Indomethacin-associated neutropenia with subsequent Gram-negative sepsis in a preterm infant. Cause or coincidence?

A preterm male infant with a patent ductus arteriosus developed neutropenia during treatment with indomethacin. Afterward, the mother described her own history of indomethacin-associated neutropenia. During the recovery from the neutropenia, the infant became septic with bacteremia caused by Enterobacter cloacae. Although indomethacin-related neutropenia has been described in adults, no case in a neonate has been reported. If neutropenia occurs after indomethacin therapy in a neonate, a familial history of indomethacin-associated neutropenia should be sought and the increased risk of infection should be considered.

Urinary effects of morphine in preterm infants.

AIM: To describe the association between morphine administration in preterm infants, hydronephrosis, and renal dysfunction. METHODS: The findings were based on serial ultrasound examinations and blood studies. RESULTS: Two preterm infants had bladder distension and hydronephrosis after they received intravenous morphine for analgesia. Morphine was used at a low dose. Each patient had a normal urine output and normal serum creatinine before the signs and symptoms of urinary retention were observed. Within 24 h of morphine administration, each infant concurrently developed oliguria and elevation of the serum creatinine. Cessation of morphine and urinary drainage resulted in rapid and complete resolution of the hydronephrosis and the elevated creatinine. CONCLUSION: Morphine, even at low dosages, can be associated with hydronephrosis in hospitalized preterm infants.

HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells.

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.

Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.

Lactoferrin protects neonatal rats from gut-related systemic infection.

Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection. Proof for this concept is limited and was addressed during in vivo and in vitro studies. Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli. Control animals had 1,000-fold more colony-forming units of E. coli per milliliter of blood than treated animals (P < 0.001). Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02). Oral therapy with rh-LF + FeSO(4) did not alter the protective effect. In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages. An in vitro assay showed that rh-LF did not kill E. coli, but a combination of rh-LF + lysozyme was microbicidal. In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF. The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E. coli in vivo.

HIV-1 actively replicates in naive CD4(+) T cells residing within human lymphoid tissues.

Although HIV-1 gene expression is detected in naive, resting T cells in vivo, such cells are resistant to productive infection in vitro. However, we found that the endogenous microenvironment of human lymphoid tissues supports de novo infection and depletion of this population. Cell cycle analysis and DNA labeling experiments established that these cells were definitively quiescent and thus infected de novo. Quantitation of the "burst size" within naive cells further demonstrated that these cells were productively infected and contributed to the local viral burden. These findings demonstrate that lymphoid tissues support active HIV-1 replication in resting, naive T cells. Moreover, these cells are not solely reservoirs of latent virus but are permissive hosts for viral replication that likely targets them for elimination.

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.

A randomized controlled trial of interleukin-1 receptor antagonist in a rabbit model of ascending infection in pregnancy.

OBJECTIVE: To determine whether treatment with interleukin-1 receptor antagonist (IL1-ra) would affect amniotic fluid concentrations of tumor necrosis factor alpha (TNF-alpha) and prostaglandins or clinical or microbiological outcomes in a model of ascending bacterial infection in pregnancy. METHODS: Timed pregnant New Zealand white rabbits at 70% of gestation underwent endoscopic inoculation of the cervices with 10(6) - 10(7) cfu Escherichia coli. Animals were randomly assigned in a blinded manner to a 5-h intravenous infusion of human IL1-ra (10 mg/kg) or placebo beginning 1-2 h after inoculation. Blood was drawn from the does for assay of serum IL1-ra concentration before inoculation, at mid-infusion, after the infusion ended and at necropsy. At necropsy, temperature and cultures were taken, and aspirated amniotic fluid was pooled for assays of TNF-aalpha, prostaglandin E2 (PGE2) and ILI-ra. RESULTS: Serum IL1-ra concentrations rose to a mean of 2 microg/ml at mid-infusion and fell markedly after the infusion to concentrations barely detectable at necropsy. Between the two groups, there were no significant differences in the rates of fever or positive cultures or in amniotic fluid concentrations of PGE2 or TNF-alpha. One unique finding was the demonstration that administration of human IL1-ra to the does resulted in measurable concentrations of human IL1-ra in the amniotic fluid. CONCLUSIONS: Treatment with an intravenous infusion of human IL1-ra after cervical inoculation with E. coli did not affect clinical or microbiological outcomes or amniotic fluid concentrations of TNF-alpha or PGE2. This experiment providesthefirstdemonstration of passage of human IL1-ra from the maternal bloodstream to the amniotic fluid.

Human immunodeficiency virus type 1 Vpr contains two leucine-rich helices that mediate glucocorticoid receptor coactivation independently of its effects on G(2) cell cycle arrest.

Human immunodeficiency virus type 1 (HIV-1) Vpr participates in nuclear targeting of the viral preintegration complex in nondividing cells and induces G(2) cell cycle arrest in proliferating cells, which creates an intracellular milieu favorable for viral replication. Vpr also activates the transcription of several promoters and enhancers by a poorly understood mechanism. Vpr enhances glucocorticoid receptor (GR) signaling and may mediate the effects of steroids on HIV replication. More specifically, recombinant Vpr can potentiate virion production from U937 cells, downregulate NF-kappaB induction, and enhance programmed cell death, all effects also mediated by glucocorticoids. Vpr has been proposed to act as a GR coactivator, although other studies suggest that these enhancing effects are merely a consequence of G(2) cell cycle arrest. We now demonstrate that Vpr functions as a GR coactivator and that this activity is independent of cell cycle arrest. In addition, we show that the Vpr-induced coactivation requires an intact glucocorticoid response element, that it is dependent on the presence of hormone and the corresponding receptor, and that it is mediated by the two highly conserved leucine-rich domains within Vpr that resemble the GR coactivator signature motif.

Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G2 cell cycle arrest.

Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G(2) cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts alpha-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water. (1)H NMR spectroscopy allows the signal assignment of N- and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G(2) cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.

Acute intrauterine infection results in an imbalance between pro- and anti-inflammatory cytokines in the pregnant rabbit.

PROBLEM: Intrauterine infection results in an increase in cytokines. This study compared the time courses for the pro- and anti-inflammatory cytokine responses in 33 pregnant rabbits at 70% gestation. Pro-inflammatory markers were activated nuclear factor-kappa B (NF-kappaB) in placenta and tumor necrosis factor-alpha (TNF-alpha) in amniotic fluid. These were compared to the anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra), in placenta and uterus. METHOD OF STUDY: Does were endoscopically inoculated with Escherichia coli through their cervices and sacrificed at six intervals between 0 and 30 hr post-inoculation. RESULTS: Activated NF-kappaB, determined by electromobility gel shift assay, increased significantly 16 hr after bacterial inoculation (P < or = 0.05). This was directly mirrored by TNF-alpha concentrations, determined by bioassay, in the amniotic fluid. However, IL-1ra levels, determined by enzyme-linked immunosorbent assay, did not increase in response to infection. CONCLUSIONS: Intrauterine infection results in an imbalance between pro- and anti-inflammatory cytokines that may potentiate infection-induced preterm delivery.

Production and excretion of nitrate by human newborn infants: neonates are not little adults.

Endothelium-derived relaxing factor, identified as nitric oxide or its adducts, is metabolized to nitrate and excreted in the urine. Since blood pressures are lower in newborn infants compared to adults, we hypothesized that newborn infants would have increased excretion of nitrate on the day of birth. Neonatal urine was collected before 24 h of age when exogenous intake of nitrate was low. Two different analytical methods showed that nitrate accounted for >99% of nitrogen oxides in urine of healthy neonates and adults. The absolute micromolar concentration of nitrate in urine from infants was significantly below that of adults. When nitrate content was standardized for the reduced renal function in the newborn infant (creatinine content) and body mass (kilogram weight), the concentration of nitrate in neonatal urine was significantly higher than that of adults. Nitrate concentrations in the urine of prematurely born infants were twice that of nitrate measured in urine from term infants. These findings suggested that nitric oxide is produced in larger intravascular quantities in newborn infants versus adults. Thus, we postulated that nitric oxide released from a nitrosothiol would be metabolized to nitrate more readily by neonatal erythrocytes compared to red blood cells obtained from adults. Neonatal erythrocytes, suspended at concentrations of 8, 12, or 16 g per deciliter of hemoglobin, produced 1.7- to 2.1-fold more nitrate than equivalent hemoglobin concentrations of adult erythrocytes that were each incubated with S-nitroso-N-acetylpenicillamine (100 microM) over a 2-h period. Taken together, the studies of urinary nitrate in newborn infants and the ability of neonatal erythrocytes to generate nitrate are consistent with a robust production of nitric oxide immediately after birth.

Transplacental passage of iohexol.

We describe the appearance of an iodinated, low molecular weight radiographic contrast agent, iohexol, in the intestines of twin neonates after administration to a pregnant mother during angiography. Nonionic contrast agents cross the human placenta and enter the fetus in significant concentrations and in this case facilitated identification of an omphalomesenteric duct cyst in one twin.

Induction of nitric oxide synthase in macrophages: inhibition by fructose-1,6-diphosphate.

Intravenous fructose-1,6-diphosphate (FDP) is reported to reverse shock and improves survival in animals given systemic lipopolysaccharide (LPS), although the mechanism is incompletely understood. Since endotoxin-related shock is associated with increased nitric oxide (NO) production, LPS-stimulated macrophages were treated with FDP, and the NO metabolite, nitrite, was measured 24 h later. Treatment of LPS-stimulated macrophages with 1, 5, or 10 mM FDP caused a dose-dependent reduction in mRNA expression for inducible NO synthase by Northern analysis and decreased the micromolar concentrations of nitrite produced by 17, 42, and 68%, respectively. Neither fructose nor sodium phosphate had these effects in LPS-exposed macrophages. Electrophoretic mobility shift assays revealed that FDP did not inhibit LPS-mediated activation of nuclear factor kappa B. Viability analysis showed that the FDP effect was not caused by cytotoxicity. Overall, these results suggest that fructose-1,6-diphosphate, a glycolytic intermediate with potential clinical use, may mitigate the adverse effects of LPS by regulating the generation of NO.

Nitric-oxide-induced reoxygenation injury in the cyanotic immature heart is prevented by controlling oxygen content during initial reoxygenation.

Reintroduction of high levels of molecular oxygen after a hypoxic period is followed by a burst of nitric oxide (NO), peroxynitrite, and oxygen free radicals (OFR), which are highly cytotoxic. This study indicates that hyperoxic reoxygenation of cyanotic immature hearts on cardiopulmonary bypass (CPB) induces a reoxygenation injury and that, by reducing NO and OFR production during institution of CPB with subsequent reoxygenation under blood cardioplegic arrest, this oxygen-related damage can be avoided and biochemical and functional status improved. Of 25 immature piglets (3-5 kg, two to three weeks old), 6 underwent one hour of CPB including thirty minutes of aortic clamping with substrate-enriched modified blood cardioplegia (hypocalcemic, alkalotic, and hyperosmolar; warm induction-cold replenishment-warm reperfusion) without preceding hypoxia (controls). Nineteen others were made hypoxic (arterial [Po2] 20-30 mmHg) for up to two hours by lowering the fraction of inspired oxygen (FIO2) on ventilator. These hypoxic piglets were then reoxygenated on CPB at different Po2 levels (hyperoxic, normoxic, or hypoxic) for five minutes, followed by the aforementioned blood cardioplegic (BCP) arrest regimen. Myocardial conjugated diene (CD) production as a marker of lipid peroxidation, and NO production, determined as its spontaneous oxidation products, nitrite (NO2-) and nitrate (NO3-), were assessed during blood cardioplegic induction, and antioxidant reserve capacity was determined by incubating myocardium in the oxidant t-butylhydroperoxide (t-BHP). Myocardial function was evaluated from end-systolic elastance (Ees, conductance catheter). Blood cardioplegic arrest caused no functional or biochemical changes in normoxic control immature piglets. In contrast, brief reoxygenation at PO2 > 400 mmHg, followed by BCP-arrest (hyperoxic) resulted in marked CD production (42 +/- 4 vs 3 +/- 1 A233 nm/minute/100 g; P < 0.05), and NO production (4500 +/- 500 vs 450 +/- 32 mmol/minute/100 g; P < 0.05) during blood cardioplegic induction, reduced antioxidant reserve capacity (malondialdehyde [MDA] at 4.0 mM of t-BHP: 1342 +/- 59 vs 958 +/- 50 nM/g protein; P < 0.05), and caused profound myocardial dysfunction; Ees recovered only 21 +/- 2% (vs 104 +/- 7; P < 0.05), despite the blood cardioplegic regimen shown to be cardioprotective in control normoxic piglets. Conversely, controlling initial PO2 to normoxic (100 mmHg) or hypoxic (20-30 mmHg) levels reduced lipid peroxidation (CD production 16 +/- 2, 2 +/- 1 A233nm/minute/100 g) and NO production (1264 +/- 736, 270 +/- 182 mmol/minute/100 g), restored antioxidant reserve capacity (MDA at 4.0 mM of t-BHP: 940 +/- 95, 982 +/- 88 nM/g protein), and allowed significant functional recovery (58 +/- 11% and 83 +/- 8%), in a PO2-dependent fashion. The authors conclude that reoxygenation of hypoxemic immature hearts by initiating hyperoxic CPB causes oxidant-related damage characterized by lipid peroxidation, enhanced NO production, and reduced antioxidants, leading to functional depression that nullifies the cardioprotective effects of blood cardioplegia. These detrimental effects can be reduced in a PO2-dependent fashion by controlling initial PO2 on CPB and subsequent reoxygenation during blood cardioplegic arrest.

Transmission of human T cell leukemia virus type I to sheep: antibody profile and detection of viral DNA sequences.

Lambs were inoculated intraperitoneally with either 1.8 x 10(7) live peripheral blood cells from an HTLV-I-infected person (five lambs) or with 8 x 10(7) live cells from the HTLV-I-producing cell lines MT-2 (four lambs) or C10 MJ (five lambs). Four control lambs were inoculated with minimal essential medium supplemented with fetal calf serum. The animals were monitored during a period of 24 months. Beginning at 5 to 12 months after inoculation, four of the five lambs inoculated with the fresh HTLV-I-infected peripheral blood cells began to develop detectable levels of antibodies to a recombinant HTLV-I gp21env antigen, as determined by an enzyme-linked immunoassay (ELISA). The anti-gp21 antibodies persisted for the remaining observation period. These antibodies were not detected in the sera from the other sheep. Absorption and blocking experiments demonstrated the specificity of the gp21 reactivity. This reactivity was also confirmed by Western blot (WB). With the exception of the serum of an MT-2-inoculated sheep that formed a weak band with p19 by WB, none of the sera of the four gp21-positive sheep or of the other experimental sheep reacted with other structural or regulatory HTLV-I proteins, as determined by ELISA, WB, and radioimmunoassay. PCR analyses demonstrated the presence of the HTLV-I provirus in peripheral blood leukocytes of the four sheep showing antibodies to gp21env. The remaining sheep were negative. PCR analyses failed to detect BLV sequences in any of the experimental sheep. None of the sheep showed clinical abnormalities during the observation period. The potential value of the sheep model for studying atypical virus-host interactions in infected people is discussed.

Pulmonary vasoconstriction due to impaired nitric oxide production after cardiopulmonary bypass.

BACKGROUND: Pulmonary hypertension is a serious complication after cardiopulmonary bypass (CPB). This study tests the hypothesis that CPB provokes oxidant-mediated pulmonary endothelial dysfunction, leading to reduced nitric oxide (NO) production and pulmonary vasoconstriction. METHODS: Twelve piglets underwent 2 hours of CPB. In 6 of them, CPB prime was supplemented with N-mercaptopropionylglycine and catalase, whereas the others were not treated. Left and right ventricular function were evaluated from end-systolic elastance and Starling analysis. Pulmonary vascular resistance and transpulmonary NO production (measuring NO2-, NO3-) were determined to assess pulmonary endothelial function. RESULTS: Cardiopulmonary bypass caused a significant increase in pulmonary vascular resistance (83 +/- 12 to 212 +/- 30 dynes.cm-5.s kg-1, p < 0.05), associated with a reduction of NO production (8.8 +/- 1.4 to 2.5 +/- 0.5 mumol/min, p < 0.05) and depressed right ventricular function (56 +/- 12% of control), whereas N-mercaptopropionylglycine and catalase added to the CPB allowed a substantial improvement of these deleterious effects of CPB. CONCLUSIONS: Cardiopulmonary bypass impairs pulmonary NO production, resulting in pulmonary vasoconstriction and right ventricular dysfunction, which can be reduced by antioxidants. These findings imply the validity of NO inhalation therapy for postoperative pulmonary hypertension as a supplementation of endogenous endothelium-derived relaxing factor.