Introduction to Personalized Medicine in Pediatrics.

Exciting new developments in biomedical and computational sciences provide an extraordinary and unparalleled opportunity to compile, connect, and analyze multiple types of "big data," driving the development of personalized medicine. These insights must begin in early life (ie, pregnancy, neonatal, and infancy) and focus on early prevention, diagnosis, and intervention-areas of medicine where pediatricians are poised to lead the way to a personalized medicine future. The rapid growth of genomics (including pharmacogenomics), transcriptomics, and related "omics" has revolutionized the diagnosis of rare monogenic disorders. It is now clarifying the pathogenesis of complex conditions ranging from autism spectrum disorder to asthma. Collaborations between clinicians and basic scientists integrating multiomics approaches in evaluating children with severe illness are transforming the fields of perinatal, neonatal, and pediatric critical care medicine. Improvements in rapid diagnostic and prognostic information suggest that pediatric personalized medicine is under way and has an exciting future. [Pediatr Ann. 2022;51(10):e381-e386.].

High-Density Blood Transcriptomics Reveals Precision Immune Signatures of SARS-CoV-2 Infection in Hospitalized Individuals.

The immune response to COVID-19 infection is variable. How COVID-19 influences clinical outcomes in hospitalized patients needs to be understood through readily obtainable biological materials, such as blood. We hypothesized that a high-density analysis of host (and pathogen) blood RNA in hospitalized patients with SARS-CoV-2 would provide mechanistic insights into the heterogeneity of response amongst COVID-19 patients when combined with advanced multidimensional bioinformatics for RNA. We enrolled 36 hospitalized COVID-19 patients (11 died) and 15 controls, collecting 74 blood PAXgene RNA tubes at multiple timepoints, one early and in 23 patients after treatment with various therapies. Total RNAseq was performed at high-density, with >160 million paired-end, 150 base pair reads per sample, representing the most sequenced bases per sample for any publicly deposited blood PAXgene tube study. There are 770 genes significantly altered in the blood of COVID-19 patients associated with antiviral defense, mitotic cell cycle, type I interferon signaling, and severe viral infections. Immune genes activated include those associated with neutrophil mechanisms, secretory granules, and neutrophil extracellular traps (NETs), along with decreased gene expression in lymphocytes and clonal expansion of the acquired immune response. Therapies such as convalescent serum and dexamethasone reduced many of the blood expression signatures of COVID-19. Severely ill or deceased patients are marked by various secondary infections, unique gene patterns, dysregulated innate response, and peripheral organ damage not otherwise found in the cohort. High-density transcriptomic data offers shared gene expression signatures, providing unique insights into the immune system and individualized signatures of patients that could be used to understand the patient's clinical condition. Whole blood transcriptomics provides patient-level insights for immune activation, immune repertoire, and secondary infections that can further guide precision treatment.

Skull osteomyelitis as a rare complication of cat scratch disease.

Bartonella henselae, the causative agent of cat scratch disease (CSD), is one of the most common causes of regional lymphadenitis in children. Other less common manifestations of B. henselae infection including fever of unknown origin, neuroretinitis, and osteomyelitis are being increasingly recognized. We describe a 3-year-old female with a recent history of typical CSD involving lymph nodes who developed osteomyelitis of the skull, a very rarely recognized complication of this infection.

Group B Streptococci Induce Proinflammatory Responses via a Protein Kinase D1-Dependent Pathway.

Group B streptococci (GBS) are one of the leading causes of life-threatening illness in neonates. Proinflammatory responses to GBS mediated through host innate immune receptors play a critical role in the disease manifestation. However, the mechanisms involved in proinflammatory responses against GBS, as well as the contribution of signaling modulators involved in host immune defense, have not been fully elucidated. In the present study, we investigated the role of protein kinase D (PKD)1 in the proinflammatory responses to GBS. We found that both live and antibiotic-killed GBS induce activation of PKD1 through a pathway that is dependent on the TLR signaling adaptor MyD88 and its downstream kinase IL-1R-associated kinase 1, but independent of TNFR-associated factor 6. Our studies using pharmacological PKD inhibitors and PKD1-knockdown macrophages revealed that PKD1 is indispensable for GBS-mediated activation of MAPKs and NF-κB and subsequent expression of proinflammatory mediators. Furthermore, systemic administration of a PKD inhibitor protects d-galactosamine-sensitized mice from shock-mediated death caused by antibiotic-killed GBS. These findings imply that PKD1 plays a critical regulatory role in GBS-induced proinflammatory reactions and sepsis, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates could be an effective way to control GBS diseases.

Limitations of beta-lactam therapy for infections caused by susceptible Gram-positive bacteria.

Penicillin and related beta-lactam agents have been the most widely used and most important antimicrobials in medical history, and remain the recommended therapy for many infectious diseases 85 years after the discovery of penicillin by Alexander Fleming. Yet the efficacy of these agents has been undermined by two factors - the emergence of clinically significant resistance to the antimicrobial activity of these agents, and clinical situations in which these drugs may be suboptimal (even though the bacterial pathogens are not "resistant" to the drugs). Observations in experimental infection models in animals (group A streptococcal myositis, pneumococcal meningitis and pneumonia, group B streptococcal sepsis) and in some cases clinical studies suggest that monotherapy with beta-lactam antibiotics may be inferior to treatment with other types of antibiotics, alone or in combination with beta-lactams - even in situations where the bacterial pathogens remain fully "susceptible" to beta-lactams in vitro.

Azithromycin Inhibits Macrophage Tumor Necrosis Factor Secretion in Response to Both Azithromycin-Susceptible and Azithromycin-Resistant Pneumococci.

We studied the effect of azithromycin (AZM) on macrophage responses to pneumococci. We found that exposure of pneumococci to AZM led to reduced tumor necrosis factor (TNF) secretion by macrophages; this effect was observed in response to both AZM-susceptible and AZM-resistant (AZM-R) pneumococci.

Rapamycin Augments the NMDA-Mediated TNF Suppression of MRSA-Stimulated RAW264.7 Murine Macrophages.

Background. Methicillin-resistant Staphylococcus aureus (MRSA) can stimulate massive cytokine release. Ketamine suppresses tumor necrosis factor (TNF) secretion by MRSA-stimulated RAW264.7 macrophages, and the mechanism likely involves N-methyl-D-aspartic acid (NMDA) receptor antagonism. The downstream effects of NMDA-mediated TNF suppression, specifically the PI3K/Akt and mTOR modulation, have not been described. Methods. RAW264.7 cells were stimulated for 18 hrs with 10(5) to 10(7) CFU/mL inocula of either of two prototypical community-acquired- (CA-) MRSA isolates, USA300 strain LAC and USA400 strain MW2. Then we added the NMDA inhibitors ketamine or 2R-amino-5-phosphonopentanoate (AP5), NMDA substrate, LY294002, and rapamycin in various combinations. Results. NMDA inhibition suppressed TNF secretion by almost a third compared to the no-ketamine control. When NMDA substrate was added, the TNF secretion increased by 10%. Addition of LY294002 suppressed TNF production by macrophages by 20%. Rapamycin exhibited a concentration-dependent TNF induction-suppression response: induction at doses of 0.1 and 1 ng/mL and suppression at 10 and 100 ng/mL. Induction of TNF was abolished when LY294002 was added and the suppression became uniform. Ketamine-induced suppression of TNF secretion was intensified 10-15% when rapamycin was added, but not when LY294002 was added. Conclusion. These findings suggest that NMDA-induced TNF suppression can be augmented by concurrent mTOR inhibition.

Role of bacterial components in macrophage activation by the LAC and MW2 strains of community-associated, methicillin-resistant Staphylococcus aureus.

We tested the contribution of four staphylococcal components - PSM-α, PSM-ß, δ-toxin, and PVL - in triggering macrophage secretion of tumor necrosis factor (TNF) and interleukins 6 (IL-6) and 12 (IL-12) by two prominent, circulating strains of community-associated, methicillin-resistant Staphylococcus aureus (CA-MRSA): LAC, USA300; MW2, USA400. RAW 264.7 murine macrophages were stimulated with live, antibiotic-exposed bacteria, and cytokine secretion was quantitated in supernatants. Deletion of PSM-α expression in LAC led to >50% reduction in macrophage TNF and IL-6 secretion and a 20% reduction in IL-12 secretion, while PSM-α deletion in MW2 did not significantly reduce macrophage TNF secretion but resulted in a 15-20% reduction in IL-6 and IL-12 secretion. Deletion of δ-toxin in either strain led to more than 50% reduction in macrophage IL-6 secretion and smaller reductions in macrophage TNF and IL-12 secretion (8-25%). Our data implicate both PSM-α and δ-toxin in stimulating macrophage cytokine responses to CA-MRSA bacteria.

Ketamine inhibits tumor necrosis factor secretion by RAW264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant Staphylococcus aureus.

BACKGROUND: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA. RESULTS: RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion. CONCLUSIONS: Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.

Nitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells.

Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing L-arginine (L-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM L-leucine [L-leu; a competitive inhibitor of L-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by L-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with L-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by L-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with L-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with L-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular L-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.

Treatment with protein synthesis inhibitors improves outcomes of secondary bacterial pneumonia after influenza.

Pneumonia occurring as a secondary infection after influenza is a major cause of excess morbidity and mortality, despite the availability and use of antibiotics active against Streptococcus pneumoniae. We hypothesized that the use of a bacteriostatic protein synthesis inhibitor would improve outcomes by reducing the inflammatory response. BALB/cJ mice infected with influenza virus and superinfected with S. pneumoniae were treated with either the cell-wall-active antibiotic ampicillin or the protein synthesis inhibitor clindamycin or azithromycin. In the model, ampicillin therapy performed significantly worse (survival rate, 56%) than (1) clindamycin therapy used either alone (82%) or in combination with ampicillin (80%) and (2) azithromycin (92%). Improved survival appeared to be mediated by decreased inflammation manifested as lower levels of inflammatory cells and proinflammatory cytokines in the lungs and by observation of less-severe histopathologic findings. These data suggest that beta-lactam therapy may not be optimal as a first-line treatment for community-acquired pneumonia when it follows influenza.

Role of bacterial DNA in macrophage activation by group B streptococci.

Bacterial DNA (CpG DNA) induces macrophage activation and the production of inflammatory mediators, including tumor necrosis factor (TNF) and nitric oxide (NO) by these cells. However, the role of bacterial DNA in the macrophage response to whole bacteria is unknown. We used overlapping strategies to estimate the relative contribution of bacterial DNA to the upregulation of TNF and NO production in macrophages stimulated with antibiotic-treated group B streptococci (GBS). Selective inhibitors of the bacterial DNA/TLR9 pathway (chloroquine, an inhibitory oligonucleotide, and DNase I) consistently inhibited GBS-induced TNF secretion by 35-50% in RAW 264.7 macrophages and murine splenic macrophages, but had no effect on inducible nitric oxide synthase (iNOS) accumulation or NO secretion. Similarly, splenic and peritoneal macrophages from mice lacking TLR9 expression secreted 40% less TNF than macrophages from control mice after GBS challenge but accumulated comparable amounts of iNOS protein. Finally, studies in both RAW 264.7 cells and macrophages from TLR9-/- mice implicated GBS DNA in the upregulation of interleukins 6 (IL-6) and 12 (IL-12) but not interferon-beta (IFNbeta), a key intermediary in macrophage production of iNOS/NO. Our data suggest that the bacterial DNA/TLR9 pathway plays an important role in stimulating TNF rather than NO production in macrophages exposed to antibiotic-treated GBS, and that TLR9-independent upregulation of IFNbeta production by whole GBS may account for this difference.

Improving therapeutic strategies for secondary bacterial pneumonia following influenza.

Secondary bacterial pneumonia following influenza is an old problem, which is re-emerging. Despite rapid advances in our armamentarium of antimicrobials, the case-fatality rate for this frequent complication of influenza remains high. In some settings, common treatment options may actually contribute to poor outcomes, as rapid lysis of pathogenic bacteria on the backdrop of an activated immune system responding to influenza may lead to inflammatory damage in the lung. An understanding of the inter-related contributions of the antecedent viral infection, the invading bacteria and the host immune response is necessary to formulate an appropriate therapeutic approach. Prevention and resolution of these fulminant infections will require new approaches, including alternate treatment strategies, combination therapies targeting several aspects of the pathogenic process and, potentially, immunomodulation. In the not-so-distant future, strategies aimed at disarming pathogens without eliminating them may be more effective than our current treatment paradigms.

Cytokine-induced arginase activity in pulmonary endothelial cells is dependent on Src family tyrosine kinase activity.

We hypothesized that the Src family tyrosine kinases (STKs) are involved in the upregulation of arginase and inducible nitric oxide synthase (iNOS) expression in response to inflammatory stimuli in pulmonary endothelial cells. Treatment of bovine pulmonary arterial endothelial cells (bPAEC) with lipopolysaccharide and tumor necrosis factor-alpha (L/T) resulted in increased urea and nitric oxide (NO) production, and this increase in urea and NO production was inhibited by the STK inhibitor PP1 (10 microM). The STK inhibitors PP2 (10 microM) and herbimycin A (10 microM) also prevented the L/T-induced expression of both arginase II and iNOS mRNA in bPAEC. Together, the data demonstrate a central role of STK in the upregulation of both arginase II and iNOS in bPAEC in response to L/T treatment. To identify the specific kinase(s) required for the induction of urea and NO production, we studied human pulmonary microvascular endothelial cells (hPMVEC) so that short interfering RNA (siRNA) techniques could be employed. We found that hPMVEC express Fyn, Yes, c-Src, Lyn, and Blk and that the protein expression of Fyn, Yes, c-Src, and Lyn could be inhibited with specific siRNA. The siRNA targeting Fyn prevented the cytokine-induced increase in urea and NO production, whereas siRNAs specifically targeting Yes, c-Src, and Lyn had no appreciable effect on cytokine-induced urea and NO production. These findings support our hypothesis that inflammatory stimuli lead to increased urea and NO production through a STK-mediated pathway. Furthermore, these results indicate that the STK Fyn plays a critical role in this process.

Gastroenteritis caused by Edwardsiella tarda in a pediatric renal transplant recipient.

Edwardsiella tarda, a member of the family Enterobacteriaceae, is a Gram-negative bacillus that is most often pathogenic in aquatic environments. Human infections with Edwardsiella are rare, with most occurring in immunocompromised or immunosuppressed hosts. Reported infections include meningitis, cholecystitis, endocarditis, osteomyelitis, soft tissue infections, bacteremia and septicemia, dysentery, and gastroenteritis. This report describes a case of E. tarda gastroenteritis in a renal transplant patient receiving immunosuppressive therapy. The epidemiology, diagnosis, clinical presentation, and treatment options pertaining to E. tarda infections are examined.

Knockout of Mkp-1 enhances the host inflammatory responses to gram-positive bacteria.

MAPK phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAPK. We have previously demonstrated that MKP-1 acts as a negative regulator of p38 and JNK in immortalized macrophages after stimulation with peptidoglycan isolated from Gram-positive bacteria. To define the physiological function of MKP-1 during Gram-positive bacterial infection, we studied the innate immune responses to Gram-positive bacteria using Mkp-1 knockout (KO) mice. We found that Mkp-1(-/-) macrophages exhibited prolonged activation of p38 and JNK, but not of ERK, following exposure to either peptidoglycan or lipoteichoic acid. Compared with wild-type (WT) macrophages, Mkp-1(-/-) macrophages produced more proinflammatory cytokines such as TNF-alpha and IL-6. Moreover, after challenge with peptidoglycan, lipoteichoic acid, live or heat-killed Staphylococcus aureus bacteria, Mkp-1 KO mice also mounted a more robust production of cytokines and chemokines, including TNF-alpha, IL-6, IL-10, and MIP-1alpha, than did WT mice. Accordingly, Mkp-1 KO mice also exhibited greater NO production, more robust neutrophil infiltration, and more severe organ damage than did WT mice. Surprisingly, WT and Mkp-1 KO mice exhibited no significant difference in either bacterial load or survival rates when infected with live S. aureus. However, in response to challenge with heat-killed S. aureus, Mkp-1 KO mice exhibited a substantially higher mortality rate compared with WT mice. Our studies indicate that MKP-1 plays a critical role in the inflammatory response to Gram-positive bacterial infection. MKP-1 serves to limit the inflammatory reaction by inactivating JNK and p38, thus preventing multiorgan failure caused by exaggerated inflammatory responses.

MKP-1 switches arginine metabolism from nitric oxide synthase to arginase following endotoxin challenge.

L-Arginine (L-arg) is metabolized to nitric oxide (NO) by inducible NO synthase (iNOS) or to urea and L-ornithine (L-orn) by arginase. NO is involved in the inflammatory response, whereas arginase is the first step in polyamine and proline synthesis necessary for tissue repair and wound healing. Mitogen-activated protein kinases (MAPK) mediate LPS-induced iNOS expression, and MAPK phosphatase-1 (MKP-1) plays a crucial role in limiting MAPK signaling in macrophages. We hypothesized that MKP-1, by attenuating iNOS expression, acts as a switch changing L-arg metabolism from NO production to L-orn production after endotoxin administration. To test this hypothesis, we performed studies in RAW264.7 macrophages stably transfected with an MKP-1 expression vector in thioglyollate-elicited peritoneal macrophages harvested from wild-type and Mkp-1(-/-) mice, as well as in vivo in wild-type and Mkp-1(-/-) mice. We found that overexpression of MKP-1 resulted in lower iNOS expression and NO production but greater urea production in response to LPS. Although deficiency of MKP-1 resulted in greater iNOS expression and NO production and lower urea production in response to LPS, neither the overexpression nor the deficiency of MKP-1 had any substantial effect on the expression of the arginases.

Gene expression profiling of the response of Streptococcus pneumoniae to penicillin.

OBJECTIVES: The aim of this study was to identify changes in the gene expression profile of Streptococcus pneumoniae in response to a subinhibitory concentration of penicillin in an effort to better understand mechanisms by which this organism copes with this stress. METHODS: S. pneumoniae serotype 2 strain D39 was grown for 1 h in the presence or absence of penicillin at a concentration equivalent to half the MIC (0.03 mg/L). RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time RT-PCR. RESULTS: A total of 386 genes were found to be responsive to penicillin. Up-regulated genes included those of the ciaR-ciaH operon, luxS, genes encoding cell envelope proteins and genes of the pst locus. Down-regulated genes included genes involved in competence, genes encoding capsular polysaccharide biosynthesis proteins, genes involved in fatty acid chain elongation and genes of the polyamine transporter operon. CONCLUSIONS: Altered expression of these genes reflects a protective response to perturbation of the bacterial cell wall by penicillin. Such genes may represent potential therapeutic targets for enhancing the activity of penicillin against this organism and provide insight into novel mechanisms of penicillin resistance.