Mucosal CCL28 Chemokine Improves Protection against Genital Herpes through Mobilization of Antiviral Effector Memory CCR10+CD44+ CD62L-CD8+ T Cells and Memory CCR10+B220+CD27+ B Cells into the Infected Vaginal Mucosa.

Four major mucosal-associated chemokines, CCL25, CCL28, CXCL14, and CXCL17, play an important role in protecting mucosal surfaces from infectious pathogens. However, their role in protection against genital herpes remains to be fully explored. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). In this study, we investigated the role of the CCL28/CCR10 chemokine axis in mobilizing protective antiviral B and T cell subsets into the VM site of herpes infection. We report a significant increase in the frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10, in herpes-infected asymptomatic (ASYMP) women compared with symptomatic women. Similarly, a significant increase in the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP C57BL/6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected ASYMP mice. Inversely, compared with wild-type C57BL/6 mice, the CCL28 knockout (CCL28-/-) mice (1) appeared to be more susceptible to intravaginal infection and reinfection with HSV type 2, and (2) exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. These findings suggest a critical role of the CCL28/CCR10 chemokine axis in the mobilization of antiviral memory B and T cells within the VM to protect against genital herpes infection and disease.

Plasma-Derived HIV-1 Virions Contain Considerable Levels of Defective Genomes.

Genetically-characterizing full-length HIV-1 RNA is critical for identifying genetically-intact genomes and for comparing these RNA genomes to proviral DNA. We have developed a method for sequencing plasma-derived RNA using long-range sequencing (PRLS assay; ∼8.3 kb from gag to the 3' end or ∼5 kb from integrase to the 3' end). We employed the gag-3' PRLS assay to sequence HIV-1 RNA genomes from ART-naive participants during acute/early infection (n = 6) or chronic infection (n = 2). On average, only 65% of plasma-derived genomes were genetically-intact. Defects were found in all genomic regions but were concentrated in env and pol. We compared these genomes to near-full-length proviral sequences from paired peripheral blood mononuclear cell (PBMC) samples for the acute/early group and found that near-identical (>99.98% identical) sequences were identified only during acute infection. For three participants who initiated therapy during acute infection, we used the int-3' PRLS assay to sequence plasma-derived genomes from an analytical treatment interruption and identified 100% identical genomes between pretherapy and rebound time points. The PRLS assay provides a new level of sensitivity for understanding the genetic composition of plasma-derived HIV-1 RNA from viremic individuals either pretherapy or after treatment interruption, which will be invaluable in assessing possible HIV-1 curative strategies. IMPORTANCE We developed novel plasma-derived RNA using long-range sequencing assays (PRLS assay; 8.3 kb, gag-3', and 5.0 kb, int-3'). Employing the gag-3' PRLS assay, we found that 26% to 51% of plasma-derived genomes are genetically-defective, largely as a result of frameshift mutations and deletions. These genetic defects were concentrated in the env region compared to gag and pol, likely a reflection of viral immune escape in env during untreated HIV-1 infection. Employing the int-3' PRLS assay, we found that analytical treatment interruption (ATI) plasma-derived sequences were identical and genetically-intact. Several sequences from the ATI plasma samples were identical to viral sequences from pretherapy plasma and PBMC samples, indicating that HIV-1 reservoirs established prior to therapy contribute to viral rebound during an ATI. Therefore, near-full-length sequencing of HIV-1 particles is required to gain an accurate picture of the genetic landscape of plasma HIV-1 virions in studies of HIV-1 replication and persistence.

Utilizing a High-Fidelity COVID-19 Simulation to Develop Collaboration Skills Among Multispecialty Cohorts.

ABSTRACT: Nursing students rarely have the opportunity to witness nurse-to-nurse collaboration with nurses from differing specialties. Simulation can provide students with an opportunity to practice this important skill. In this simulation, students from a critical care course and students from a maternal/newborn course worked together to care for a pregnant client with COVID-19. The client's condition required expertise from both an obstetrical nurse and a critical care nurse. Although the design of the simulation was aimed at collaboration, various other important observations and teaching moments were uncovered during the implementation of the simulation.

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

Neonatal neutrophils stimulated by group B Streptococcus induce a proinflammatory T-helper cell bias.

BackgroundGroup B Streptococcus (GBS) infection causes inflammatory comorbidities in newborns. While the mechanisms remain unclear, evidence suggests that impaired innate-adaptive immune interactions may be contributory. We hypothesized that GBS-stimulated neonatal neutrophils provide a milieu that may drive proinflammatory T-helper (Th) cell programming.MethodsNeutrophils were stimulated with Type III GBS (COH1); supernatants or intact neutrophils were cocultured with CD4+ T cells or regulatory T cells (Tregs). Resulting intracellular cytokines and nuclear transcription factors were determined by multicolor flow cytometry.ResultsGBS-stimulated neutrophils released soluble mediators that induced greater interleukin-17 (IL-17) responses in neonatal vs. adult CD4+ T cells in the absence of added polarizing cytokines. GBS-stimulated neonatal neutrophils also induced robust expression of the canonical nuclear transcription factors for Th1 (Tbet) and Th17 (IL-17) cells in CD4+ T cells. Following GBS stimulation, both intact neutrophils and neutrophil-derived mediators promoted the generation of Tregs with Th1 and Th17 characteristics.ConclusionGBS-stimulated neonatal neutrophils bias the in vitro Th differentiation program of neonatal CD4+ T cells and promote proinflammatory Th1 and Th17 phenotypes in Tregs. Our data suggest that developmental modifications of innate-adaptive immune cross-talk mechanisms may contribute to the inflammatory complications associated with neonatal GBS infection.

Identification of a second substrate-binding site in solute-sodium symporters.

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ~1. In addition, the related and more experimentally tractable SSS member PutP (the Na(+)/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.

Safety and diagnostic value of brain biopsy in HIV patients: a case series and meta-analysis of 1209 patients.

Early brain biopsy may be indicated in HIV patients with focal brain lesion. This study aimed to evaluate and compare the safety and diagnostic value of brain biopsy in HIV patients in the pre-highly active antiretroviral therapy (HAART) versus post-HAART era via meta-analysis. Appropriate studies were identified per search criteria. The local database was retrospectively reviewed to select a similar patient cohort. Patient demographics, brain biopsy technique, histopathology and patient outcomes were extracted from each study. Study-specific outcomes were combined per random-effects model. Outcomes were compared between the pre-HAART and post-HAART era. Correlations between outcomes and baseline characteristics were assessed via meta-regression analysis. The proportions of histopathological diagnosis were tabulated and compared between the pre- and post-HAART era. Survival analysis was performed for patients in the post-HAART era. A total of 26 studies (including the local database) with 1209 patients were included in this meta-analysis. The most common indications for brain biopsy were diagnosis unlikely to be toxoplasmosis (n=8, 42.1%), focal brain lesion (n=5, 26.3%) or both (n=3, 15.8%). The weighted proportions for diagnostic success were 92% (95% CI 90.0% to 93.8%), change in management 57.7% (45.9% to 69.1%) and clinical improvement 36.6% (26.3% to 47.5%). Morbidity and mortality were 5.7% (3.6% to 8.3%) and 0.9% (0.3% to 1.9%), respectively. Diagnostic success rate was significantly higher in the post-HAART than the pre-HAART era (97.5% vs 91.9%, p=0.047). The odds ratio (OR) for diagnostic success in patients with contrast-enhanced lesions was 2.54 ((1.25 to 5.15), p<0.01). The median survival for HIV patients who underwent biopsy in the post-HAART era was 225 days (90-2446). Brain biopsy in HIV patients is safe with high diagnostic yield. Early brain biopsy should be considered in patients without classic presentation of toxoplasmosis encephalitis.

Percutaneous Image-Guided Cryoablation of Musculoskeletal Metastases: Pain Palliation and Local Tumor Control.

PURPOSE: To evaluate the safety and effectiveness of cryoablation of musculoskeletal metastases in terms of achieving pain palliation and local tumor control. MATERIALS AND METHODS: A retrospective review was performed of 92 musculoskeletal metastases in 56 patients treated with percutaneous image-guided cryoablation. Mean age of the cohort was 53.9 y ± 15.1, and cohort included 48% (27/56) men. Median tumor volume was 13.0 cm3 (range, 0.5-577.2 cm3). Indications for treatment included pain palliation (41%; 38/92), local tumor control (15%; 14/92), or both (43%; 40/92). Concurrent cementoplasty was performed after 28% (26/92) of treatments. RESULTS: In 78 tumors treated for pain palliation, median pain score before treatment was 8.0. Decreased median pain scores were reported 1 day (6.0; P < .001, n = 62), 1 week (5.0; P < .001, n = 70), 1 month (5.0; P < .001, n = 63), and 3 months (4.5; P = .01, n = 28) after treatment. The median pain score at 6-month follow-up was 7.5 (P = .33, n = 11). Radiographic local tumor control rates were 90% (37/41) at 3 months, 86% (32/37) at 6 months, and 79% (26/33) at 12 months after treatment. The procedural complication rate was 4.3% (4/92). The 3 major complications included 2 cases of hemothorax and 1 transient foot drop. CONCLUSIONS: Cryoablation is an effective treatment for palliating painful musculoskeletal metastases and achieving local tumor control.

Effect of the 2010 task force criteria on reclassification of cardiovascular magnetic resonance criteria for arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: We sought to evaluate the effect of application of the revised 2010 Task Force Criteria (TFC) on the prevalence of major and minor Cardiovascular Magnetic Resonance (CMR) criteria for Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) versus application of the original 1994 TFC. We also assessed the utility of MRI to identify alternative diagnoses for patients referred for ARVC evaluation. METHODS: 968 consecutive patients referred to our institution for CMR with clinical suspicion of ARVC from 1995 to 2010, were evaluated for the presence of major and minor CMR criteria per the 1994 and 2010 ARVC TFC. CMR criteria included right ventricle (RV) dilatation, reduced RV ejection fraction, RV aneurysm, or regional RV wall motion abnormalities. When quantitative measures of RV size and function were not available, and in whom abnormal size or function was reported, a repeat quantitative analysis by 2 qualified CMR physicians in consensus. RESULTS: Of 968 patients, 220 (22.7%) fulfilled either a major or a minor 1994 TFC, and 25 (2.6%) fulfilled any of the 2010 TFC criterion. Among patients meeting any 1994 criteria, only 25 (11.4%) met at least one 2010 criterion. All patients who fulfilled a 2010 criteria also satisfied at least one 1994 criterion. Per the 2010 TFC, 21 (2.2%) patients met major criteria and 4 (0.4%) patients fulfilled at least one minor criterion. Eight patients meeting 1994 minor criteria were reclassified as satisfying 2010 major criteria, while 4 patients fulfilling 1994 major criteria were reclassified to only minor or no criteria under the 2010 TFC.Eighty-nine (9.2%) patients had alternative cardiac diagnoses, including 43 (4.4%) with clinically significant potential ARVC mimics. These included cardiac sarcoidosis, RV volume overload conditions, and other cardiomyopathies. CONCLUSIONS: Application of the 2010 TFC resulted in reduction of total patients meeting any diagnostic CMR criteria for ARVC from 22.7% to 2.6% versus the 1994 TFC. CMR identified alternative cardiac diagnoses in 9.2% of patients, and 4.4% of the diagnoses were potential mimics of ARVC.

Coronary computed tomography angiography at 140 kV versus 120 kV: assessment of image quality and radiation exposure in overweight and moderately obese patients.

BACKGROUND: Although a tube potential of 140 kV is available on most computed tomography (CT) scanners, its incremental diagnostic value versus 120 kV has been controversial. PURPOSE: To retrospectively evaluate the image quality and radiation exposure of cardiac computed tomography angiography (CCTA) performed at 140 kV in comparison to CCTA at 120 kV in overweight and moderately obese patients. MATERIAL AND METHODS: Eighty-eight patients who were referred for CCTA between January 2010 and May 2012 were included. Forty-four patients who were overweight or moderately obese (body mass index [BMI], 25-35 kg/m(2)) underwent CCTA with dual-source CT (DSCT) scanner at 140 kV. Forty-four match controls who underwent CCTA with DSCT at 120 kV were identified per BMI, average heart rate, scan indication, and scan acquisition mode. All scans were performed per routine protocols with direct physician supervision. Quantitative image metrics (CT attenuation, image noise, contrast-to-noise ratio [CNR], and signal-to-noise ratio [SNR] of left main [LM] and proximal right coronary artery [RCA]) were assessed. Effective radiation dose was compared between the two groups. RESULTS: Overall, all scans were diagnostic without any non-evaluable coronary segment per clinical report. 140 kV had a lower attenuation and image noise versus 120 kV (P<0.01). Both SNR and CNR of proximal coronary arteries were similar between 140 kV and 120 kV (SNR, LM P=0.93, RCA P=0.62; CNR, LM P=0.57, RCA P=0.77). 140 kV was associated with a 35.3% increase in effective radiation dose as compared with 120 kV (5.1 [3.6-8.2] vs. 3.3 [2.0-5.1] mSv, respectively; P<0.01). CONCLUSION: 140 kV CCTA resulted in similar image quality but a higher effective radiation dose in comparison to 120 kV CCTA. Therefore, in overweight and moderately obese patients, a tube potential of 120 kV may be sufficient for CCTA with diagnostic image quality.

Radiation dose reduction in pediatric cardiac computed tomography: experience from a tertiary medical center.

Cardiac CT angiography (cCTA) has become an established method for the assessment of congenital heart disease. However, the potential harmful effects of ionizing radiation must be considered, particularly in younger, more radiosensitive patients. In this study, we sought to assess the temporal change in radiation doses from pediatric cCTA during an 8-year period at a tertiary medical center. This retrospective study included all patients ≤18 years old who were referred to electrocardiography (ECG)-gated cCTA for the assessment of congenital heart disease or inflammatory disease (Kawasaki disease) from November 2004 to September 2012. During the study period, 95 patients were scanned using 3 different scanner models-64-slice multidetector CT (64-MDCT) and first- (64-DSCT) and second-generation (128-DSCT) dual-source CT-and 3 scan protocols-retrospective ECG-gated helical scanning (RG), prospective ECG-triggered axial scanning (PT), or prospective ECG-triggered high-pitch helical scanning (HPH). Effective dose (ED) was calculated with the dose length product method with a conversion factor (k) adjusted for age. ED was then compared among scan protocols. Image quality was extracted from clinical cCTA reports when available. Overall, 94 % of scans were diagnostic (80 % for 64-slice MDCT, 93 % for 64-slice DSCT, and 97 % for 128-slice DSCT).With 128-DSCT, median ED (1.0 [range 0.6-2.0] mSv) decreased by 85.8 % and 66.8 % compared with 64-MDCT (6.8 [range 2.9-13.6] mSv) and 64-DSCT (2.9 [range 0.9-4.1] mSv), respectively. With HPH, median ED (0.9 [range 0.6-1.8] mSv) decreased by 59.4 % and 85.4 % compared with PT (2.2 [range 0.9-3.4] mSv) and RG (6.1 [range 2.5-10.6] mSv). cCTA can now be obtained at very low radiation doses in pediatric patients using the latest dual-source CT technology in combination with prospective ECG-triggered HPH acquisition.

Automatic extraction of comprehensive cardiac CT angiography parameters: a novel program with high accuracy and efficiency.

We tested the accuracy and efficiency of a novel automated program capable of extracting 15 cardiac computed tomography angiography (CTA) parameters from clinical CTA reports. Five hundred cardiac CTA reports were retrospectively collected and processed. All reports were pre-populated with a structured template per guideline. The program extracted 15 parameters with high accuracy (97.3 %) and efficiency (84 s). This program may be used at other institutions with similar accuracy if its report format follows the Society of Cardiovascular Computed Tomography (SCCT) guideline recommendation.

Addressing Health Equity in Food Allergy.

Social determinants of health can lead to poor health outcomes for food-allergic patients, including limited access to allergen-free foods and specialty care. Housing and transportation limitations can worsen social factors including food insecurity, poor early food introduction, increased reactivity to foods, lower tertiary/allergy care utilization, and increased emergency department utilization. A key component of addressing health equity involves valuing all people with sustained, focused efforts to address social determinants of health. In this clinical commentary, we discuss the current state of heath equity for food-allergic patients, highlighting the disparities in emergency care, food allergy prevention, and food insecurity. Solutions to improve health equity through clinical practice are proposed. Currently available funding opportunities through the National Institutes of Health for health equity initiatives are outlined. Gaps in health equity for food-allergic patients include the lack of documented successful implementation of effective solutions to food insecurity, poor early food introduction uptake, poor access to specialist care, and unequal distribution of educational resources. The availability of research funding and legislative policies supporting access to food and education bolster the impetus to move toward health equity for 20 million people in the United States with food allergy.

A GPS map for pulmonary hypertension: a review of imaging modalities.

Pulmonary hypertension (PH) is a lethal disease with a prevalence of 10-20 % in the general population. The current gold standard diagnosis approach is right heart catheterization, directly detecting pulmonary artery pressure. Imaging for PH patients plays an important role in the etiological diagnosis and evaluation of right heart (RV) function, which is a key determinant of morbidity and mortality in PH patients. Currently, echocardiography is the first-line imaging modality for screening PH and evaluating RV function. CMRI is the gold standard method to evaluate RV function. MDCT, radionuclide ventriculography and pulmonary angiography are mostly used in the differential diagnosis of the cause of PH. In this review article, we also mention several newly studied imaging modalities, such as three-dimensional echocardiography, intravascular ultrasound (IVUS) and optical coherence tomography (OCT), which showed promise for early diagnosis of PH.

Epitope-Based IgE Assays and Their Role in Providing Diagnosis and Prognosis of Food Allergy.

With advances in molecular and computational science, epitope-specific IgE antibody profiling has been developed and recently brought into clinical practice. Epitope-based testing detects IgE antibodies that directly bind to antigenic sites of an allergen, providing increased resolution specificity and fewer false-positive results for diagnosing food allergy. Epitope-binding profiles may also serve as prognostic markers of food allergy and help predict quantities of allergen that would provoke a reaction (ie, eliciting dose, possible severity of a reaction after allergen ingestion, and outcomes of treatment options such as oral immunotherapy [OIT]). Future studies are under way to discover additional applications of epitope-specific antibodies for multiple food allergens.

An unusual presentation of chronic recurrent multifocal osteomyelitis involving bones of the skull.

Chronic recurrent multifocal osteomyelitis is a rare, multisystemic inflammatory disease that affects children and adolescents. We present the case of an African-American adolescent male who presented with recurrent swelling of the temporal region with skull involvement on head imaging, which is atypical for chronic recurrent multifocal osteomyelitis. He had clinical and laboratory improvement after initiation of indomethacin and pamidronate.

Highly Networked SARS-CoV-2 Peptides Elicit T Cell Responses with Enhanced Specificity.

Identifying SARS-CoV-2-specific T cell epitope-derived peptides is critical for the development of effective vaccines and measuring the duration of specific SARS-CoV-2 cellular immunity. In this regard, we previously identified T cell epitope-derived peptides within topologically and structurally essential regions of SARS-CoV-2 spike and nucleocapsid proteins by applying an immunoinformatics pipeline. In this study, we selected 30 spike- and nucleocapsid-derived peptides and assessed whether these peptides induce T cell responses and avoid major mutations found in SARS-CoV-2 variants of concern. Our peptide pool was highly specific, with only a single peptide driving cross-reactivity in people unexposed to SARS-COV-2, and immunogenic, inducing a polyfunctional response in CD4+ and CD8+ T cells from COVID-19 recovered individuals. All peptides were immunogenic and individuals recognized broad and diverse peptide repertoires. Moreover, our peptides avoided most mutations/deletions associated with all four SARS-CoV-2 variants of concern while retaining their physicochemical properties even when genetic changes are introduced. This study contributes to an evolving definition of individual CD4+ and CD8+ T cell epitopes that can be used for specific diagnostic tools for SARS-CoV-2 T cell responses and is relevant to the development of variant-resistant and durable T cell-stimulating vaccines.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging.

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.