Severe COVID-19 initially presenting as mesenteric adenopathy.

Coronavirus disease 2019 (COVID-19) can present with abdominal pain in children and adults. Most imaging findings have been limited to characteristic lung findings, as well as one report of bowel-ischemia-related findings in adults. We report a case of COVID-19 in a healthy teenager who initially presented with isolated mesenteric adenopathy, typically a self-limited illness, which progressed to severe illness requiring intensive care before complete recovery. The boy tested negative for COVID-19 twice by polymerase chain reaction (PCR) from upper respiratory swabs before sputum PCR resulted positive. A high index of suspicion should be maintained for COVID-19 given the continued emergence of new manifestations of the disease.

Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.

The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis, depends on its ability to invade the host, replicate, and transmit infection. At its initial peripheral infection site in the distal lung airways, M. tuberculosis infects macrophages, which transport it to deeper tissues. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show in mice and zebrafish that M. tuberculosis, and its close pathogenic relative Mycobacterium marinum, preferentially recruit and infect permissive macrophages while evading microbicidal ones. This immune evasion is accomplished by using cell-surface-associated phthiocerol dimycoceroserate (PDIM) lipids to mask underlying pathogen-associated molecular patterns (PAMPs). In the absence of PDIM, these PAMPs signal a Toll-like receptor (TLR)-dependent recruitment of macrophages that produce microbicidal reactive nitrogen species. Concordantly, the related phenolic glycolipids (PGLs) promote the recruitment of permissive macrophages through a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence, and PGL, which (along with CCR2) is known to be associated with human tuberculosis. Our findings also suggest an explanation for the longstanding observation that M. tuberculosis initiates infection in the relatively sterile environment of the lower respiratory tract, rather than in the upper respiratory tract, where resident microflora and inhaled environmental microbes may continually recruit microbicidal macrophages through TLR-dependent signalling.

The relation between body image perceptions, parental messages, and depressive symptoms among Latinx college students.

OBJECTIVES: This study examines the role of parental messages about body image in relation to body image dissatisfaction (BID) and depressive symptoms among Latinx college students. We assessed negative and positive messages about body image from mothers and fathers to examine the indirect effect of BID in explaining links from parental communication to depressive symptoms. METHOD: The sample included 198 Latinx college students in the southeastern United States (age range 18-25, 70% female). We used four mediation models, whereby parental comments were modeled to affect depressive symptoms through BID. RESULTS: Results indicated that although there was no direct effect between parental messages and depressive symptoms, both negative maternal and paternal comments had indirect effects on depressive symptoms via BID. CONCLUSIONS: Parental messages about body image have significant implications for understanding the etiology of BID and concomitant depressive symptoms among Latinx college students. The findings highlight the important role of parental communication in Latinx student health and the need for future studies to better understand Latinx college students' interpretations of their parents' positive and negative comments. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Diverse Clinical Isolates of Mycobacterium tuberculosis Develop Macrophage-Induced Rifampin Tolerance.

The Mycobacterium tuberculosis lineage 4 strains CDC1551 and H37Rv develop tolerance to multiple antibiotics upon macrophage residence. To determine whether macrophage-induced tolerance is a general feature of clinical M. tuberculosis isolates, we assessed macrophage-induced drug tolerance in strains from lineages 1-3, representing the other predominant M. tuberculosis strains responsible for tuberculosis globally. All 3 lineages developed isoniazid tolerance. While lineage 1, 3, and 4 strains developed rifampin tolerance, lineage 2 Beijing strains did not. Their failure to develop tolerance may be explained by their harboring of a loss-of-function mutation in the Rv1258c efflux pump that is linked to macrophage-induced rifampicin tolerance.

In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish.

Introduction: Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug-drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. Methods: To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae via microscopy. We used PEPITA and confocal microscopy to characterize in vivo the consequences of drug-drug interactions on ototoxic drug uptake and cellular damage of zebrafish lateral line hair cells. Results and discussion: By applying PEPITA to measure ototoxic drug interaction outcomes, we discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

Seroprevalence and clinical characteristics of SARS-CoV-2 infection in children with cystic fibrosis.

BACKGROUND: People with cystic fibrosis (PwCF) have chronic lung disease and may be at increased risk of coronavirus disease 2019 (COVID-19)-related morbidity and mortality. This study aimed to determine seroprevalence and clinical characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children with cystic fibrosis (CF), and to assess antibody responses following SARS-CoV-2 infection or vaccination. METHODS: Children and adolescents with CF followed at Seattle Children's Hospital were enrolled between July 20, 2020 and February 28, 2021. SARS-CoV-2 serostatus was determined on enrollment at 6 and 11 months (±2 months) for nucleocapsid and spike IgG. Participants completed intake and weekly surveys inquiring about SARS-CoV-2 exposures, viral/respiratory illnesses, and symptoms. RESULTS: Of 125 PwCF enrolled, 14 (11%) had positive SARS-CoV-2 antibodies consistent with recent or past infection. Seropositive participants were more likely to identify as Hispanic (29% vs. 8%, p = 0.04) and have pulmonary exacerbations requiring oral antibiotics in the year prior (71% vs. 41%, p = 0.04). Five seropositive individuals (35.7%) were asymptomatic, while six (42.9%) reported mild symptoms, primarily cough and nasal congestion. Antispike protein IgG levels were approximately 10-fold higher in participants following vaccination compared with participants who had natural infection alone (p < 0.0001) and resembled levels previously reported in the general population. CONCLUSIONS: A majority of PwCF have mild or no symptoms of SARS-CoV-2 making it difficult to distinguish from baseline respiratory symptoms. Hispanic PwCF may be disproportionately impacted, consistent with racial and ethnic COVID-19 disparities among the general US population. Vaccination in PwCF generated antibody responses similar to those previously reported in the general population.

In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish.

Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae. By applying PEPITA to characterize ototoxic drug interaction outcomes, we have discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

Impairment in inflammasome signaling by the chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients results in an increase in inflammatory response.

Pseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1ß by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1ß expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.

Carbapenems drive the collateral resistance to ceftaroline in cystic fibrosis patients with MRSA.

Chronic airways infection with methicillin-resistant Staphylococcus aureus (MRSA) is associated with worse respiratory disease cystic fibrosis (CF) patients. Ceftaroline is a cephalosporin that inhibits the penicillin-binding protein (PBP2a) uniquely produced by MRSA. We analyzed 335 S. aureus isolates from CF sputum samples collected at three US centers between 2015-2018. Molecular relationships demonstrated that high-level resistance of preceding isolates to carbapenems were associated with subsequent isolation of ceftaroline resistant CF MRSA. In vitro evolution experiments showed that pre-exposure of CF MRSA to meropenem with further selection with ceftaroline implied mutations in mecA and additional mutations in pbp1 and pbp2, targets of carbapenems; no effects were achieved by other ß-lactams. An in vivo pneumonia mouse model showed the potential therapeutic efficacy of ceftaroline/meropenem combination against ceftaroline-resistant CF MRSA infections. Thus, the present findings highlight risk factors and potential therapeutic strategies offering an opportunity to both prevent and address antibiotic resistance in this patient population.

Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.

Mycobacterium tuberculosis is the leading worldwide cause of death due to a single infectious agent. Existing anti-tuberculous therapies require long treatments and are complicated by multi-drug-resistant strains. Host-directed therapies have been proposed as an orthogonal approach, but few have moved into clinical trials. Here, we use the zebrafish-Mycobacterium marinum infection model as a whole-animal screening platform to identify FDA-approved, host-directed compounds. We identify multiple compounds that modulate host immunity to limit mycobacterial disease, including the inexpensive, safe, and widely used drug clemastine. We find that clemastine alters macrophage calcium transients through potentiation of the purinergic receptor P2RX7. Host-directed drug activity in zebrafish larvae depends on both P2RX7 and inflammasome signaling. Thus, targeted activation of a P2RX7 axis provides a novel strategy for enhanced control of mycobacterial infections. Using a novel explant model, we find that clemastine is also effective within the complex granulomas that are the hallmark of mycobacterial infection.

Delay of Initial Feeding of Zebrafish Larvae Until 8 Days Postfertilization Has No Impact on Survival or Growth Through the Juvenile Stage.

The use of early-stage zebrafish for biomedical research spans early organogenesis to free-swimming larva. A key benefit of this model organism is that repeated assessments spanning several days can be performed of individual larvae within a single experiment, often in conjunction with administered drugs. However, the initiation of feeding, typically at 5 days postfertilization (dpf), can make serial assessments challenging. Therefore, delayed feeding would increase the utility of the model. To ask whether feeding could be delayed without adversely affecting larval growth and development up to 39 dpf, we systematically raised zebrafish and introduced feeding at 5 dpf or delayed initial feeding up to 9 dpf. We assessed survival into the juvenile stage (39 dpf) and anterior-posterior length at this age as proxies for growth and development. Delaying feeding initiation up to 8 dpf did not decrease baseline survival of greater than 90%; survival decreased to 66% only when delayed to 9 dpf. Larval length was no different under any of these conditions. Our findings define 9 dpf as the critical age before which larval zebrafish must be fed when raising to 39 dpf. The option to delay feeding to 8 dpf will broaden experimental applications for the zebrafish larval model.

Macrophage-Dependent Cytoplasmic Transfer during Melanoma Invasion In Vivo.

Interactions between tumor cells and tumor-associated macrophages play critical roles in the initiation of tumor cell motility. To capture the cellular interactions of the tumor microenvironment with high-resolution imaging, we directly visualized tumor cells and their interactions with macrophages in zebrafish. Live imaging in zebrafish revealed that macrophages are dynamic, yet maintain sustained contact with tumor cells. In addition, the recruitment of macrophages to tumor cells promotes tumor cell dissemination. Using a Cre/LoxP strategy, we found that macrophages transfer cytoplasm to tumor cells in zebrafish and mouse models. Remarkably, macrophage cytoplasmic transfer correlated with melanoma cell dissemination. We further found that macrophages transfer cytoplasm to tumor cells upon cell contact in vitro. Thus, we present a model in which macrophage/tumor cell contact allows for the transfer of cytoplasmic molecules from macrophages to tumor cells corresponding to increased tumor cell motility and dissemination.

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development.

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.

vhnf1 integrates global RA patterning and local FGF signals to direct posterior hindbrain development in zebrafish.

The vertebrate hindbrain is transiently divided along the anterior-posterior axis into seven morphologically and molecularly distinct segments, or rhombomeres, that correspond to Hox expression domains. The establishment of a proper 'hox code' is required for the development of unique rhombomere identities, including specification of neuronal fates. valentino (val), the zebrafish ortholog of mafB/Kreisler (Kr), encodes a bZip transcription factor that is required cell autonomously for the development of rhombomere (r) 5 and r6 and for activation of Hox group 3 gene expression. Recent work has demonstrated that the expression of val itself depends on three factors: retinoic acid (RA) signals from the paraxial mesoderm; fibroblast growth factor (Fgf) signals from r4; and variant hepatocyte nuclear factor 1 (vhnf1, also known as tcf2), a homeodomain transcription factor expressed posterior to the r4-5 boundary. We have investigated the interactions between these inputs onto val expression in the developing zebrafish hindbrain. We show that RA induces val expression via activation of vhnf1 expression in the hindbrain. Fgf signals from r4, acting through the MapK pathway, then cooperate with Vhnf1 to activate val expression and subsequent r5 and r6 development. Additionally, vhnf1 and val function as part of a multistep process required for the repression of r4 identity in the posterior hindbrain. vhnf1 acts largely independently of val to repress the r4 'hox code' posterior to the r4-5 boundary and therefore to block acquisition of r4-specific neuronal fates in the posterior hindbrain. However, vhnf1 is not able to repress all aspects of r4 identity equivalently. val is required downstream of vhnf1 to repress r4-like cell-surface properties, as determined by an 'Eph-ephrin code', by repressing ephrin-B2a expression in r5 and r6. The different requirements for vhnf1 and val to repress hoxb1a and ephrin-B2a, respectively, demonstrate that not all aspects of an individual rhombomere's identity are regulated coordinately.