Gardnerella Revisited: Species Heterogeneity, Virulence Factors, Mucosal Immune Responses, and Contributions to Bacterial Vaginosis.

Gardnerella species are associated with bacterial vaginosis (BV) and have been investigated as etiological agents of the condition. Nonetheless, the isolation of this taxon from healthy individuals has raised important questions regarding its etiological role. Recently, using advanced molecular approaches, the Gardnerella genus was expanded to include several different species that exhibit differences in virulence potential. Understanding the significance of these different species with respect to mucosal immunity and the pathogenesis and complications of BV could be crucial to solving the BV enigma. Here, we review key findings regarding the unique genetic and phenotypic diversity within this genus, virulence factors, and effects on mucosal immunity as they stand. We also comment on the relevance of these findings to the proposed role of Gardnerella in BV pathogenesis and in reproductive health and identify key gaps in knowledge that should be explored in the future.

Sustained attention and behavioral ratings of attention in struggling readers.

Attention is correlated with reading, but the extent to which behavioral ratings and sustained attention relate to reading skills is unclear. We assessed 245 4th and 5th grade struggling readers (mean age = 10.3 years) on behavioral ratings of attention, sustained attention, and reading over a school year. Contributions of behavioral ratings and sustained attention were considered cross-sectionally and longitudinally in the context of other important predictors of reading. Results suggest that sustained measures and behavioral ratings assess distinct, yet overlapping, aspects of attention. Both types of attention accounted for unique variance in comprehension, but not word reading accuracy or fluency, when evaluated cross-sectionally. Results also support the role of behavioral ratings of attention in fluency and in comprehension growth. Findings suggest that multidimensional assessment of attention is useful when considering its relation to reading, and highlights the need to integrate conceptualizations of attention that arise from different theoretical approaches.

Neurocognitive functioning in individuals with congenital central hypoventilation syndrome.

BACKGROUND: Congenital central hypoventilation syndrome (CCHS) is a rare disorder characterized by respiratory system abnormalities, including alveolar hypoventilation and autonomic nervous system dysregulation. CCHS is associated with compromised brain development and neurocognitive functioning. Studies that evaluate cognitive skills in CCHS are limited, and no study has considered cognitive abilities in conjunction with psychosocial and adaptive functioning. Moreover, the roles of pertinent medical variables such as genetic characteristics are also important to consider in the context of neurocognitive functioning. METHODS: Seven participants with CCHS ranging in age from 1 to 20 years underwent neuropsychological evaluations in a clinic setting. RESULTS: Neurocognitive testing indicated borderline impaired neurocognitive skills, on average, as well as relative weaknesses in working memory. Important strengths, including good coping skills and relatively strong social skills, may serve as protective factors in this population. CONCLUSION: CCHS was associated with poor neurocognitive outcomes, especially with some polyalanine repeat expansion mutations (PARMS) genotype. These findings have important implications for individuals with CCHS as well as medical providers for this population.

Live Attenuated Zoster Vaccine Boosts Varicella Zoster Virus (VZV)-Specific Humoral Responses Systemically and at the Cervicovaginal Mucosa of Kenyan VZV-Seropositive Women.

Background: Attenuated varicella zoster virus (VZV) is a promising vector for recombinant vaccines. Because human immunodeficiencyvirus (HIV) vaccines are believed to require mucosal immunogenicity, we characterized mucosal VZV-specific humoral immunity following VZVOka vaccination. Methods: Adult Kenyan VZV-seropositive women (n = 44) received a single dose of the live zoster VZVOka vaccine. The anamnestic responses to the virus were followed longitudinally in both plasma and mucosal secretions using an in-house glycoprotein enzyme-linked immunosorbent assay and safety and reactogenicity monitored. VZV seroprevalence and baseline responses to the virus were also characterized in our cohorts (n = 288). Results: Besides boosting anti-VZV antibody responses systemically, vaccination also boosted anti-VZV immunity in the cervicovaginal mucosa with a 2.9-fold rise in immunoglobulin G (P < .0001) and 1.6-fold rise in immunoglobulin A (IgA) (P = .004) from the time before immunization and 4 weeks postvaccination. Baseline analysis demonstrated high avidity antibodies at the gastrointestinal and genital mucosa of VZV-seropositive women. Measurement of VZV-specific IgA in saliva is a sensitive tool for detecting prior VZV infection. Conclusions: VZVOka vaccine was safe and immunogenic in VZV-seropositive adult Kenyan women. We provided compelling evidence of VZV ability to induce genital mucosa immunity. Clinical Trials Registration: NCT02514018.

A novel strain of cynomolgus macaque cytomegalovirus: implications for host-virus co-evolution.

BACKGROUND: Cytomegaloviruses belong to a large, ancient, genus of DNA viruses comprised of a wide array of species-specific strains that occur in diverse array of hosts. METHODS: In this study we sequenced the ~217 Kb genome of a cytomegalovirus isolated from a Mauritius cynomolgus macaque, CyCMV Mauritius, and compared it to previously sequenced cytomegaloviruses from a cynomolgus macaque of Filipino origin (CyCMV Ottawa) and two from Indian rhesus macaques (RhCMV 180.92 and RhCMV 68-1). RESULTS: Though more closely related to CyCMV Ottawa, CyCMV Mauritius is less genetically distant from both RhCMV strains than is CyCMV Ottawa. Several individual genes, including homologues of CMV genes RL11B, UL123, UL83b, UL84 and a homologue of mammalian COX-2, show a closer relationship between homologues of CyCMV Mauritius and the RhCMVs than between homologues of CyCMV Mauritius and CyCMV Ottawa. A broader phylogenetic analysis of 12 CMV strains from eight species recovers evolutionary relationships among viral strains that mirror those amongst the host species, further demonstrating co-evolution of host and virus. CONCLUSIONS: Phylogenetic analyses of rhesus and cynomolgus macaque CMV genome sequences demonstrate co-speciation of the virus and host.

Selection for a CEACAM receptor-specific binding phenotype during Neisseria gonorrhoeae infection of the human genital tract.

Infections by Neisseria gonorrhoeae are increasingly common, are often caused by antibiotic-resistant strains, and can result in serious and lasting sequelae, prompting the reemergence of gonococcal disease as a leading global health concern. N. gonorrhoeae is a human-restricted pathogen that primarily colonizes urogenital mucosal surfaces. Disease progression varies greatly between the sexes: men usually present with symptomatic infection characterized by a painful purulent urethral discharge, while in women, the infection is often asymptomatic, with the most severe pathology occurring when the bacteria ascend from the lower genital tract into the uterus and fallopian tubes. Classical clinical studies demonstrated that clinically infectious strains uniformly express Opa adhesins; however, their specificities were unknown at the time. While in vitro studies have since identified CEACAM proteins as the primary target of Opa proteins, the gonococcal specificity for this human family of receptors has not been addressed in the context of natural infection. In this study, we characterize a collection of low-passage-number clinical-specimen-derived N. gonorrhoeae isolates for Opa expression and assess their CEACAM-binding profiles. We report marked in vivo selection for expression of phase-variable Opa proteins that bind CEACAM1 and CEACAM5 but selection against expression of Opa variants that bind to the neutrophil-restricted decoy receptor CEACAM3. This is the first study showing phenotypic selection for distinct CEACAM-binding phenotypes in vivo, and it supports the opposing functions of CEACAMs that facilitate infection versus driving inflammation within the genital tract.

Experimental infection of Cynomolgus Macaques (Macaca fascicularis) with human varicella-zoster virus.

Varicella-zoster virus (VZV) is a member of the alphaherpesvirus family and the causative agent of chickenpox and shingles. To determine the utility of cynomolgus macaques (Macaca fascicularis) as a nonhuman primate model to evaluate VZV-based simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) vaccines, we experimentally inoculated 10 animals with the parental Oka (Oka-P) strain of VZV derived from MeWo or Telo-RF cells. VZV DNA could be detected in the lungs as late as 4 days postinfection, with replicating virus detected by shell vial culture assay in one case. Infection did not result in any overt clinical symptoms but was characterized by humoral and cell-mediated immunity in a time frame and at a magnitude similar to those observed following VZV vaccination in humans. The cell line source of VZV inoculum influenced both the magnitude and polyfunctionality of cell-mediated immunity. Animals mounted a vigorous anamnestic antibody response following a second inoculation 12 weeks later. Inoculations resulted in transient increases in CD4(+) T-cell activation and proliferation, as well as a sustained increase in CD4(+) T cells coexpressing CCR5 and α4ß7 integrin. In contrast to previous failed attempts to successfully utilize attenuated VZV-Oka as an SIV vaccine vector in rhesus macaques due to suboptimal infectivity and cellular immunogenicity, the ability to infect cynomolgus macaques with Oka-P VZV should provide a valuable tool for evaluating VZV-vectored SIV/HIV vaccines.

Establishment of an immortal cynomolgus macaque fibroblast cell line for propagation of cynomolgus macaque cytomegalovirus (CyCMV).

Cynomolgus macaques are widely used as an animal model in biomedical research. We have established an immortalized cynomolgus macaque fibroblast cell line (MSF-T) by transducing primary dermal fibroblasts isolated from a 13-year-old male cynomolgus macaque with a retrovirus vector expressing human telomerase reverse transcriptase (hTERT). The MSF-T cells showed increased telomerase enzyme activity and reached over 200 in vitro passages compared to the non-transduced dermal fibroblasts, which reached senescence after 43 passages. The MSF-T cell line is free of mycoplasma contamination and is permissive to the newly identified cynomolgus macaque cytomegalovirus (CyCMV). CyCMV productively infects MSF-T cells and induces down-regulation of MHC class I expression. The MSF-T cell line will be extremely useful for the propagation of CyCMV and other cynomolgus herspesviruses in host-derived fibroblast cells, allowing for the retention of host-specific viral genes. Moreover, this cell line will be beneficial for many in vitro experiments related to this animal model.

"Because of COVID ": The impacts of COVID-19 on First Nation people accessing the HIV cascade of care in Manitoba, Canada.

BACKGROUND: The COVID-19 pandemic (March 2020-May 2023) had a profound effect around the world with vulnerable people being particularly affected, including worsening existing health inequalities. This article explores the impact of the pandemic on health services for First Nations people living with HIV (FN-PWLE) in Manitoba, Canada. This study investigated perceptions of both health care providers and FN-PWLE through qualitative interviews occurring between July 2020 and February 2022 to understand their experience and identify lessons learned that could be translated into health system changes. METHODS: Using a qualitative, participatory-action, intentional decolonizing approach for this study we included an Indigenous knowledge keeper and Indigenous research associates with lived experience as part of the study team. A total of twenty-five [25] in-depth semi-structured interviews were conducted with eleven healthcare providers (HCPs) and fourteen First Nation people with lived HIV experience (FN-PWLE). In total, 18/25 or 72% of the study participants self-identified as First Nation people. RESULTS: The COVID-19 pandemic negatively impacted health services access for FN-PWLE, a) disrupted relationships between FN-PWLE and healthcare providers, b) disrupted access to testing, in-person appointments, and medications, and c) intersectional stigma was compounded. Though, the COVID-19 pandemic also led to positive effects, including the creation of innovative solutions for the health system overall. CONCLUSIONS: The COVID-19 pandemic exaggerated pre-existing barriers and facilitators for Manitoba FN-PWLE accessing and using the healthcare system. COVID-19 impacted health system facilitators such as relationships and supports, particularly for First Nation people who are structurally disadvantaged and needing more wrap-around care to address social determinants of health. Innovations during times of crisis, included novel ways to improve access to care and medications, illustrated how the health system can quickly provide solutions to long-standing barriers, especially for geographical barriers. Lessons learned from the COVID-19 pandemic should be considered for improvements to the health system's HIV cascade of care for FN-PWLE and other health system improvements for First Nations people with other chronic diseases and conditions. Finally, this study illustrates the value of qualitative and First Nation decolonizing research methods. Further studies are needed, working together with First Nations organizations and communities, to apply these recommendations and innovations to change health care and people's lives.

Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus.

Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.

Characterization of English and Spanish language proficiency among middle school English learners with reading difficulties.

Among bilinguals, language-related variables such as first and second language proficiency and balance may be related to important cognitive and academic outcomes, but approaches to characterizing these variables are inconsistent, particularly among at-risk samples of children. The current study employed comprehensive language assessment of English and Spanish language skills and contrasted various approaches to the characterization of language among at-risk ELs in middle school (N = 161). Specifically, we contrasted variable-centered and person-centered approaches, and convergence between objective and self-report measures. Findings support a two-factor structure of English and Spanish language skills in this population, three profiles of students (balanced, moderately unbalanced-higher Spanish, and very unbalanced-higher English), convergence between variable-centered and person-centered approaches, and mixed support for subjective indices of usage. Results provide a foundation from which to examine the roles of L1 and L2 proficiency as well as balance in important cognitive and academic outcomes in this at-risk and understudied population.

Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling.

BACKGROUND: The microbiota of the lower female genital tract plays an important role in women's health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. METHODS: DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. RESULTS: DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

Gardnerella subgroup dominant microbiomes are associated with divergent cervicovaginal immune responses in a longitudinal cohort of Kenyan women.

Most cervicovaginal microbiome-immunology studies to date have relied on 16S rDNA microbial profiling which does not resolve the molecular subgroups of Gardnerella, believed to be central to the pathogenesis of bacterial vaginosis (BV) and subsequent risk of HIV acquisition. Here we used the cpn60 universal target which in addition to other microbial taxa, resolves four Gardnerella subgroups, for cervicovaginal microbial profiling in a longitudinal cohort of Kenyan women to examine associations with cellular and soluble markers of inflammation and HIV susceptibility. Participants (N = 41) were sampled, contributing 362 samples for microbiome analysis. All non-Lactobacillus dominant microbial communities were associated with high pro-inflammatory cytokine levels. Divergent associations were observed among different Gardnerella subgroup dominated communities with respect to the chemokine IP-10. Specifically, Gardnerella subgroup A dominant and polymicrobial communities were associated with reduced concentrations of IP-10 in adjusted linear mixed models (p<0.0001), compared to microbial communities dominated by Lactobacillus (non-iners) species. However, these associations did not translate to significant differences in the proportion or absolute number of CCR5, HLA-DR and CD38 expressed on cervical CD4+ T- cells. These findings suggest that some associations between Gardnerella subgroup dominant microbiomes and mucosal immunity differ and are relevant for the study of BV-pathogenesis and understanding the mechanisms of BV-associated HIV risk.

Multi-low-dose mucosal simian immunodeficiency virus SIVmac239 challenge of cynomolgus macaques immunized with "hyperattenuated" SIV constructs.

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Delta5-CMV and Delta6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10(8) 50% tissue culture infective doses (TCID(50)) of Delta5-CMV or Delta6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Delta5-CMV vaccine group compared to the Delta6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Delta5-CMV-vaccinated animals (3.7 x 10(5) copies/ml) was approximately 1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 x 10(4) copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.

Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression.

The IL-1 family cytokines are regulated on transcriptional and posttranscriptional levels. Pattern recognition and cytokine receptors control pro-IL-1beta transcription whereas inflammasomes regulate the proteolytic processing of pro-IL-1beta. The NLRP3 inflammasome, however, assembles in response to extracellular ATP, pore-forming toxins, or crystals only in the presence of proinflammatory stimuli. How the activation of gene transcription by signaling receptors enables NLRP3 activation remains elusive and controversial. In this study, we show that cell priming through multiple signaling receptors induces NLRP3 expression, which we identified to be a critical checkpoint for NLRP3 activation. Signals provided by NF-kappaB activators are necessary but not sufficient for NLRP3 activation, and a second stimulus such as ATP or crystal-induced damage is required for NLRP3 activation.

Internal herniation through the foramen of Winslow during pregnancy: MR findings [corrected].

We present a unique case of jaundice in pregnancy, secondary to common bile duct obstruction by an unusual internal colonic hernia. We also illustrate the benefits of using non-ionizing imaging modalities in the diagnosis and pre-operative planning of this rare condition.

The Role of Reading Anxiety among Struggling Readers in Fourth and Fifth Grade.

Cognitive predictors of reading are well known, but less is understood about the roles of "noncognitive" factors, including emotional variables such as anxiety. While math anxiety has been a focus of study, its analogue in the reading literature is understudied. We assessed struggling fourth and fifth graders (n = 272) on reading anxiety in the context of general anxiety, cognitive predictors (working memory, verbal knowledge), and demographics. Regressions tested for unique contributions to three reading outcomes: word reading accuracy, oral reading fluency, and reading comprehension. Reading anxiety and general anxiety correlated moderately (r = .63) but were differentially related to reading. Reading anxiety predicted comprehension when all other predictors were considered, and predicted oral reading fluency until word reading accuracy was added to the model. Results offer a more nuanced understanding of the nature of reading anxiety, and its implications for struggling readers.

Epidemiology, clinical characteristics, and virologic features of COVID-19 patients in Kazakhstan: A nation-wide retrospective cohort study.

BACKGROUND: The earliest coronavirus disease-2019 (COVID-19) cases in Central Asia were announced in March 2020 by Kazakhstan. Despite the implementation of aggressive measures to curb infection spread, gaps remain in the understanding of the clinical and epidemiologic features of the regional pandemic. METHODS: We did a retrospective, observational cohort study of patients with laboratory-confirmed COVID-19 hospitalized in Kazakhstan between February and April 2020. We compared demographic, clinical, laboratory and radiological data of patients with different COVID-19 severities on admission. Logistic regression was used to assess factors associated with disease severity and in-hospital death. Whole-genome SARS-CoV-2 analysis was performed in 53 patients. FINDINGS: Of the 1072 patients with laboratory-confirmed COVID-19 in March-April 2020, the median age was 36 years (IQR 24-50) and 484 (45%) were male. On admission, 683 (64%) participants had asymptomatic/mild, 341 (32%) moderate, and 47 (4%) severe-to-critical COVID-19 manifestation; 20 in-hospital deaths (1•87%) were reported by 5 May 2020. Multivariable regression indicated increasing odds of severe disease associated with older age (odds ratio 1•05, 95% CI 1•03-1•07, per year increase; p<0•001), the presence of comorbidities (2•34, 95% CI 1•18-4•85; p=0•017) and elevated white blood cell count (WBC, 1•13, 95% CI 1•00-1•27; p=0•044) on admission, while older age (1•09, 95% CI 1•06-1•13, per year increase; p<0•001) and male sex (5•63, 95% CI 2•06-17•57; p=0•001) were associated with increased odds of in-hospital death. The SARS-CoV-2 isolates grouped into seven phylogenetic lineages, O/B.4.1, S/A.2, S/B.1.1, G/B.1, GH/B.1.255, GH/B.1.3 and GR/B.1.1.10; 87% of the isolates were O and S sub-types descending from early Asian lineages, while the G, GH and GR isolates were related to lineages from Europe and the Americas. INTERPRETATION: Older age, comorbidities, increased WBC count, and male sex were risk factors for COVID-19 disease severity and mortality in Kazakhstan. The broad SARS-CoV-2 diversity suggests multiple importations and community-level amplification predating travel restriction. FUNDING: Ministry of Education and Science of the Republic of Kazakhstan.

The Structure of Processing Speed in Children and its Impact on Reading.

The present study had two aims. First, we set out to evaluate the structure of processing speed in children by comparing five alternative models: two conceptual models (a unitary model, a complexity model) and three methodological models (a stimulus material model, an output response model, and a timing modality model). Second, we then used the resulting models to predict multiple types of reading, a highly important developmental outcome, using other well-known predictors as covariates. Participants were 844 children enrolled in third through fifth grade in urban public elementary schools who received 16 measures of processing speed that varied in the above dimensions. A two-factor complexity model that differentiated between simple and complex processing speed was the preferred model and fit the data well. Both types of PS predicted reading fluency, and complex (but not simple) PS predicted single word reading and comprehension. Results offer insight to the structure of processing speed, its relation to closely related concepts (such as executive function), and provide nuance to the understanding of the way processing speed influences reading.