boundary of each tract .Analysis along individual tract locations was carried out by first sampling along 100 evenly spaced locations in native subject space  and thenTwo participants completed the MPF mapping scans, but not the dMRI scan. For these individuals, MPF profiles were defined as follows: MPF maps were first co-registered to a standard-space template brain, and probabilistic white matter labels were used to define initial candidate tracts . Tract lFirst, we wanted to examine whether early parental input and infants’ output measures of natural language environment, as recorded with the LENA system and manually coded in infancy are related to later emergent literacy and related skills at the age of 5 years. For the total sample of 53 participants, we calculated the simple bivariate correlations between their early LENA measures at the age of 6, 10, 14, 18, and 24 months, and their emergent literacy and related skills at the age of 5-years.N = 20).Second, we examined the correlations between the early LENA measures and 5-years measures for the smaller sub-group of participants that had brain imaging data at 26-months of age  included families at or below the federal poverty line (ratio < 1) as well as families ranging well into the upper quadrants of wealth . The summary statistics of the participants’ gender, age, and SES in each sample are presented in Planned comparisons focused on the following white matter tracts: The left and right AF, SLF, inferior longitudinal fasciculus (ILF), and inferior fronto-occipital fasciculus (IFOF). Two regression models were tested within each tract: (1) 26-month MPF values were regressed on each of the LENA language variables of interest , and (2) pre-reading measures collected at 5 years  were regressed on 26-month MPF values. Average MPF values were then extracted from regions with significant bivariate effects for both infant LENA measures and the 5-year language measures. These values were entered into a mediation analysis designed to test whether 26-month white matter organization accounts for the longitudinal relationship between infant language measures and 5-year pre-reading skills see .For the whole sample of 53 participants, the simple bivariate Pearson correlations between 6- and 24-months and 5-years of age measures revealed some significant results for both parental input and infants’ output measures. Parental input, as measured by parentese (PT) when infants were 14-months-old is positively correlated with all skills measured at 5-years of age  at the age of 5-years. In addition, PT at 6-months and at 18-months old is positively correlated with lowercase letters knowledge at 5-years. However, there are no significant correlations between conversational turn-taking (CT) at 6–24-months-old and the 5-years-old measures.Infants’ output, as measured by children’s production of speech-like vocalizations (CS) at the age of 14-months old is positively correlated with uppercase and lowercase letter-sound knowledge at 5-years. However, CS at 6-months old is negatively correlated with all 5-years measures. In addition, CS at 24-months old is negatively correlated with uppercase and lowercase letters knowledge. The results are summarized in When examining the behavioral results for the sub-group of 20 participants that had also brain imaging data at the age of 26-months, the simple bivariate Pearson correlations between 14-months and 5-years of age measures revealed significant results for both parental input and infants’ output measures. As found for the larger sample, PT at 14-months is positively correlated with uppercase and lowercase letters and sounds knowledge at the age of 5-years. However, it is not significantly correlated with phonological awareness, and vocabulary. PT at 6, 10, 18, and 24 months is not significantly correlated with any 5-years measures. Similarly, as found in the larger sample, CS at 14-months is positively correlated with uppercase and lowercase letter-sound knowledge at 5-years. In addition, CS at 24-months old is negatively correlated with uppercase and lowercase letters knowledge, as found in the bigger sample. In contrast to the larger sample, we also found a significant correlation between CT at 14-months-old and ULS knowledge, and between CT at 6-months and ULS and LLS knowledge (but not with other 5-years-old measures). The results are summarized in For the subgroup of 20 participants with MRI data at 26 months, significant bivariate correlations  were observed within the left AF and left SLF for PT and CT at 14 months, within the left AF for CS at 14 months, and within the left AF for LLN and ULS at 5 years . The sigTo further assess the relationship between 14-month language measures, 26-months MPF, and 5-years pre-reading measures (ULS and LLN), average MPF values were extracted from regions with significant bivariate effects at both 14 months and 5 years . Mean MPF values were then tested as mediators for each significant correlation between 14-months and 5-years behavioral measures. As shown in The goal of the current study was to examine how parent and child language variables measured in infancy relate to later emergent literacy skills, and whether white matter development mediates these relationships. We first examined correlations between early parental input and child output measures in the natural language environment from 6 to 24 months, and emergent literacy skills at the age of 5 years. We then examined correlations between quantitative MPF estimates of white matter myelination at 26 months and the longitudinal behavioral measures. Finally, we tested whether MPF values at 26 months mediated the relationships between early language measures and later literacy skills. Parental input and infants’ speech and speech-like vocalizations were found to predict emergent literacy skills at 5-years of age. Furthermore, myelin density estimates in the left AF were found to mediate the correlations between the early language measures and later emergent literacy skills. Together, these longitudinal results add to the literature relating to the long-term effect of early language skills and parental input and suggest that parental input and parent-infant interactions support the development of emergent literacy skills partly through myelination of the left arcuate pathway.Previous studies have demonstrated that expressive and receptive language skills measured in the first 3 years of life predict literacy skills in kindergarten and elementary school, and emergent literacy skills in preschool . These rParentese at 14-months of age predicted emergent literacy skills at 5 years of age. Specifically, all of the 5-year measures, including letter names and sound knowledge, phonological awareness, and expressive vocabulary, in the full sample, and letter name and sound knowledge in the smaller sample of participants who had MRI data. These results are consistent with previous studies reporting that parental input and home language environment in the second year of life are important for literacy skill development , and extThe results of the behavioral analysis further indicate that Infants’ speech and speech-like vocalizations at 14-months predict letter-sound knowledge at age 5 years. Recent work has demonstrated that speech-sound production in pre-readers uniquely predicts word later identification in 2nd grade, with additional mediating effects of phonological awareness and letter-knowledge skills . HoweverChild speech at 6 and 24 months was negatively correlated with later reading measures, which was unexpected. Importantly, the child speech measure does not differentiate among categories of speech-like vocalization, such as canonical vs. non-canonical babbling. Children with lower 5-year vocabulary skills might therefore produce more, but less sophisticated, vocalizations at 6 and 24 months. Alternatively, child output at these ages might be less reliably measured, especially at 6 months, where children produce fewer vocalizations, overall. Notably, within the MRI sample, negative effects at 6 months were smaller, and non-significant. Future work, using a finer-grained manual coding of the LENA recordings at each age, will be needed to evaluate these possibilities. In contrast, at 14 months, children can be expected to produce canonical babbling (syllables produced with adult-like consonant vowel transitions), as well as a small number of early words. This time point may therefore contain greater individual variability related to expressive language development.Finally, the results of the behavioral correlations indicate that parent-infant interactions, indexed by conversational turns at 14-months, predict letter-sounds knowledge at the age of 5-years. This effect was strongest for letter-sound knowledge in the subset of participants with MRI data. Importantly, the MRI group primarily included families who previously participated in a parental language intervention , 2020, aThe current study is the first to demonstrate long-term effects of parent-infant turn-taking on letter-sounds knowledge at the age of 5-years. However, we note that the effect was strongest in the subset of participants with MRI data, and therefore needs further examination in a larger sample. Importantly, the MRI group primarily included families who previously participated in a parental language intervention , 2020, aAltogether, the behavioral results from the current study tie together the conclusions from previous studies and illuminate that early parental input, infants’ speech production, and parent-child interactions support not only language development, but the development of emergent literacy skills as well.Infant vocalizations, parentese speech input, and parent-child conversational turns at 14-months correlated with estimates of myelin density within left AF 26-months. Parent-child conversational turns, but not infant vocalizations, also correlated with left SLF myelination at 26-months. No significant correlations with early language measures were found for the right hemisphere white matter tracts, or for ventral pathways (IFOF and ILF), reinforcing the idea that the emerging language network is left-hemisphere dominant very early in development. These results extend the findings from previous studies indicating that parent-child conversational turns and language skills in 4–6-year-olds correlate with concurrent structural connectivity in the left AF and SLF , storyteFurther, letter name and sound knowledge at 5-years correlated with estimated myelin density in the left arcuate and the left and right ILF at 26-months. These results extend previous studies indicating that brain structure and function in the left dorsal pathways in 5-year-olds predict later reading skills including letter-word identification , phonoloAltogether, the results of the brain-behavior correlations from the current study indicate that the effects of early experience on the left dorsal pathways may have implications for later development of specific emergent literacy skills.Myelination of the left AF, estimated using quantitative MPF mapping at 26-months, was found to mediate the relationship between parent-child conversational turns and child speech at 14 months, and letter sound knowledge at 5 years. Myelin density estimates in the left arcuate were also found to mediate the relationship between parentese at 14 months and letter name knowledge at 5 years. These findings suggest a potential biological mechanism underpinning for the longitudinal relationship between parent-child interactions and later decoding skills.The left AF and SLF have previously been found to be related to the development of expressive and receptive language skills , and emeIt has been shown that parent-child conversational turns and parentese enhance the infant’s speech and speech-like production, which in turn, encourage caregivers to respond and provide contingent feedback, and lead to a positive social feedback loop . The curn = 20). Future work is therefore needed to clarify whether the current findings generalize to a larger and more demographically diverse sample, with a greater range of conversational exposure, and whether these results are specifically related to the environmental enrichment provided by the intervention.There are number of limitations in the current study which need to be mentioned. First, the sample is limited to native English speakers and children without known environmental or genetic risk factors, such as lower SES or family history of dyslexia. Further, the sample includes many families who participated a parental language intervention from 6 to 18 months , which was previously found to increase parentese speech and parent-child conversational turn taking , 2020. FFuture studies should examine how additional variables, such as lexical diversity in parent and child speech, relate to both emergent and longer-term literacy skills, and how measures of brain structure and function might relate to these effects. These goals can be achieved by larger longitudinal studies that follow participants from infancy to school-age using multiple approaches to measure brain structure and function, and behavioral language and literacy skills. Furthermore, as mentioned in the Introduction, according to SVR , both deIn the current study we demonstrate for the first time a relationship between parental language input and parent-child interaction during late infancy and later emergent literacy skills in 5-year-olds, with an additional brain measure that suggests a biological mechanism for these effects, namely, developmental myelination of specific components of the left hemisphere’s emerging language network, the left dorsal pathways. The key findings are that first, Infants’ emergent speech production, together with parental use of parentese speech style and their conversational interactions with their infants directly predict emergent literacy skills, including letter names and sounds knowledge, phonological awareness, and expressive vocabulary in preschool. Second, Infants’ emergent speech production, together with parental use of parentese speech style and their conversational interactions with their infants directly relate to myelination of left dorsal pathways  at the age of 26-months. Third, emergent literacy skills, and specifically letter names and sounds knowledge in 5-year-olds directly relate to myelination of the left dorsal pathways  at the age of 26-months. Fourth, left AF myelination at 26-months may account for the relationship between measures of emergent speech production, parental input, and parent-child interactions in infancy and letter name and sounds knowledge in 5-year-olds.These findings contribute to our understanding of the brain mechanisms involved in reading development and break new ground by suggesting a potential mechanism by which language experience early in life scaffolds later reading acquisition. Further research is needed to test the mechanism hypothesis.The raw data supporting the conclusions of this article will be made available by the authors, following reasonable request.The studies involving human participants were reviewed and approved by The University of Washington Human Subjects Division IRB Committee. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.YW, EH, and PK contributed to the conception and design of the study. NF collected and analyzed the LENA data. NC and VY processed the quantitative MRI data to produce MPF maps. EH performed the diffusion MRI analysis. YW and EH organized the database, performed the statistical analysis, and wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


Background: Domestic workers, flight crews, and sailors are three vulnerable population subgroups who were required to travel due to occupational demand in Hong Kong during the COVID-19 pandemic. Objective: The aim of this study was to explore the social networks among three vulnerable population subgroups and capture temporal changes in their probability of being exposed to SARS-CoV-2 via mobility. Methods: We included 652 COVID-19 cases and utilized Exponential Random Graph Models to build six social networks: one for the cross-sectional cohort, and five for the temporal wave cohorts, respectively. Vertices were the three vulnerable population subgroups. Edges were shared scenarios where vertices were exposed to SARS-CoV-2. Results: The probability of being exposed to a COVID-19 case in Hong Kong among the three vulnerable population subgroups increased from 3.38% in early 2020 to 5.78% in early 2022. While domestic workers were less mobile intercontinentally compared to flight crews and sailors, domestic workers were 1.81-times in general more likely to be exposed to SARS-CoV-2. Conclusions: Vulnerable populations with similar ages and occupations, especially younger domestic workers and flight crew members, were more likely to be exposed to SARS-CoV-2. Social network analysis can be used to provide critical information on the health risks of infectious diseases to vulnerable populations. Vulnerability is the state in which a person is exposed to the possibility of physical or emotional harm and cannot protect himself or herself from the harm . VulneraThe coronavirus disease COVID-19) pandemic started in mid-December 2019  pandemic. At the In network science, social network is defined as a network that not only refers to online social media platforms, but also has a broader meaning in which the vertices represent human beings, and the edges stand for shared connections or relationships between people . In publSocial network analysis has been utilized in a wide range of public health research for decades, from human immunodeficiency virus (HIV) infection  and syphIn this study, we utilized publicly available COVID-19 case daily reports and contact tracing records from the Center for Health Protection in Hong Kong Department of Health for domestic workers, flight crews, sailors, and their close contacts. Our study aims include exploring social networks among the three vulnerable population subgroups and capturing temporal changes in their probability of being exposed to SARS-CoV-2 via mobility. We hypothesize that vulnerable populations with increasing mobility will be at a higher risk of exposure to SARS-CoV-2. This research fills a significant knowledge gap on the social networks of vulnerable populations and their mobility in Hong Kong during the COVID-19 pandemic. Our findings can be utilized to understand SARS-CoV-2 exposure via social networks and risk among vulnerable populations, which could contribute to inform comprehensive public health policies and provide necessary health resources to protect vulnerable populations from bearing disproportionate health risks during the COVID-19 pandemic.We obtained COVID-19 case reports of 13,400 confirmed COVID-19 cases in Hong Kong from 23 January 2020 to 24 January 2022. Specifically, we retrieved daily situation reports of the COVID-19 cases published by the Center for Health Protection in the Hong Kong Department of Health . We thenThis study was designed to measure each vulnerable population’s social networks in relation to mobility in Hong Kong during the COVID-19 pandemic. We manually filtered 652 cases out of the 13,400 cases pool by reviewing all the situation reports and contact tracing records. The 652 cases in this study met our definition of inclusion in one of the following vulnerable populations during the COVID-19 pandemic: (1) domestic workers, (2) flight crews, and (3) sailors. We included these three vulnerable population subgroups and their reported close contacts as the study population in this study. We stratified the study population into five cohorts by COVID-19 temporal waves in Hong Kong see  based onWe built six observed empirical networks for this study: one for the cross-sectional overall cohort, and five for the temporal wave cohorts, respectively. All six networks were undirected and unweighted. A social network is composed of two components: vertices and edges . VerticeDescriptive statistical analysis was conducted to examine the demographic characteristics of the study population. We calculated and reported the mean value for continuous variables  and proportion for categorical variables . For descriptive statistical analysis on social networks, we computed and reported average density as well as three measures of centrality: degree centrality, betweenness centrality, and eigenvector centrality. Density represents the proportion of observed edges to the maximum of possible edges in a network ,51. A deWe utilized Exponential Random Graph Models (ERGMs) to build and examine our networks. Firstly, we built a null model for the cumulative cross-sectional network with one term, the edges. This null model is a random graph model that shares the same size of vertices as our observed empirical cross-sectional network. We used this null model as a baseline model to examine how the fit models improved by comparing their Akaike Information Criterion (AIC) values with the AIC in the baseline model. The smaller AIC value the better. We then fitted models by adding terms on four vertex attributes: (1) age (continuous variable in years); (2) gender ; (3) COVID-19 symptomatic status prior to diagnoses ; and (4) vulnerable population category . Monte Carlo Markov Chain (MCMC) maximum likelihood estimation (MLE) was conducted for each fit model. We selected the fit model with the lowest AIC value as our best-fit-model for the cross-sectional network. We extracted the parameter estimates from the best-fit-model and carried out logistic transformation to estimate the probability of edge formation. We also obtained odds ratios through the exponentiation of the parameter estimates of interest. To capture the temporal changes in the probability of edge formation, we built null and fit ERGMs for each of the five waves and their subnetworks, respectively. Subsequently, we ran MCMC MLE and selected the best-fit-models for each of the five subnetworks.p-values on each of our predictors. A Monte Carlo empirical p-value less than 0.05 indicates poor model fit [A total of 1000 random networks were simulated for each of the six observed empirical networks based on the best-fit-models. In total, we simulated 6000 networks. We plotted randomly selected simulated networks to visually compare them with the observed empirical networks. In order to examine the quality of our model fit, we conducted goodness-of-fit examination and reported the Monte Carlo empirical odel fit . After eodel fit . SpecifiThe mobility of our study population was captured based on publicly available contact tracing reports . Tempora® studio version , respectively.We evaluated statistical significance in this study at Descriptive statistics for the demographic characteristics of our study population are shown in For the distribution of the vulnerable population category, 54.5% of the study population were domestic workers (n = 355), 18.4% were flight crews (n = 120), 16.1% were sailors (n = 105), and 11% were close contacts (n = 72) of these three vulnerable population subgroups. For temporal changes in the distribution of the vulnerable population category, the proportion of domestic workers increased dramatically from 19% in wave 1 to 77% in wave 4 and 67% in early wave 5. The proportions of flight crews and sailors fluctuated across the five waves. The proportion of close contacts of the three vulnerable population subgroups dropped sharply from 60% in wave 1 to 1% in early wave 5.r = 0.775). Degree and eigenvector centrality indicated strong positive correlations in wave 3, wave 4, early wave 5, and cross-sectional overall networks . However, no strong correlations among centralities were found in the wave 2 network.Descriptive statistics for network characteristics are presented in We observed clusters of connected vertices in each observed network and a large component in the early wave 5 network F. Since To further explore the clusters and large component in detail, we zoomed in on each network and displayed the vertices with high degree in p-values indicated that all the predictors that we added into the best-fit-models were an adequate fit, since all of the p-values were greater than 0.05. If the Monte Carlo empirical p-value was less than 0.05, it meant that the predictor was a poor fit.For model fit, we examined how our models fit the observed network data. The best-fit-models’ terms and statistics are displayed in The goodness-of-fit plots for degree distribution of the best-fit-models are presented in p = 0.046). Working as domestic workers (p = 0.009) or flight crews (p = 0.003) were positively and significantly associated with the likelihood of edge formation. For homophily effects, being symptomatic prior to a COVID-19 diagnosis increased the likelihood of being connected by an edge with one another (p = 0.022). Additionally, if the vulnerable individual fell into the same vulnerable population category, they were more likely to be connected by edges. All of the four vulnerable population categories  were significant for homophily effects, respectively (p = 0.001).We initially examined the basic association between age and vertex degree by plotting a scatterplot . The scap = 0.004) was positively and significantly associated with the likelihood of edge formation, while working as part of a flight crew (p = 0.023) had a negative and significant association. Findings for homophily effects were similar with the cross-sectional network, except working as a domestic worker was not significant in terms of the wave 2 network homophily effect. In the wave 3 network, domestic workers were found to be more likely to be connected by edges (p = 0.004). In the wave 4 network, no significant main effects nor homophily effects were found. In the early wave 5 network, age (p = 0.001) and working as a sailor (p = 0.047) were found to be negatively and significantly associated with the likelihood of two vertices being connected by edges. In contrast, working as a domestic worker (p = 0.015) or sailor (p = 0.001) were more likely to be connected by an edge with one another.We then examined each of the five subnetworks for main effects and homophily effects on temporal changes. In the wave 1 network, no significant main effects nor homophily effects were found. In the wave 2 network, working as domestic workers  or susceptible-exposed-infectious-recovered (SEIR) models.Our findings demonstrated that vulnerable populations who need to travel due to occupation during the COVID-19 pandemic showed higher risk of exposure to SARS-CoV-2 than the general population. Specially, vulnerable populations with similar ages and occupations, especially younger domestic workers and flight crew members, were more likely to be exposed to SARS-CoV-2. Our study highlights the utility of social network analysis to provide critical information on the health risks of airborne infectious diseases to hard-to-track vulnerable populations. Awareness of the disproportional health risks borne by vulnerable populations can motivates calls for health policy makers to provide additional support and resources needed to reduce risk.


The COVID-19 pandemic has caused morbidity and mortality, particularly among vulnerable populations. We aimed to assess social and demographic characteristics associated with COVID-19 severity among symptomatic participants during pregnancy.The International Registry of Coronavirus Exposure in Pregnancy is a multinational, longitudinal observational cohort study of adult participants tested for SARS-CoV-2 or who received clinical diagnosis of COVID-19 during pregnancy (NCT04366986). Disease severity status of mild, moderate, or severe was determined based on symptoms and healthcare utilization. Stratified by current versus recent pregnancy at enrollment, univariate mixed-effects logistic regression modeling was used to characterize association between social and demographic characteristics with COVID-19 severity, using a cumulative mixed effect model with country as a random effect.The odds of developing more severe COVID-19  were higher among participants with lower socioeconomic status , among participants with lower educational attainment . Participants over 25 years of age had lower odds of severe COVID-19 versus participants < 25 years . Employment in food services was also associated with increased odds of more severe COVID-19, whereas employment in healthcare and within home, and primiparity were associated with lower severity.Findings suggest that employment setting and economic status have strong associations with COVID-19 severity, which warrants considering social determinants of health in the context of assessing risk factors of more severe COVID-19 during pregnancy.IRCEP was registered with the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP) [EUPAS37360] and clinicaltrials.gov [NCT04366986].The online version contains supplementary material available at 10.1186/s12889-022-14532-8. The Coronavirus Disease 2019 (COVID-19) pandemic has exacerbated existing sociodemographic disparities during pregnancy. Previous studies have suggested that compared to non-pregnant individuals, pregnant individuals are at an increased risk of experiencing infection, severe illness, and severe maternal morbidity and mortality due to COVID-19 , which pWhile some clinical risk factors have been associated with COVID-19 severity during pregnancy, environments where people work, live, and participate in leisure or social activities have direct impacts on health, especially in disease transmission and severity . Ever siPrevious studies have demonstrated that neighborhood level characteristics such as lower educational attainment and median household income were associated with COVID-19 illness severity during pregnancy . ComparePregnant and recently pregnant participants enrolled in the International Registry of Coronavirus Exposure in Pregnancy  by completing several online survey modules. IRCEP is a multinational, observational cohort study of adult participants who were tested for SARS-CoV-2, regardless of results, or had clinical diagnosis of COVID-19 during pregnancy. Questions concerning pre-pregnancy sociodemographic factors, maternal clinical characteristics, health outcomes, pregnancy/birth outcomes, and infant characteristics were asked. IRCEP was registered with the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP) [EUPAS37360] and clinicaltrials.gov [NCT04366986], with additional design and cohort information published previously . The InsParticipants with symptomatic COVID-19 any time during pregnancy who completed the demographics panel were included in our analysis .A focus group of pregnant and recently pregnant women in the United States participated in the review of the study surveys prior to the study launch. They evaluated the questions, the flow of the surveys, and the overall design of the study and provided important feedback that was subsequently used to improve both the content and the format of the web-based data collection tool. In addition, during the conduct of the study, participants were asked to provide comments about each survey module and their responses were carefully considered by the study team for possible use in the study.Participants self-reported COVID-19 symptoms, clinical diagnoses, and SARS-CoV-2 laboratory test results and were asked to upload de-identified medical records. For primary outcome, a formal COVID-19 test was not required, and a small proportion of the cases (6%) did not have test results available. This was more common at the beginning of the pandemic in some populations and/or countries; therefore, sensitivity analysis was utilized to assess whether availability of a positive test affected the results.COVID-19 severity: mild, moderate, and severe was the primary outcome. Severity levels were determined based on symptoms and healthcare utilization using an algorithm similar to definitions provided by the Centers for Disease Control and Prevention (CDC) without clinical assessments , and medical comorbidities such as asthma, high blood pressure, and any other cardiovascular condition  were included. Information about pregnancy and pregnancy history were also included, as they could potentially affect COVID-19 severity, including, whether pregnancy was considered high-risk, whether it was first pregnancy, and the trimester of COVID-19 exposure (reported at enrollment).Continuous variables are described as means and standard deviations (SD) and categorical variables are reported as frequencies and percentages. Stratified analyses were conducted by enrollment timing due to potential differences in COVID-19 severity between groups (pregnant vs. recently pregnant). Recently pregnant was defined as enrolling within 180 days following pregnancy. Prior to analysis, a chi-square test was conducted to examine whether pregnancy status at enrollment (pregnant vs. recently pregnant) was associated with COVID-19 severity. Participants in the recently pregnant group reported a higher frequency of severe COVID-19.To determine the crude association between social and demographic characteristics and COVID-19 severity, univariate mixed-effects ordinal logistic regression models in the form of a cumulative link mixed effect model were performed. A random effect for country was added to the model to account for global differences during the pandemic. Odds ratios (OR), Wald-type 95% confidence intervals (95% CI), and statistical significance were reported for all parameters. To adjust for multiple testing given the number of social and demographic characteristics examined, Benjamini–Hochberg corrections were used to produce adjusted p-values. Ordinal logistic regression models assumed proportional odds, where odds ratios across all COVID-19 severity categories were equivalent . Model diagnostics were performed to test against the assumptions, including underlying proportional odds, or parallel assumptions, that allows for one set of coefficients for the COVID-19 severity levels. The proportional odds model assumption was evaluated using the Brant test .n = 10,177), testing positive without any symptoms  or having inconclusive or unknown SARS-CoV-2 status (n = 248). In all 5,653 participants who were considered positive for SARS-CoV-2 via test or clinical assessment and who exhibited symptoms, 4,231 completed the demographics panel. The 4,231 composed the analytical sample used to generate the results, with the primary analysis focusing on participants who were pregnant at enrollment . Approximately, 25% of participants in the analytical sample enrolled after pregnancy  . The majority reported mild/moderate COVID-19 severity .Participants assessed for eligibility in this analysis were enrolled from June 24, 2020 to March 18, 2021. Of 19,452 participants . Furthermore, more severe COVID-19 was observed for participants who worked in food service . Participants who worked in healthcare  or from home  were less likely to experience severe COVID-19.Among participants who were still pregnant at enrollment, some demographic and economic characteristics were associated with COVID-19 severity Fig. . ParticiParticipants from lower economic status  had increased odds of more severe COVID-19 than wealthy participants, and odds increased with decreasing wealth . The same trend was seen with country-level economic status with participants from upper-middle class income countries having higher odds of more severe COVID-19 relative to those of high-income countries .A similar gradient was seen with education, where participants with high school  or less than high school education  had higher odds of more severe COVID-19 relative to participants with a graduate education  on = 1063) were participants enrolled after pregnancy (the recently pregnant group). Generally, participant characteristics associated with greater COVID-19 severity were similar in participants in this category to those who were pregnant at enrollment. Having a high pre-pregnancy BMI (≥ 30 kg/m2), having poor economic status, a high-risk pregnancy, fair/poor pre-pregnancy health and having more than one previous pregnancy significantly increased the odds of more severe COVID-19 in this subgroup , lower educational attainment, and employment setting (food service vs. unemployed) were associated with increased odds of more severe COVID-19. In addition, we found several co-morbidities associated with COVID-19 severity: having a prior pregnancy vs. first pregnancy, pre-pregnancy higher BMI, asthma, and high-risk pregnancy all increase the odds of more severe COVID-19.These results are similar to other studies in the non-pregnant population which have found a disproportionate burden of SARS-CoV-2 infection among persons living in poverty and those with lower educational attainment , 22. In Similar to data from Sakowicz , our stuWhile not the focus of our analysis, our results on comorbidities are congruent with data compiled in a meta-analysis reporting higher BMI (> 30), asthma, and any other pre-existing maternal comorbidity are factors associated with severe COVID-19 disease in pregnancy .Epidemiological studies have reported a high risk of SARS-CoV-2 infection among food workers as a result of contact with one another for extended periods, including at work and shared housing and transportation . The sigIndividual lower income and lower educational attainment were associated with COVID-19 severity in Prasannan et al. . In our Lastly, all the factors highlighted in this analysis  contribute in one way or another to perpetuating the cycle of disease in populations already at risk. COVID-19 has had a profound impact on our society and has exacerbated the maternal health crisis existing in many areas of the world. Pregnant individuals, already facing disadvantaged situations as a result of unfavorable sociodemographic conditions, are placed at higher risk of COVID-19 severity . RecogniA strength of our analysis is its size and breadth, contributing important information on the relationships between social and behavioral characteristics with COVID-19 severity in a multinational registry with 4,231 participants in 41 countries. To our knowledge, IRCEP is the largest COVID-19 pregnancy registry. Although our sample is large, it may have limited generalizability to the general population of pregnant people, in that enrollment was conducted on line and require access to the Internet via computer/mobile phone.Selection into the study population may be associated with the severity of COVID-19, and enrollment was allowed following birth or a pregnancy loss. We stratified by timing of enrollment (pregnant vs. recently pregnant) given differences in COVID-19 severity and disproportionate numbers of severe COVID-19 in the recently pregnant group. The recently pregnant group is composed of participants who enrolled retrospectively after pregnancy, including those who safely delivered their baby or experienced a pregnancy loss. The retrospective enrollment could have been related to SDHs and/or COVID-19 severity, which could explain any differences across analyses by enrollment timing. Also, those with severe COVID-19 late in pregnancy may not have had the ability to participate in the study until they recovered and, by then, they had delivered. In addition, given that the majority of the sample were those 25 to 34 years old (66%), selection bias may have affected the association between younger age and more severe COVID-19.Our planned analysis focused on demographic and social characteristics. All of the data was self-reported, and the study had a potential for information bias, including COVID-19 severity and participant COVID-19 risk minimizing behaviors. Participant-reported behaviors to lower risk of SARS-CoV-2 infection seemed to increase the odds of more severe COVID-19, as the analysis conditions on having a symptomatic infection. This finding was thought to be related to collider bias , where tThere were some limitations on the inclusion of SDHs. About 25% of patients eligible for this analysis were excluded due to missing social and demographic characteristics, as the demographic module was optional Fig. . While sIn examining the relationship between social and demographic characteristics with COVID-19 severity at enrollment, we selected characteristics prior to pregnancy and prior to COVID-19 diagnosis where possible. However, temporality may not always be precisely known or available . Finally, COVID-19 testing was not available in all regions, and hence SARS-CoV-2 infection status is subject to misclassification. Restricting to those with COVID-19 testing (94% of sample) did not appreciably modify the results. Despite the potential for misclassification, our findings are robust given the requirement for participants in this analysis to exhibit COVID-19 symptoms.The social and demographic characteristics examined in this study are expected to be correlated with one another and with underlying risk factors for COVID-19 severity. Fitting an adjusted model combining social and demographic characteristics was limited to including occupation, economic class, and maternal age when exploring the increased risk of more severe infection among food service workers given that our primary interest was on individual characteristics. In addition, sample size limitations in the severe COVID-19 category (~ 100 participants) limited the number of characteristics that could be adjusted in multivariable modeling.Despite the limitations, this study provides important insights into the relationship between demographic factors and SDHs at increased odds of more severe COVID-19 during pregnancy prior to introduction of COVID-19 vaccines.Demographic, social, and structural determinants of health that have been reported in the general population to be associated with an increased odds of COVID-19 severity were also associated with a higher odd of COVID-19 severity in pregnancy. Further exploration of structural factors related to socioeconomic inequity is required to improve the delivery of timely intervention and optimal healthcare for all pregnant populations.Additional file 1.