Karen Li

Proinflammatory and anti-inflammatory cytokine responses in preterm infants with systemic infections.

Objective: A prospective study to investigate the pattern of proinflammatory and anti-inflammatory cytokine responses in preterm infants with systemic infection. Methods: Very low birthweight infants in whom infection was suspected when they were > 72 hours of age were eligible. A full sepsis screen was performed in each episode. Key cytokines of both proinflammatory and anti-inflammatory pathways, including interleukin (IL) 2, IL4, IL5, IL6, IL10, interferon (IFN) γ, and tumour necrosis factor (TNF) α, were measured at 0 (at the time of sepsis evaluation), 24, and 48 hours by flow cytometric analysis or immunoassay. Results: Thirty seven of the 127 episodes of suspected clinical sepsis were proven infection or necrotising enterocolitis. Both proinflammatory (IL2, IL6, IFNγ, TNFα) and anti-inflammatory (IL4, IL10) cytokines were significantly increased in infected infants compared with non-infected infants. Significant correlations were observed between IL6 and TNFα or IL10 as well as IL10 and IFNγ in infected infants. In the subgroup analysis, plasma IL6, IL10, and TNFα concentrations, and IL10/TNFα and IL6/IL10 ratios were significantly elevated in patients with disseminated intravascular coagulation compared with infected infants without. The IL10/TNFα ratios had decreased significantly 48 hours after the onset, whereas the IL6/IL10 ratio showed only a non-significant decreasing trend. Further, the IL6/IL10 ratio in the deceased infant was disproportionally increased at presentation and continued to increase despite treatment. Conclusion: The results indicate that the counter-regulatory mechanism between the proinflammatory and anti-inflammatory cytokine pathways is probably operational in preterm infants of early gestation. High plasma IL6, IL10, and TNFα concentrations, and IL10/TNFα and IL6/IL10 ratios signify severe infection, but transiently elevated plasma IL10 concentration or IL10/TNFα ratio does not necessarily indicate a poor prognosis.

Neutrophil CD64 expression: A sensitive diagnostic marker for late-onset nosocomial infection in very low birthweight infants

This study aims to evaluate the diagnostic utilities of four leukocyte surface antigens—two lymphocyte antigens (CD25 and CD45RO) and two neutrophil antigens (CD11b and CD64)—for identification of late-onset nosocomial bacterial infection in preterm, very low birthweight infants, and to define the optimal cutoff value for each marker so that it may act as a reference with which future studies can be compared. Very low birthweight infants in whom infection was suspected when they were >72 h of age were eligible for the study. A full sepsis screen was performed in each episode. IL-6, C-reactive protein, and leukocyte surface antigens (CD25, CD45RO, CD11b, and CD64) were measured at 0 (at the time of sepsis evaluation), 24, and 48 h by standard biochemical methods and quantitative flow cytometric analysis. The diagnostic utilities including sensitivity, specificity, and positive and negative predictive values of each marker and combination of markers for predicting late-onset neonatal infection were determined. One hundred twenty-seven episodes of suspected clinical sepsis were investigated in 80 infants. Thirty-seven episodes were proven infection. The calculated optimal cutoff values for CD25, CD45RO, CD11b, and CD64 were 3,100, 2,900, 10,450, and 4,000 phycoerythrin-molecules bound per cell, respectively. An interim analysis of data after 68 episodes suggested that CD25 and CD45RO were poor predictors of neonatal infection with sensitivity or specificity <75% during a single measurement. Thus, these two markers were excluded from further investigation. In the final analysis, CD64 has the highest sensitivity (95–97%) and negative predictive value (97–99%) at 0 and 24 h after the onset. The addition of IL-6 or C-reactive protein (0 h) to CD64 (24 h) further enhanced the sensitivity and negative predictive value to 100%, and has the specificity and positive predictive value exceeding 88% and 80%, respectively. Neutrophil CD64 expression is a very sensitive marker for diagnosing late-onset nosocomial infection in very low birthweight infants. If further validated, the use of CD64 as an infection marker should allow early discontinuation of antibiotic treatment at 24 h without waiting for the definitive microbiologic culture results. The quantitative flow cytometric analysis applied in this study could be developed into a routine clinical test with high comparability and reproducibility across different laboratories.

Neutrophil CD64 is a sensitive diagnostic marker for early-onset neonatal infection

This prospective study aimed to evaluate the diagnostic utilities of neutrophil CD64 expression for the identification of early-onset clinical infection and pneumonia in term infants and to define the optimal cutoff value so that it may act as a reference with which future studies can be compared. Term newborns in whom infection was suspected when they were <72 h of age were recruited into the study. C-reactive protein (CRP) and expression of CD64 on neutrophils were measured at 0 h (at the time of sepsis evaluation) and 24 h. The sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of CRP, CD64, and the combination of these two markers for predicting neonatal sepsis were determined. A total of 338 infants with suspected clinical sepsis were investigated, 115 of whom were found to be clinically infected. CRP and CD64 in infected infants were both significantly elevated at 0 and 24 h compared with noninfected infants (p < 0.001). The calculated optimal cutoff value for CD64 was 6136 antibody-phycoerythrin molecules bound/cell. CD64 has a very high sensitivity (96%) and NPV (97%) at 24 h. The addition of CRP only marginally enhanced the sensitivity and NPV (97 and 98%, respectively). In conclusion, neutrophil CD64 is a very sensitive diagnostic marker for the identification of early-onset clinical infection and pneumonia in term newborns. The results strongly suggest that measurement of neutrophil CD64 may allow neonatal clinicians to discontinue antibiotic treatment at 24 h in infants who are clinically stable and whose CD64 expressions are below the optimal cutoff level.

Thrombopoietin Protects Against In Vitro and In Vivo Cardiotoxicity Induced by Doxorubicin

Background— Doxorubicin (DOX) is an important antineoplastic agent. However, the associated cardiotoxicity, possibly mediated by the production of reactive oxygen species, has remained a significant and dose-limiting clinical problem. Our hypothesis is that the hematopoietic/megakaryocytopoietic growth factor thrombopoietin (TPO) protects against DOX-induced cardiotoxicity and might involve antiapoptotic mechanism exerted on cardiomyocytes. Methods and Results— In vitro investigations on H9C2 cell line and spontaneously beating cells of primary, neonatal rat ventricle, as well as an in vivo study in a mouse model of DOX-induced acute cardiomyopathy, were performed. Our results showed that pretreatment with TPO significantly increased viability of DOX-injured H9C2 cells and beating rates of neonatal myocytes, with effects similar to those of dexrazoxane, a clinically approved cardiac protective agent. TPO ameliorated DOX-induced apoptosis of H9C2 cells as demonstrated by assays of annexin V, active caspase-3, and mitochondrial membrane potential. In the mouse model, administration of TPO (12.5 &mgr;g/kg IP for 3 alternate days) significantly reduced DOX-induced (20 mg/kg) cardiotoxicity, including low blood cell count, cardiomyocyte lesions (apoptosis, vacuolization, and myofibrillar loss), and animal mortality. Using Doppler echocardiography, we observed increased heart rate, fractional shortening, and cardiac output in animals pretreated with TPO compared with those receiving DOX alone. Conclusions— These data have provided the first evidence that TPO is a protective agent against DOX-induced cardiac injury. We propose to further explore an integrated program, incorporating TPO with other protocols, for treatment of DOX-induced cardiotoxicity and other forms of cardiomyopathy.

Host-response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants

Preterm infants are highly susceptible to life-threatening infections that are clinically difficult to detect, such as late-onset septicemia and necrotizing enterocolitis (NEC). Here, we used a proteomic approach to identify biomarkers for diagnosis of these devastating conditions. In a case-control study comprising 77 sepsis/NEC and 77 nonsepsis cases (10 in each group being monitored longitudinally), plasma samples collected at clinical presentation were assessed in the biomarker discovery and independent validation phases. We validated the discovered biomarkers in a prospective cohort study with 104 consecutively suspected sepsis/NEC episodes. Proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid A (SAA) were identified as the most promising biomarkers. The ApoSAA score computed from plasma apoC2 and SAA concentrations was effective in identifying sepsis/NEC cases in the case-control and cohort studies. Stratification of infants into different risk categories by the ApoSAA score enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of nonsepsis infants. The negative predictive value of this antibiotic policy was 100%. The ApoSAA score could potentially allow early and accurate diagnosis of sepsis/NEC. Upon confirmation by further multicenter trials, the score would facilitate rational prescription of antibiotics and target infants who require urgent treatment.

IP-10 is an early diagnostic marker for identification of late-onset bacterial infection in preterm infants

Very low birth weight (VLBW) infants with suspected late-onset infection requiring sepsis screening were enrolled in a prospective study to evaluate the diagnostic utilities of a comprehensive panel of key chemokines and cytokines, both individually and in combination, to identify diagnostic markers for early recognition of bacterial sepsis and necrotizing enterocolitis (NEC). Plasma chemokines interleukin (IL)-8, interferon-γ–inducible protein 10 (IP-10), monokine induced by interferon-γ (MIG), monocyte chemoattractant protein 1 (MCP-1), growth-related oncogene-α (GRO-α), and regulated upon activation of normal T cell expressed and secreted (RANTES) and cytokines IL-1β, IL-6, IL-10, IL-12p70, and tumor necrosis factor α (TNF-α) were measured at the onset of sepsis (0 h) and 24 h later. Of 155 suspected infection episodes, 44 were classified as infected. Concentrations of all studied inflammatory mediators (except IL-1β and RANTES) were significantly higher in the infected than in the noninfected group at 0 h, but the levels decreased precipitously by 24 h. IP-10 with a plasma cutoff concentration ≥1250 pg/mL could identify all septicemic and NEC cases and had the highest overall sensitivity (93%) and specificity (89%) at 0 h. We conclude that preterm infants have the ability to induce a robust chemokine and cytokine response during sepsis, and IP-10 is a sensitive early marker of infection.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: Implication in the migration

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC‐ and P‐MSC possess immunophenotypic and functional characteristics similar to BM‐MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM‐ and P‐MSC was found 5.9‐ and 3.2‐folds higher than that of UC‐MSC, respectively. By the use of 2‐DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC‐MSC when compared with those in BM‐ and P‐MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor‐1 (PAI‐1) and manganese superoxide dismutase, higher expression was found in the UC‐MSC. We also showed that the overexpression of the PAI‐1 impaired the migration capacity of BM‐ and P‐MSC while silencing of PAI‐1 enhanced the migration capacity of UC‐MSC. Our study indicates that PAI‐1 and other migration‐related proteins are pivotal in governing the migration capacity of MSC.

Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice.

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This studys purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum‐free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.

Promoting effects of serotonin on hematopoiesis : Ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5‐hydroxytryptamine (5‐HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony‐forming unit‐fibroblast (CFU‐F) formation, and antiapoptosis of megakaryoblastic M‐07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony‐forming unit‐mixed [CFU‐GEMM]) and multilineage committed progenitors (burst‐forming unit/colony‐forming unit‐erythroid [BFU/CFU‐E], colony‐forming unit‐granulocyte macrophage, colony‐forming unit‐megakaryocyte, CD61+CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU‐E, and CFU‐GEMM engraftment in BM of animals 6 weeks post‐transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet‐derived growth factor, or vascular endothelial growth factor stimulated BM CFU‐F formation. In M‐07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase‐3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5‐HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation.

Partial neuroprotective effect of pretreatment with tanshinone IIA on neonatal hypoxia-ischemia brain damage.

Tanshinone IIA is a compound purified from the Chinese herb Danshen (Radix Salviae Miltiorrhiza Bge). The neuroprotective effect of tanshinone IIA was investigated in a neonatal rat model of hypoxia-ischemia brain damage. Hypoxia-ischemia encephalopathy was induced in rats at day 7 of postnatal age by ligation of the right common carotid artery, followed by 2 h of hypoxia. Tanshinone IIA (10 mg/kg, i.p.) was injected daily from day 2 before surgery for 9 or 16 d. Our results demonstrated significant and sustained brain damage in the hypoxia-ischemia– and vehicle-treated groups at 1 and 3 wk after surgery. Treatment with tanshinone IIA significantly reduced the severity of brain injury, as indicated by the increase in ipsilateral brain weight and neuron density, compared with those of sham-operated animals. The recovery of sensorimotor function and histology was observed in animals that received tanshinone IIA. The plasma of tanshinone IIA–treated rats exhibited higher antioxidant activities, as reflected by the oxygen radical absorbance capacity assay, compared with the vehicle-treated rats. In the neural progenitor cell line C17.2 that was subjected to 2,2′-azobis (2-amidino propane hydrochloride)–induced oxidative stress, tanshinone IIA increased cell viability and protected against mitochondrial damage (JC-1 assay). Our results suggest that tanshinone IIA has antioxidative activities and that treatment that is started before a hypoxic-ischemic insult is partially neuroprotective. Further studies are required to elucidate whether rescue treatment with tanshinone IIA is effective and to determine whether its protective effect is also associated with secondary cooling of the brain.