Facile Preparation of N-Glycosylated 10-Piperazinyl Artemisinin Derivatives and Evaluation of Their Antimalarial and Cytotoxic Activities.

According to the precepts that C-10 amino-artemisinins display optimum biological activities for the artemisinin drug class, and that attachment of a sugar enhances specificity of drug delivery, polarity and solubility so as to attenuate toxicity, we assessed the effects of attaching sugars to N-4 of the dihydroartemisinin (DHA)-piperazine derivative prepared in one step from DHA and piperazine. N-Glycosylated DHA-piperazine derivatives were obtained according to the Kotchetkov reaction by heating the DHA-piperazine with the sugar in a polar solvent. Structure of the D-glucose derivative is secured by X-ray crystallography. The D-galactose, L-rhamnose and D-xylose derivatives displayed IC50 values of 0.58⁻0.87 nM against different strains of Plasmodium falciparum (Pf) and selectivity indices (SI) >195, on average, with respect to the mouse fibroblast WEHI-164 cell line. These activities are higher than those of the amino-artemisinin, artemisone (IC50 0.9⁻1.1 nM). Notably, the D-glucose, D-maltose and D-ribose derivatives were the most active against the myelogenous leukemia K562 cell line with IC50 values of 0.78⁻0.87 µM and SI > 380 with respect to the human dermal fibroblasts (HDF). In comparison, artemisone has an IC50 of 0.26 µM, and a SI of 88 with the same cell lines. Overall, the N-glycosylated DHA-piperazine derivatives display antimalarial activities that are greatly superior to O-glycosides previously obtained from DHA.

Lethal coinfection of influenza virus and Streptococcus pneumoniae lowers antibody response to influenza virus in lung and reduces numbers of germinal center B cells, T follicular helper cells, and plasma cells in mediastinal lymph Node.

UNLABELLED: Secondary Streptococcus pneumoniae infection after influenza is a significant clinical complication resulting in morbidity and sometimes mortality. Prior influenza virus infection has been demonstrated to impair the macrophage and neutrophil response to the subsequent pneumococcal infection. In contrast, how a secondary pneumococcal infection after influenza can affect the adaptive immune response to the initial influenza virus infection is less well understood. Therefore, this study focuses on how secondary pneumococcal infection after influenza may impact the humoral immune response to the initial influenza virus infection in a lethal coinfection mouse model. Compared to mice infected with influenza virus alone, mice coinfected with influenza virus followed by pneumococcus had significant body weight loss and 100% mortality. In the lung, lethal coinfection significantly increased virus titers and bacterial cell counts and decreased the level of virus-specific IgG, IgM, and IgA, as well as the number of B cells, CD4 T cells, and plasma cells. Lethal coinfection significantly reduced the size and weight of spleen, as well as the number of B cells along the follicular developmental lineage. In mediastinal lymph nodes, lethal coinfection significantly decreased germinal center B cells, T follicular helper cells, and plasma cells. Adoptive transfer of influenza virus-specific immune serum to coinfected mice improved survival, suggesting the protective functions of anti-influenza virus antibodies. In conclusion, coinfection reduced the B cell response to influenza virus. This study helps us to understand the modulation of the B cell response to influenza virus during a lethal coinfection. IMPORTANCE: Secondary pneumococcal infection after influenza virus infection is an important clinical issue that often results in excess mortality. Since antibodies are key mediators of protection, this study aims to examine the antibody response to influenza virus and demonstrates that lethal coinfection reduced the B cell response to influenza virus. This study helps to highlight the complexity of the modulation of the B cell response in the context of coinfection.

Use and safety of enteral nutrition protocols in acute care: A scoping review of literature and retrospective audit of practice.

AIM: Standardised enteral nutrition protocols are recommended in critical care, however their use and safety are not well described in other inpatient populations. This mixed methods study reports on the use and safety of enteral nutrition protocols for non-critically ill adults. METHODS: A scoping review of published literature was conducted. In addition a retrospective audit of practice at an Australian tertiary teaching hospital with an existing hospital-wide standardised enteral nutrition protocol was performed. Data on use, safety and adequacy of enteral nutrition prescription were collected from medical records for patients receiving enteral nutrition on acute wards (January-March 2020). RESULTS: Screening of 9298 records yielded six primary research articles. Studies were generally low quality. Published literature suggested that protocols may reduce time to enteral nutrition initiation and goal rate, and improve adequacy of nutrition provision. No adverse outcomes were reported. From the local audit of practice (105 admissions, 98 patients), enteral nutrition commencement was timely (median 0 (IQR 0-1) days from request; goal rate: median 1 (IQR 0-2) days from commencement and adequate (nil underfeeding), without prior dietitian review in 82% of cases. Enteral nutrition was commenced per protocol in 61% of instances. No adverse events, including refeeding syndrome, were observed. CONCLUSIONS: Most inpatients requiring enteral nutrition can be safely and adequately managed on enteral nutrition protocols. Evaluation of protocols outside of the critical care setting remains a gap in the literature. Standardised enteral nutrition protocols may improve delivery of nutrition to patients, whilst allowing dietitians to focus on those with specialised nutrition support needs.

Integrative plasma and fecal metabolomics identify functional metabolites in adenoma-colorectal cancer progression and as early diagnostic biomarkers.

Changes in plasma and fecal metabolomes in colorectal cancer (CRC) progression (normal-adenoma-CRC) remain unclear. Here, plasma and fecal samples were collected from four independent cohorts of 1,251 individuals (422 CRC, 399 colorectal adenoma [CRA], and 430 normal controls [NC]). By metabolomic profiling, signature plasma and fecal metabolites with consistent shift across NC, CRA, and CRC are identified, including CRC-enriched oleic acid and CRC-depleted allocholic acid. Oleic acid exhibits pro-tumorigenic effects in CRC cells, patient-derived organoids, and two murine CRC models, whereas allocholic acid has opposing effects. By integrative analysis, we found that oleic acid or allocholic acid directly binds to α-enolase or farnesoid X receptor-1 in CRC cells, respectively, to modulate cancer-associated pathways. Clinically, we establish a panel of 17 plasma metabolites that accurately diagnoses CRC in a discovery and three validation cohorts (AUC = 0.848-0.987). Overall, we characterize metabolite signatures, mechanistic significance, and diagnostic potential of plasma and fecal metabolomes in CRC.

Dendritic and T cell response to influenza is normal in the patients with X-linked agammaglobulinemia.

INTRODUCTION: Influenza virus is a potential cause of severe disease in the immunocompromised. X-linked agammaglobulinemia(XLA) is a primary immunodeficiency characterized by the lack of immunoglobulin, B cells, and plasma cells,secondary to mutation in Bruton's tyrosine kinase (Btk) gene. Btk is expressed in both B and dendritic cells (DC). However, little is known about the immune response of DC and T cells to influenza virus in XLA patients. METHODS: The in vitro maturation and antigen presenting function of monocyte-derived immature DC (im DC) from 12 XLA patients and 23 age-matched normal controls in response to influenza virus were examined. Influenza virus specific CD4 and CD8 T cell responses in the patients and controls were further determined after administration of inactivated trivalent influenza vaccine. RESULTS: im DC from XLA patients had normal maturation based on major histocompatibility complex (MHC)-I, MHCII, CD83 and CD86 expression, and interferon (IFN)-α and interleukin-12 production upon influenza virus stimulation.They also had a normal capacity to induce allogeneic T cell proliferation in response to influenza virus. TIV was well tolerated in XLA patients. Influenza virus-specific CD4+IFN-γ+ and CD8+ IFN-γ+ T cells and HLA-A2/M158­66-tetramer+ CTLs could be induced by TIV in XLA patients, and the levels and duration of maintaining these virus-specific cells in XLA patients are comparable to that in normal controls. CONCLUSION: We demonstrated for the first time that XLA patients have fully competent DC and T cell immune responses to influenza virus. TIV is safe and could be an option for providing T cell-mediated protection against influenza virus infection in XLA patients.

Successive influenza virus infection and Streptococcus pneumoniae stimulation alter human dendritic cell function.

BACKGROUND: Influenza virus is a major cause of respiratory disease worldwide and Streptococcus pneumoniae infection associated with influenza often leads to severe complications. Dendritic cells are key antigen presenting cells but its role in such co-infection is unclear. METHODS: In this study, human monocyte derived-dentritic cells were either concurrently or successively challenged with the combination of live influenza virus and heat killed pneumococcus to mimic the viral pneumococcal infection. Dendritic cell viability, phenotypic maturation and cytokine production were then examined. RESULTS: The challenge of influenza virus and pneumococcus altered dendritic cell functions dependent on the time interval between the successive challenge of influenza virus and pneumococcus, as well as the doses of pneumococcus. When dendritic cells were exposed to pneumococcus at 6 hr, but not 0 hr nor 24 hr after influenza virus infection, both virus and pneumococcus treated dendritic cells had greater cell apoptosis and expressed higher CD83 and CD86 than dendritic cells infected with influenza virus alone. Dendritic cells produced pro-inflammatory cytokines: TNF-α, IL-12 and IFN-γ synergistically to the successive viral and pneumococcal challenge. Whereas prior influenza virus infection suppressed the IL-10 response independent of the timing of the subsequent pneumococcal stimulation. CONCLUSIONS: Our results demonstrated that successive challenge of dendritic cells with influenza virus and pneumococcus resulted in synergistic up-regulation of pro-inflammatory cytokines with simultaneous down-regulation of anti-inflammatory cytokine, which may explain the immuno-pathogenesis of this important co-infection.

Type I and III Interferon Productions Are Impaired in X-Linked Agammaglobulinemia Patients Toward Poliovirus but Not Influenza Virus.

Background: X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) mutation. Patients are susceptible to severe enterovirus infections. The underlying mechanism remains unknown. BTK is involved in toll-like receptors pathway, which initiates antiviral responses including interferon (IFN) productions. Objective: To demonstrate type I and III IFN productions in dendritic cells of XLA patients is decreased in response to oral poliovirus vaccine (OPV) but not H1N1 virus. Methods: Monocyte-derived dendritic cells (MoDCs) were derived from nine XLA patients aged 22-32 years old and 23 buffy coats from Hong Kong Red Cross blood donors. LFM-A13 was used to inhibit BTK. OPV Sabin type 1 and H1N1 influenza virus were used to stimulate MoDCs with RPMI as mock stimulation. The antiviral cytokine productions and phenotypic maturation of MoDCs were determined 24 h post-stimulation. OPV RNA was determined at 0, 6, 12, and 24 h post-stimulation. Results: Upon OPV stimulation, IFN-α2, IFN-ß, and IFN-λ1 productions in MoDCs from XLA patients and BTK-inhibited MoDCs of healthy controls were significantly lower than that from healthy controls. Whereas upon H1N1 stimulation, the IFN-α2, IFN-ß, and IFN-λ1 productions were similar in MoDCs from XLA patients, BTK-inhibited MoDCs of healthy controls and healthy controls. The mean fluorescent intensities (MFI) of CD83, CD86, and MHC-II in MoDCs from XLA patients in response to OPV was similar to that in response to mock stimulation, while the MFI of CD83, CD86, and MHC-II were significantly higher in response to H1N1 stimulation than that in response to mock stimulation. Whereas, the MFI of CD83, CD86, and MHC-II in MoDCs of healthy controls were significantly higher in response to both OPV and H1N1 stimulation compared to that in response to mock stimulation. Conclusion: Production of type I and III IFN in response to OPV was deficient in MoDCs from XLA patients, but was normal in response to H1N1 due to deficient BTK function. Moreover, phenotypic maturation of MoDCs from XLA patients was impaired in response to OPV but not to H1N1. These selective impairments may account for the unique susceptibility of XLA patients toward severe enterovirus infections.

Methylene Homologues of Artemisone: An Unexpected Structure-Activity Relationship and a Possible Implication for the Design of C10-Substituted Artemisinins.

We sought to establish if methylene homologues of artemisone are biologically more active and more stable than artemisone. The analogy is drawn with the conversion of natural O- and N-glycosides into more stable C-glycosides that may possess enhanced biological activities and stabilities. Dihydroartemisinin was converted into 10ß-cyano-10-deoxyartemisinin that was hydrolyzed to the α-primary amide. Reduction of the ß-cyanide and the α-amide provided the respective methylamine epimers that upon treatment with divinyl sulfone gave the ß- and α-methylene homologues, respectively, of artemisone. Surprisingly, the compounds were less active in vitro than artemisone against P. falciparum and displayed no appreciable activity against A549, HCT116, and MCF7 tumor cell lines. This loss in activity may be rationalized in terms of one model for the mechanism of action of artemisinins, namely the cofactor model, wherein the presence of a leaving group at C10 assists in driving hydride transfer from reduced flavin cofactors to the peroxide during perturbation of intracellular redox homeostasis by artemisinins. It is noted that the carba analogue of artemether is less active in vitro than the O-glycoside parent toward P. falciparum, although extrapolation of such activity differences to other artemisinins at this stage is not possible. However, literature data coupled with the leaving group rationale suggest that artemisinins bearing an amino group attached directly to C10 are optimal compounds.

Outcome of Encephalitis in Pediatric Intensive Care Unit.

OBJECTIVE: To review pathogens, morbidity and mortality in pediatric intensive care unit (PICU) patients with viral and infectious encephalitis. METHODS: Retrospective chart review of all patients with encephalitis admitted to the PICU between 2002 and 2014 was done. RESULTS: Encephalitis (n = 46) accounted for 2.7 % of PICU admissions, but 11.8 % PICU mortality over a 12-y period. A microorganism (primarily virus) was identified in 59 % of encephalitis patients in the PICU. Enteroviruses and herpes viruses were isolated from the cerebrospinal fluid (CSF). Respiratory viruses [such as respiratory syncytial virus (RSV) and influenza viruses] and enteric viruses (such as rotavirus and norovirus) were obtained in the nasopharyngeal aspirate and stool respectively, but undetectable from the CSF. More than one-fourth patients with encephalitis died in the PICU. Boys accounted for 85 % of nonsurvivors and 52 % survivors (p = 0.038). Mechanical ventilation, inotrope, intravenous immunoglobulin (IVIG) and corticosteroid usage were significantly higher among non-survivors (p 0.001-0.044). Binomial logistic regression showed that patients who received corticosteroid had a lower chance of survival than those who did not after adjusting for gender, IVIG and mechanical ventilation (adjusted odd ratio = 0.071, 95 % CI 0.006-0.881; p 0.039). Eighteen (55 %) of the survivors had moderate-to-severe neurodevelopmental impairments. CONCLUSIONS: Encephalitis is associated with significant mortality despite intensive care. Over 25 % case died and 55 % of survivors had moderate-to-severe neurodevelopmental impairments. There appeared to be no emerging outbreaks of encephalitis during the 15-y study period.