Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021-2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Effect of rotavirus vaccine implementation on the prevalence of coinfections with enteric viruses in Norway.

Acute gastroenteritis (AGE) is a common illness in both adults and children worldwide and is caused by several microorganisms including viruses, bacteria, and parasites. Rotavirus (RV), which is the main cause of AGE, can occur as a mixed infection with other viruses. The aim of this study is to assess the molecular epidemiology of viral enteric viruses and assess RV coinfections with other enteric viruses and their influence on disease severity before and after RV vaccine introduction in children under 5 years of age. A total of 600 samples collected from children hospitalized for AGE in five large hospitals in Norway, and were analyzed for viral gastroenteritis agents by enzyme immunoassay and quantitative real-time polymerase chain reaction (qRT-PCR). Positive results confirmed either by Sanger sequencing or genotyped by multiplex semi-nested RT-PCR. In total, 243 of the 300 (81%) samples, collected from the prevaccine cohort, were positive for at least one of the four viruses tested in this study. RV was most frequently identified in 82.6% of the samples. In the postvaccine cohort, 114 of the 300 (38%) samples were positive for at least one of the viruses tested. RV found in 36.5% of the samples. Coinfections found less frequently in the postvaccine cohort. Among circulating enteric viruses in Norway, RV is the most important cause of viral gastrointestinal infection. As expected, there were fewer RV positive and fewer coinfections after RV vaccine implementation. The results provide valuable data that can aid in further evaluation of the vaccine impact.

Epitope specific T-cell responses against influenza A in a healthy population.

Pre-existing human CD4(+) and CD8(+) T-cell-mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross-reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database (www.iedb.org), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super-type coverage. Optimized libraries of CD4(+) and CD8(+) T-cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T-cell responses in healthy donors using interferon-γ ELISPOT assays. Upon stimulation, significant CD4(+) and CD8(+) T-cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4(+) and CD8(+) T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T-cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines.

Receipt of hepatitis E vaccine and fetal loss in rural Bangladesh: further analysis of a double-blind, cluster-randomised, controlled trial.

BACKGROUND: Vaccination constitutes an attractive control measure for hepatitis E virus (HEV), a major cause of maternal and perinatal mortality globally. Analysis of pregnant participants in an effectiveness trial of the HEV vaccine HEV239 showed possible HEV239-associated fetal losses. We aimed to conduct a detailed analysis of this safety signal. METHODS: In a double-blind, cluster-randomised trial, 67 villages in Matlab, Bangladesh, were randomly allocated (1:1) to two vaccine groups, in which non-pregnant women aged 16-39 years received either HEV239 (HEV239 group) or Hepa-B (a hepatitis B vaccine; control group). We implemented weekly surveillance for pregnancy detection, and follow-up of pregnancies once every 2 weeks, using physician-confirmed diagnoses to evaluate fetal loss outcomes (miscarriage [spontaneous abortion], stillbirth, and elective termination). Data from a parallel system of reproductive health surveillance in Matlab were used to clarify study diagnoses when necessary. Miscarriage was assessed only among participants whose first positive pregnancy test and vaccination date (for whichever dose was closest to the date of last menstrual period [LMP]) were before 20 weeks' gestation. We defined the following analysis periods of interest: from 90 days before the LMP until the pregnancy outcome (the proximal period); from the LMP date until the pregnancy outcome (the pregnancy period); from 90 days before the LMP until the LMP date (90 days pre-LMP period); and from enrolment until 90 days before the LMP (the distal period). Both Poisson and Cox regression models were used to assess the associations between receipt of HEV239 and fetal loss outcomes. The trial was registered with ClinicalTrials.gov (NCT02759991). FINDINGS: Among the 19 460 non-pregnant participants enrolled in the trial, 5011 were identified as having pregnancies within 2 years following vaccination and met the criteria for analysis (2407 in the HEV239 group and 2604 in the control group). Among participants vaccinated in the proximal period and evaluated for miscarriage, miscarriage occurred in 54 (8·9%) of 607 in the HEV239 group and 32 (4·5%) of 719 in the control group (adjusted relative risk [aRR] 2·0 [95% CI 1·3-3·1], p=0·0009). Similarly, the risk of miscarriages was increased in the HEV239 group versus the control group among participants inadvertently vaccinated during pregnancy (22 [10·5%] miscarriages among 209 participants in the HEV239 group vs 14 [5·3%] of 266 in the control group; aRR 2·1 [95% CI 1·1-4·1], p=0·036) and among those vaccinated within 90 days pre-LMP (32 [8·0%] of 398 vs 18 [4·0%] of 453; 1·9 [1·1-3·2], p=0·013). No increased risk of miscarriage was observed in those who received HEV239 in the distal period (93 [5·6%] of 1647 vs 80 [4·5%] of 1773; 1·3 [0·8-1·9], p=0·295). Stillbirth and elective termination showed no increased risk among women administered HEV239 versus those administered Hepa-B in any of the analysis periods. INTERPRETATION: HEV239 given shortly before or during pregnancy was associated with an elevated risk of miscarriage. This association poses a possible safety concern for programmatic use of HEV239 in women of childbearing age. FUNDING: Research Council of Norway and Innovax.

Safety and effectiveness of a recombinant hepatitis E vaccine in women of childbearing age in rural Bangladesh: a phase 4, double-blind, cluster-randomised, controlled trial.

BACKGROUND: Hepatitis E virus (HEV) leads to high mortality in pregnant women in low-income countries. We aimed to evaluate the safety of a HEV vaccine and its effectiveness in preventing hepatitis E during pregnancy. METHODS: In this phase 4, double-blind, cluster-randomised trial, 67 villages in Matlab, Bangladesh, were randomised 1:1 to receive HEV239 (a recombinant HEV vaccine) or a control vaccine (Hepa-B, a hepatitis B vaccine), using block randomisation with random number tables and blocks of size eight, stratified by cluster population size. Eligible non-pregnant women (aged 16-39 years) were vaccinated intramuscularly on day 0, at 1 month, and at 6 months, and followed up for 2 years after the last immunisation. The primary endpoint was hepatitis E in the pregnant, per-protocol population (those who received all three doses within 2 days of the scheduled dates), while safety was a secondary endpoint, assessed in the intention-to-treat (ITT) population (participants who received at least one dose). Solicited adverse events were recorded for the first 7 days after each dose, and unsolicited events until 2 years after a participant's final dose. Pregnancy-related safety outcomes were assessed in the pregnant ITT population. This study is registered with ClinicalTrials.gov (NCT02759991). FINDINGS: Between Oct 2, 2017, and Feb 28, 2019, 19 460 participants were enrolled and received either HEV239 (9478 [48·7%] participants, 33 clusters) or Hepa-B (9982 [51·3%] participants, 34 clusters), of whom 17 937 (92·2%) participants received three doses and 17 613 (90·5%) were vaccinated according to protocol (8524 [48·4%] in the HEV239 group and 9089 [51·6%] in the control group). No pregnant participants were confirmed to have hepatitis E in either treatment group. HEV239 showed a mild safety profile, similar to Hepa-B, with no difference in the proportion of solicited adverse events between groups and no severe solicited events. Pain was the most common local symptom (1215 [12·8%] HEV239 recipients and 1218 [12·2%] Hepa-B recipients) and fever the most common systemic symptom (141 [1·5%] HEV239 recipients and 145 [1·5%] Hepa-B recipients). None of the serious adverse events or deaths were vaccine related. Among pregnant participants, the HEV239 group had a higher risk of miscarriage (136 [5·7%] of 2407 pregnant participants) compared with the control group (102 [3·9%] of 2604; adjusted odds ratio 1·54 [95% CI 1·15-2·08]). INTERPRETATION: The effectiveness of HEV239 in pregnant women remains uncertain. HEV239 was safe and well tolerated in non-pregnant women, but findings regarding miscarriage warrant further investigation. FUNDING: Research Council of Norway; Innovax.

Tumor stroma engraftment of gene-modified mesenchymal stem cells as anti-tumor therapy against ovarian cancer.

BACKGROUND AIMS: Many ovarian cancers originate from ovarian surface epithelium, where they develop from cysts intermixed with stroma. The stromal layer is critical to the progression and survival of the neoplasm and consequently is recruited into the tumor microenvironment. METHODS: Using both syngeneic mouse tumors (ID8-R) and human xenograft (OVCAR3, SKOV3) tumor models, we first confirmed that intraperitoneally injected circulating mesenchymal stem cells (MSCs) could target, preferentially engraft and differentiate into α-smooth muscle actin-positive myofibroblasts, suggesting their role as "reactive stroma" in ovarian carcinoma development and confirming their potential as a targeted delivery vehicle for the intratumoral production of interferon-ß (IFN-ß). Mice with ovarian carcinomas then received weekly intraperitoneal injections of IFN-ß expressing MSCs. RESULTS: Intraperitoneal injections of IFN-ß expressing MSCs resulted in complete eradication of tumors in 70% of treated OVCAR3 mice (P = 0.004) and an increased survival of treated SKOV3 mice compared with controls (P = 0.01). Similar tumor growth control was observed using murine IFN-ß delivered by murine MSCs in ID8-R ovarian carcinoma. As a potential mechanism of tumor killing, MSCs produced IFN-ß-induced caspase-dependent tumor cell apoptosis. CONCLUSIONS: Our results demonstrate that ovarian carcinoma engrafts MSCs to participate in myofibrovascular networks and that IFN-ß produced by MSCs intratumorally modulates tumor kinetics, resulting in prolonged survival.

Immunogenicity and safety of a two-dose regimen with hepatitis E virus vaccine in healthy adults in rural Bangladesh: A randomized, double-blind, controlled, phase 2/pilot trial.

BACKGROUND: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide and it contributes to considerable maternal and neonatal mortality and morbidity in many low-income countries like Bangladesh. A three-dose regimen of a vaccine against HEV (HEV 239) has shown promising results in China. The effects and safety of this vaccine in other populations and with different dosing regimens remains uncertain. OBJECTIVES: Investigate the immune response and safety of a two-dose regimen with the HEV 239 vaccine among healthy adults. Examine the feasibility of conducting a larger HEV 239 vaccine trial in rural Bangladesh. METHODS: One-hundred healthy men and non-pregnant women 16-39 years old were randomized in a 1:1 ratio to receive two doses of either the study (HEV) or control (Hepatitis B virus, HBV) vaccine (at 0, 1 month). Blood samples were collected at day 0, day 60 and 2 years after vaccination. The primary endpoints were the proportion and severity of adverse events up to 2 months after dose one and the longitudinal shift in anti-HEV IgG levels from day 0 to day 60 and 2 years after vaccination. RESULTS: Adverse events to HEV 239 were comparable to the control vaccine, mild in severity and resolved within one to nine days. All participants in the study group seroconverted and achieved high levels of HEV IgG antibodies that remained positive for two years in all but one. A T-cell response was detected one month after HEV 239 vaccination. CONCLUSION: Our results show that two doses of the HEV 239 vaccine produces broad and likely functional immune responses against HEV that remain for at least two years. The safety profile was acceptable and a phase four study of HEV 239 in rural Bangladesh is feasible. CLINICALTRIALS: gov Identifier: NCT02759991.

The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells.

Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

Hepatitis E Virus (HEV) Synopsis: General Aspects and Focus on Bangladesh.

HEV is the most common cause of acute hepatitis globally. This review summarizes the latest knowledge on the epidemiology, clinical characteristics, testing, and treatment of HEV infection. We also focused on Bangladesh to highlight the distinct challenges and the possible remedies. In low-income settings, the virus is mainly transmitted between people by fecal contamination of drinking water causing large outbreaks, and sporadic cases. The disease is usually mild and self-limiting acute hepatitis. Still, pregnant women and their offspring in low-income countries are at particular risk for severe disease, with up to 20% maternal mortality. Despite the high burden of the disease, HEV remains a relatively neglected virus, with detection hampered by costly tests and a lack of suitable treatments. Molecular PCR diagnostics, together with ELISA antibody tests, remain the preferred methods for diagnosis of HEV; however, rapid bedside diagnostics are available and could offer a practical alternative, especially in low-income countries. One vaccine (HEV 239) is only available in China and Pakistan, as efficacy against the other genotypes remains uncertain. The effectiveness trial conducted in Bangladesh might lead the way in gathering more efficacy data and could, together with improved surveillance and raised awareness, dramatically reduce the global burden of HEV.

Stability and Feasibility of Dried Blood Spots for Hepatitis E Virus Serology in a Rural Setting.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. In many low-income countries it causes large outbreaks and disproportionally affects pregnant women and their offspring. Surveillance studies to find effective preventive interventions are needed but are hampered by the lack of funding and infrastructure. Dried blood spots (DBS) offer an easier and more robust way to collect, transport, and store blood samples compared to plasma/serum samples, and could ease some of the barriers for such studies. In this study we optimize an HEV IgG ELISA for DBS samples and validate it on 300 paired DBS and plasma samples collected in rural areas of Bangladesh from participants in a HEV vaccine study. We demonstrate that HEV IgG in blood stored as DBS is stable for two months at up to 40 °C, and for five freeze-thaw cycles. The specificity was 97% and the overall sensitivity of the DBS assay was 81%. The sensitivity was higher in samples from vaccinated participants (100%) compared to previously infected participants (59%), reflecting a positive correlation between IgG titer and sensitivity. We found a strong correlation between DBS and plasma samples with an r2 of 0.90, but with a higher degree of difference between individual paired samples. Our study shows that DBS offers a stable alternative to plasma/serum for HEV IgG measurements and can facilitate serological studies, particularly in resource limited areas.

Diversity of Rotavirus Strains in Children; Results From a Community-Based Study in Nepal.

Objective: The objectives of this study were to describe the incidence and genetic diversity of Rotavirus (RV) infection among children up to 3 years of age in a community in Nepal. Methods: We investigated community-acquired cases of asymptomatic and symptomatic RV infections in children from birth to 36 months of age in a community-based birth cohort in Bhaktapur, Nepal. Monthly surveillance and diarrheal stool samples were collected from 240 children enrolled at birth, of which 238 completed the 3 years of follow-up. Samples were screened for rotavirus by Enzyme Immuno Assay (EIA). All RV screened positives were further genotyped by reverse transcription-polymerase chain reaction for the capsid genes VP7 and VP4. Results: In total, 5,224 stool samples were collected from 238 children, followed from birth to 36 months of age. Diarrhea occurred in 92.4% (230/238) of all children in the cohort. During the 3 years study period, RV was more frequently seen in children with symptoms (7.6%) than in non-symptomatic children (0.8%). The highest RV detection rate was found in younger children between 3 and 21 months of age. Although rotavirus is known as winter diarrhea, it was detected throughout the year except in August. The highest positivity rate was observed in the months between December and March, with a peak in January. Four common G types were seen: G2 (30%), G1 (29%), G12 (19%), and G9 (16%). The most predominant genotypes seen were G2P[4] (30%), followed by G1P[8] (27.0%), G12P[6] (14.0%), G9P[8] (10%), and remaining were mixed, partial, and untyped. Conclusion: Our study confirms that rotavirus is a common cause of gastroenteritis in young children in the community. The prevalence and pathogenicity of rotavirus infection differed by age. There was substantial variability in circulating strains in the community samples compared to samples collected from hospitals. This shows the importance of including community-based surveillance systems to monitor the diversity of circulating rotavirus strains in Nepal.

Direct evidence of mesenchymal stem cell tropism for tumor and wounding microenvironments using in vivo bioluminescent imaging.

Multipotent mesenchymal stromal/stem cells (MSC) have shown potential clinical utility. However, previous assessments of MSC behavior in recipients have relied on visual detection in host tissue following sacrifice, failing to monitor in vivo MSC dispersion in a single animal and limiting the number of variables that can be observed concurrently. In this study, we used noninvasive, in vivo bioluminescent imaging to determine conditions under which MSC selectively engraft in sites of inflammation. MSC modified to express firefly luciferase (ffLuc-MSC) were injected into healthy mice or mice bearing inflammatory insults, and MSC localization was followed with bioluminescent imaging. The inflammatory insults investigated included cutaneous needle-stick and surgical incision wounds, as well as xenogeneic and syngeneic tumors. We also compared tumor models in which MSC were i.v. or i.p. delivered. Our results demonstrate that ffLuc-expressing human MSC (hMSC) systemically delivered to nontumor-bearing animals initially reside in the lungs, then egress to the liver and spleen, and decrease in signal over time. However, hMSC in wounded mice engraft and remain detectable only at injured sites. Similarly, in syngeneic and xenogeneic breast carcinoma-bearing mice, bioluminescent detection of systemically delivered MSC revealed persistent, specific colocalization with sites of tumor development. This pattern of tropism was also observed in an ovarian tumor model in which MSC were i.p. injected. In this study, we identified conditions under which MSC tropism and selective engraftment in sites of inflammation can be monitored by bioluminescent imaging over time. Importantly, these consistent findings were independent of tumor type, immunocompetence, and route of MSC delivery.

Debate on Bacille Calmette-Guérin vaccination against COVID-19: Is it worth performing clinical trials?

The non-specific beneficial effects of Bacille Calmette-Guérin (BCG) vaccination suggest that this vaccine might play a role in protecting individuals against severe coronavirus disease 2019 (COVID-19). Several studies propose that BCG vaccination may increase the body's immunity, thereby preventing respiratory infections caused by other respiratory pathogens. As the number of deaths due to COVID-19 is increasing rapidly and there is no specific treatment available to date, scientists are evaluating the effectiveness of already approved drugs as therapies against COVID-19, and the results were found to vary widely: from no significant effect being observed to a reduction in the time taken for clinical improvement. This study thus aims to evaluate whether it is worth performing clinical trials to examine the effects of the BCG vaccine on COVID-19. We herein emphasize the need to conduct phase III randomized controlled trials with adequate sample size and quality to investigate the effects of the BCG vaccine on COVID-19. In the event that BCG vaccination provides non-specific protection against COVID-19, administering it could be helpful in controlling the transmission of COVID-19 and other infectious diseases during future pandemics.

Genetic Diversity of Astrovirus in Children From a Birth Cohort in Nepal.

Objective: This study describes the types of Human astroviruses detected in stool samples collected from a birth cohort of children in Nepal. Methods: Using a commercial kit (ProSpecT), a total of 5,224 diarrheal and non-diarrheal stool samples were screened for Human astrovirus by ELISA. RT-PCR was performed on ELISA positive samples (2.8%) for further confirmation. The primary RT-PCR assay used targets the ORF2 region and detects human astrovirus type 1-8. Samples that were negative in this assay were further analyzed using primers that target the ORF1b region of human astrovirus which detect both classical type (HAstV 1-8) and novel types (MLB1-5, VA 1-5). PCR positive samples were analyzed by Sanger sequencing to determine the genotype. Results: A total of 148 available ELISA positive stool samples were analyzed by RT-PCR and further genotyped. RT-PCR analysis of these samples using the ORF2 and ORF1b assay revealed that 124 (84%) were positive for classical human types (HAstV 1-8). Seven different classical HAstV genotypes based on ORF2 and ORF1a were identified (HAstV 1- HAstV 8) with the greatest prevalence of HAstV 5 genotype (42.2%), followed by HAstV 1 (34.7%), HAstV 2 and HAstV 8 (7.4%), HAstV 4 (4.1%), HAstV 3 (3.3%), and HAstV 6 (0.8%). Non-classical types were not detected in our study. Conclusion: A high diversity of circulating Astrovirus strains were detected in young children, both with and without symptoms of gastroenteritis. HAstV 5 and HAstV 1 were the most common genotypes in young children in Nepal.

Vitamin D Deficiency is Associated with Increased Use of Antimicrobials among Preschool Girls in Ethiopia.

Preschool children in Addis Ababa, Ethiopia, are highly exposed to influenza viruses. Factors related to infections, nutrition, and environmental conditions that might explain the burden of influenza among these children were investigated. Ninety-five preschool children, 48 girls and 47 boys, were followed clinically for 12 months. Illness and immune responses to influenza; three other respiratory viruses; five airway pathogenic bacteria; and levels of vitamins D, A, and B12 were assessed. Most of the children had antibodies to numerous respiratory viral and bacterial agents at study start, and many were infected during follow-up. Twenty-five girls and 25 boys fell ill during the study, and were treated with one or more courses of systemic antimicrobials. Ninety percent of both girls and boys had 25-hydroxyvitamin D [25(OH)D] levels below the recommended levels. While there was no overall difference in the levels of vitamins D, A, and B12 between girls and boys, treated girls had significantly lower 25(OH)D levels than non-treated girls and treated boys. There was a considerable number of short for age children, but only the short treated girls had significantly lower 25(OH)D levels than the non-treated children. Preschool girls with low 25(OH)D levels were more vulnerable to pathogenic microbes than boys.

Distinct T and NK cell populations may serve as immune correlates of protection against symptomatic pandemic influenza A(H1N1) virus infection during pregnancy.

Maternal influenza infection during pregnancy is associated with increased risk of morbidity and mortality. However, the link between the anti-influenza immune responses and health-related risks during infection is not well understood. We have analyzed memory T and NK cell mediated immunity (CMI) responses in pandemic influenza A(H1N1)pdm09 (pdm09) virus infected non-vaccinated pregnant women participating in the Norwegian Influenza Pregnancy Cohort (NorFlu). The cohort includes information on immunization, self-reported health and disease status, and biological samples (plasma and PBMC). Infected cases (N = 75) were defined by having a serum hemagglutination inhibition (HI) titer > = 20 to influenza pdm09 virus at the time of delivery, while controls (N = 75) were randomly selected among non-infected pregnant women (HI titer <10). In ELISpot assays cases had higher frequencies of IFNγ+ CD8+ T cells responding to pdm09 virus or conserved CD8 T cell-restricted influenza A virus epitopes, compared to controls. Within this T cell population, frequencies of CD95+ late effector (CD45RA+CCR7-) and naive (CD45RA+CCR7+) CD8+ memory T cells correlated inversely with self-reported influenza illness (ILI) symptoms. ILI symptoms in infected women were also associated with lower numbers of poly-functional (IFNγ+TNFα+, IL2+IFNγ+, IL2+IFNγ+TNFα+) CD4+ T cells and increased frequencies of IFNγ+CD3-CD7+ NK cells compared to asymptomatic cases, or controls, after stimulation with the pdm09 virus. Taken together, virus specific and functionally distinct T and NK cell populations may serve as cellular immune correlates of clinical outcomes of pandemic influenza disease in pregnant women. Our results may provide information important for future universal influenza vaccine design.

High Prevalence of Humoral and Cellular Immunity to Influenza Viruses in Preschool Children Living in Addis Ababa, Ethiopia.

BACKGROUND: Influenza in children who reside in tropical and subtropical regions has until recently been regarded as insignificant. However, new evidence suggests that it significantly impacts hospitalization and promotes secondary bacterial coinfections. Ethiopia is situated in a subtropical area where influenza viruses are likely to circulate year round. METHODS: Clinical data were recorded in a cohort of 103 healthy preschool children recruited in Addis Ababa, Ethiopia. Humoral and cellular immune responses to influenza virus were determined by hemagglutination inhibition (HI) and interferon-γ enzyme-linked immunospot assays. RESULTS: Ninety-six percent of the children (2-5 years old) had pre-existing HI antibody responses to 1 or more of the circulating influenza A subtypes, H1N1 (51%), H3N2 (86%), or influenza B (51%) strains. At the age of 4, all children had been infected with at least 1 strain, and 75% had been infected with 2-4 different viral strains. CD4+ and CD8+ T-cell responses against conserved viral antigens increased with repeated exposures, indicating boosting of cross-reactive cellular immunity. Malnutrition did not seem to affect these immune responses to influenza. CONCLUSIONS: Influenza is highly prevalent among children in this area of Ethiopia. Due to the risk of secondary bacterial pneumonia, increased influenza awareness might benefit child health.

Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment.

BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU. RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting ß-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than ß-catenin. CONCLUSION: Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.