Increased Susceptibility for Adverse Reactions to Ultrasound Enhancing Agents in Sickle Cell Disease.

BACKGROUND: Pain-related adverse events (AEs) to ultrasound enhancing agents (UEAs) have been reported in patients with sickle cell disease (SCD). The aims of this study were to characterize the scope of these AEs in the SCD population and to investigate potential mechanisms on the basis of pathways involved in SCD vaso-occlusive crisis (VOC) and pain. METHODS: The prevalence and classification of AEs were analyzed from two clinical trials in which high-dose Definity infusions were used in patients with SCD (n = 55) or matched control subjects (n = 43) to study muscle or myocardial microvascular perfusion. Because complement (C') activation can trigger VOC in SCD, C' activation and surface adhesion of C' proteins on lipid UEAs were studied in vitro. C'-mediated UEA attachment to bone marrow immune cells was assessed using flow cytometry in a murine SCD model (Townes mice). Blood from patients receiving Definity was obtained to measure specific lysophospholipid metabolites of lipids in Definity thought to mediate SCD pain. RESULTS: Moderate or greater AEs, all of which were nociceptive (back or bone pain), occurred in one control subject and nine SCD subjects (2% vs 16%, P = .02). Patients with SCD who had AEs tended to have more severe manifestations of SCD. Three of the subjects with SCD had previously received Definity without complications. In patients with SCD, four AEs were classified as severe in intensity and as serious AEs on the basis of need for medical intervention. AEs were described to be similar to SCD-related pain, but there was no evidence for VOC, hemolysis, hypotension, or hypoxemia. At baseline, markers of C' activation were greater in patients with SCD than control subjects. However, after administration of lipid UEAs, SCD and control subjects were similar with regard to C' activation response, anaphylatoxin production, bone marrow microbubble retention, and production of lysophospholipids. There was a trend toward increased deposition of C3b and C3bi on lipid UEAs exposed to serum from patients with SCD. CONCLUSIONS: Patients with SCD are particularly susceptible to nociceptive AEs when given Definity at high doses. The mechanism for these AEs remains unclear but most are not related to the triggering of classic VOC.

Differences in outcomes between surgical pericardial window and pericardiocentesis in children with postpericardiotomy syndrome.

Children with postpericardiotomy syndrome may develop hemodynamically significant pericardial effusions warranting drainage by surgical pericardial window or pericardiocentesis. The optimal approach is unknown. We performed a retrospective observational study at two pediatric cardiac centers. We included 42 children aged <18 years who developed postpericardiotomy syndrome following cardiac surgery between 2014 and 2021. Thirty-two patients underwent pericardial window and 10 underwent pericardiocentesis. Patients in the pericardial window group presented with postpericardiotomy syndrome sooner than those who underwent pericardiocentesis (median 7.5 days vs. 14.5 days, P = 0.03) and tended to undergo earlier intervention (median 8 days vs. 16 days, P = 0.16). No patient required subsequent drainage. There were no differences between groups in days of pericardial tube duration (median 4 days), complications, and subsequent days of intensive care or hospitalization. For children with postpericardiotomy syndrome with a pericardial effusion warranting drainage, these data suggest that pericardial window and pericardiocentesis have similar efficacy, safety, and resource utilization.

"We are in this together:" dyadic-level influence and decision-making among HIV serodiscordant couples in Tanzania receiving access to PrEP.

BACKGROUND: A substantial number of new HIV infections in sub-Saharan Africa occur within stable couples. Biomedical prevention (pre-exposure prophylaxis, PrEP) and treatment (antiretroviral therapy, ART) can provide benefits to sexual partners and can be used to prevent infection within HIV serodiscordant couples. However, research is typically focused on individuals, not dyads, even when the intervention may directly or indirectly impact sexual partners. Gaps remain in understanding best practices for recruitment, informed consent, and intervention implementation in studies involving HIV prevention and treatment among heterosexual serodiscordant couples. This qualitative study was undertaken to understand and describe decision-making and dyadic-level influence among members of serodiscordant couples regarding (1) participation in a dyadic-based research study involving HIV self-testing and access to PrEP, and (2) utilization of PrEP and ART. METHODS: This qualitative study was nested within an observational cohort study assessing the acceptability of home-based couples' HIV self-testing and uptake of dyadic care for serodiscordant couples involving facilitated referral for HIV-positive partners and access to PrEP for HIV-negative partners. Semi-structured in-depth interviews were conducted among a subset of study participants (n = 22) as well as individuals involved in serodiscordant relationships who chose not to participate (n = 9). Interviews focused on couples' decision-making regarding study participation and dyadic-level influence on medication use. Interviews were transcribed verbatim and translated from Kiswahili into English. Data were analyzed using thematic analysis. RESULTS: Three major themes were identified: (1) HIV as "two people's secret" and the elevated role of partner support in serodiscordant relationships; (2) the intersectional role of HIV-status and gender on decision-making; (3) the relational benefits of PrEP, including psychosocial benefits for the couple that extend beyond prevention. CONCLUSIONS: The study found that couples made joint decisions regarding study participation and uptake of HIV-related medication. Relational autonomy and dyadic-level influence should be considered within research and programs involving HIV serodiscordant couples.

Rheumatoid arthritis in patients with HIV: management challenges.

Over the past few decades, HIV has been transformed from a once-uniformly fatal disease to now a manageable but complex multisystem illness. Before highly active antiretroviral therapy (HAART), reports suggested that HIV-infected patients with rheumatoid arthritis (RA) would experience remission of their disease. It has now become clear that RA can develop in HIV-infected patients at any time, independent of HAART. Choosing the right medication to treat symptoms related to RA while avoiding excess weakening of the immune system remains a clinical challenge. Agents such as hydroxychloroquine and sulfasalazine might best balance safety with efficacy, making them reasonable first choices for therapy in HIV-infected patients with RA. More immune suppressing agents such as methotrexate may balance safety with efficacy, but data are limited. Corticosteroids such as prednisone may also be reasonable but could increase the risk of osteonecrosis. Among biologic response modifiers, tumor necrosis factor α inhibitors may balance safety with efficacy, but perhaps when HIV replication is controlled with HAART. Monitoring RA disease activity remains challenging as only one retrospective study has been published in this area. Those with HIV infection and RA can experience comorbidities such as accelerated heart disease and osteoporosis, a consequence of the chronic inflammatory state that each illness generates. Although HIV-infected patients are at risk for developing the immune reconstitution inflammatory syndrome when starting HAART, it appears that immune reconstitution inflammatory syndrome has a minimal effect on triggering the onset or the worsening of RA.

Campylobacter jejuni CsrA Regulates Metabolic and Virulence Associated Proteins and Is Necessary for Mouse Colonization.

Campylobacter jejuni infection is a leading bacterial cause of gastroenteritis and a common antecedent leading to Gullian-Barré syndrome. Our previous data suggested that the RNA-binding protein CsrA plays an important role in regulating several important phenotypes including motility, biofilm formation, and oxidative stress resistance. In this study, we compared the proteomes of wild type, csrA mutant, and complemented csrA mutant C. jejuni strains in an effort to elucidate the mechanisms by which CsrA affects virulence phenotypes. The putative CsrA regulon was more pronounced at stationary phase (111 regulated proteins) than at mid-log phase (25 regulated proteins). Proteins displaying altered expression in the csrA mutant included diverse metabolic functions, with roles in amino acid metabolism, TCA cycle, acetate metabolism, and various other cell processes, as well as pathogenesis-associated characteristics such as motility, chemotaxis, oxidative stress resistance, and fibronectin binding. The csrA mutant strain also showed altered autoagglutination kinetics when compared to the wild type. CsrA specifically bound the 5' end of flaA mRNA, and we demonstrated that CsrA is a growth-phase dependent repressor of FlaA expression. Finally, the csrA mutant exhibited reduced ability to colonize in a mouse model when in competition with the wild type, further underscoring the role of CsrA in C. jejuni colonization and pathogenesis.

Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation.

BACKGROUND: Although Campylobacter jejuni is consistently ranked as one of the leading causes of bacterial diarrhea worldwide, the mechanisms by which C. jejuni causes disease and how they are regulated have yet to be clearly defined. The global regulator, CsrA, has been well characterized in several bacterial genera and is known to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus Campylobacter. Previously, we reported data illustrating the requirement for CsrA in several virulence related phenotypes of C. jejuni strain 81-176, indicating that the Csr pathway is important for Campylobacter pathogenesis. RESULTS: We compared the Escherichia coli and C. jejuni orthologs of CsrA and characterized the ability of the C. jejuni CsrA protein to functionally complement an E. coli csrA mutant. Phylogenetic comparison of E. coli CsrA to orthologs from several pathogenic bacteria demonstrated variability in C. jejuni CsrA relative to the known RNA binding domains of E. coli CsrA and in several amino acids reported to be involved in E. coli CsrA-mediated gene regulation. When expressed in an E. coli csrA mutant, C. jejuni CsrA succeeded in recovering defects in motility, biofilm formation, and cellular morphology; however, it failed to return excess glycogen accumulation to wild type levels. CONCLUSIONS: These findings suggest that C. jejuni CsrA is capable of efficiently binding some E. coli CsrA binding sites, but not others, and provide insight into the biochemistry of C. jejuni CsrA.

Circuitry linking the Csr and stringent response global regulatory systems.

CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.

Effects of sequential Campylobacter jejuni 81-176 lipooligosaccharide core truncations on biofilm formation, stress survival, and pathogenesis.

Campylobacter jejuni is a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that a C. jejuni strain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required for C. jejuni pathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaF and lgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed DeltagalT and DeltacstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (DeltawaaF and DeltalgtF but not DeltagalT or DeltacstII mutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (DeltacstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active against C. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The DeltawaaF mutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect of C. jejuni pathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of the C. jejuni LOS in host colonization. Collectively, this study has uncovered novel roles for the C. jejuni LOS, highlights the dynamic nature of the C. jejuni cell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.

Although infection with Campylobacter jejuni is one of the leading causes of gastroenteritis worldwide, relatively little is known about the factors that are required to elicit a protective immune response. The need for a vaccine against this pathogen is well recognized and a number of vaccine candidates have been tested with varying degrees of success; however, there is still a lack of a suitable vaccine. To gain a better understanding of the outer-membrane protein components of this organism, a 'gold standard' method to purify the outer membrane is needed. Therefore, we attempted to develop a robust and reliable method which resulted in a pure outer-membrane fraction. A total of nine methodologies were examined and analysed by SDS-PAGE and immunoblotting using subcellular markers for the cytoplasm, cytoplasmic membrane and outer membrane. We found that glycine extraction, differential detergent extraction using Triton X-100, serial extraction using 1 M Tris pH 7, spheroplasting by lysozyme and sonication, and carbonate extraction did not produce pure outer-membrane preparations. However, we identified three methods that provided outer-membrane fractions free from subcellular contamination. Isopycnic centrifugation using a 30-60 % sucrose gradient produced seven fractions free from cytoplasmic or cytoplasmic membrane contamination; however, these fractions did not correspond as well as expected with the typical outer-membrane-associated peak (e.g. Escherichia coli or Salmonella). The spheroplast method using lysozyme alone also resulted in pure outer-membrane fraction, as did carbonate washing of this sample. The extraction of outer membranes using N-lauroylsarcosine (Sarkosyl) produced the purest and most reproducible sample. These outer-membrane preparations will be useful for future studies aimed at identifying C. jejuni surface proteins as vaccine components.

Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion.

The putative global posttranscriptional regulator csrA was mutated in Campylobacter jejuni 81-176. The csrA mutant was attenuated in surviving oxidative stress. CsrA also contributed to biofilm formation and adherence to and invasion of INT407 intestinal epithelial cells, suggesting a regulatory role for CsrA in C. jejuni pathogenesis.

A temperature-regulated Campylobacter jejuni gluconate dehydrogenase is involved in respiration-dependent energy conservation and chicken colonization.

Campylobacter jejuni is a gastrointestinal pathogen of humans but can asymptomatically colonize the avian gut. C. jejuni therefore grows at both 37 degrees C and 42 degrees C, the internal temperatures of humans and birds respectively. Microarray and proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 42 degrees C of two proteins, Cj0414 and Cj0415, orthologous to gluconate dehydrogenase (GADH) from Pectobacterium cypripedii. 81-176 demonstrated GADH activity, converting d-gluconate to 2-keto-d-gluconate, that was higher at 42 degrees C than at 37 degrees C. In contrast, cj0414 and cj0415 mutants lacked GADH activity. Wild-type but not cj0415 mutant bacteria exhibited gluconate-dependent respiration. Neither strain grew in defined media with d-gluconate or 2-keto-d-gluconate as a sole carbon source, revealing that gluconate was used as an electron donor rather than as a carbon source. When administered to chicks individually or in competition with wild-type, the cj0415 mutant was impaired in establishing colonization. In contrast, there were few significant differences in colonization of BALB/c-ByJ mice in single or mixed infections. These results suggest that the ability of C. jejuni to use gluconate as an electron donor via GADH activity is an important metabolic characteristic that is required for full colonization of avian but not mammalian hosts.

PKCdelta clustering at the leading edge and mediating growth factor-enhanced, but not ecm-initiated, dermal fibroblast migration.

We have previously shown that the immobilized extracellular matrices (ECMs) initiate cell migration and soluble growth factors (GFs) further enhance ECM-initiated cell migration. GFs alone cannot initiate cell migration. To further investigate the specificity of the two signaling mechanisms, we focused on the protein kinase C (PKC) family genes in primary human dermal fibroblasts (DFs). We here show that platelet-derived growth factor-BB (PDGF-BB) strongly stimulates membrane translocation and leading edge clustering of protein kinase Cdelta (PKCdelta). In contrast, attachment to collagen matrix alone does not cause the translocation. Although the kinase function of PKCdelta is dispensable for initial membrane translocation, it is critical for its sustained presence at the cells's leading edge. Blockade of endogenous PKCdelta signaling with dominant-negative kinase-defective PKC (PKCdelta-KD) or PKCdelta-small interfering RNA (siRNA) completely inhibited PDGF-BB-stimulated DF migration. In contrast, neither PKCdelta-KD nor PKCdelta-siRNA affected collagen-induced initiation of DF migration. Overexpression of a constitutively activated PKCdelta (PKCdelta-R144/145A) partially mimics the effect of PDGF-BB. However, PKCdelta-KD, PKCdelta-siRNA, or PKCdelta-R144/145A does not affect PDGF-BB-stimulated activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, or c-Jun N-terminal kinase. Instead, inhibition of PKCdelta blocks PDGF-BB-stimulated activation of signal transducer and activator of transcription 3 (Stat3). This study unveiled the specificity of PKCdelta in the control of DF migration.