SARS-CoV-2 RNA positive pediatric organ donors: A case report.

BACKGROUND: Preliminary evidence suggests that non-lung organ donation from resolved, asymptomatic or mildly symptomatic SARS-CoV-2 infected adults may be safe. However, several biological aspects of SARS-CoV-2 infection differ in children and the risk for transmission and outcomes of recipients from pediatric donors with SARS-CoV-2 infection are not well described. METHODS: We report two unvaccinated asymptomatic pediatric non-lung organ deceased donors who tested positive for SARS-CoV-2 RNA by RT-PCR. Donor One unexpectedly had SARS-CoV-2 RNA detected in nasopharyngeal swab and plasma specimens at autopsy despite several negative tests (upper and lower respiratory tract) in the days prior to organ recovery. Donor Two had SARS-CoV- 2 RNA detected in multiple nasopharyngeal swabs but not lower respiratory tract specimens (endotracheal aspirate and bronchoalveolar lavage) during routine surveillance prior to organ recovery and was managed with remdesivir and monoclonal antibodies prior to organ recovery. RESULTS: Two hearts, two livers and four kidneys were successfully transplanted into seven recipients. No donor to recipient transmission of SARS-CoV-2 was observed and graft function of all organs has remained excellent for up to 7 months of followup. CONCLUSIONS: Due to the persistent gap between organ availability and the number of children waiting for transplants, deceased pediatric patients with non-disseminated SARS-CoV-2 infection, isolated to upper and/or lower respiratory tract, should be considered as potential non-lung organ donors.

Age-Based Percentiles of Measured Mean Arterial Pressure in Pediatric Patients in a Hospital Setting.

OBJECTIVES: To develop and validate age-specific percentile curves of measured mean arterial pressure for children in a hospital setting. DESIGN: Retrospective observational study of electronic records. SETTING: Tertiary care, freestanding pediatric hospital in Seattle, WA. PATIENTS: Nonpremature children, birth to 18 years old, evaluated in the emergency room, or admitted to either acute care or critical care units. INTERVENTIONS: Oscillometric blood pressure data collected from February 2012 to June 2016 were examined for documentation of systolic, diastolic, and mean arterial pressure values. Quantile curves were developed using restricted cubic splines and validated with two sets of patient data. The effects of birth sex and behavioral state on the curves were examined. The frequency of values less than 5th percentile for mean arterial pressure within a population was compared with four published criteria for hypotension. MEASUREMENTS AND MAIN RESULTS: Eighty-five-thousand two-hundred ninety-eight patients (47% female) provided 2,385,122 mean arterial pressure readings to develop and validate age-based distributions to create percentile curves and a reference table. The behavior state of patients affected the curves, with disturbed behavior state more prevalent in toddler-aged patients. There was no clinical difference between females and males within age brackets. Mean arterial pressure quantiles identified additional hypotensive episodes as compared with systolic blood pressure thresholds and predicted mean arterial pressure values. Code and data available at: https://osf.io/upqtv/. CONCLUSIONS: This is the first study reporting age-specific quantiles of measured mean arterial pressure in children in a hospital setting. The percentile curves may guide care in illnesses when perfusion pressure is critical and serve as parameter for bedside and electronic record-based response to clinical change. Future work to correlate threshold mean arterial pressure values with outcomes would be feasible based on quantile curves.

Interaction of Critical Care Practitioners With a Decision Support Tool for Weaning Mechanical Ventilation in Children.

BACKGROUND: There is evidence that ventilator weaning protocols provide benefit to children receiving mechanical ventilation, but many protocols do not include explicit instructions for decreasing ventilator support from maximal settings. We evaluated care provider opinions on ventilator weaning recommendations made by a computerized decision support tool. METHODS: Recommendations for ventilator adjustment were generated using a computerized decision support tool based on the ARDSNet protocol using data from children with acute hypoxemic respiratory failure admitted to the pediatric ICU (PICU). Attending physicians, fellows, nurse practitioners, and respiratory therapists (RTs) caring for these patients answered a brief survey to assess whether recommendations were reasonable and whether the practitioner believed they could be implemented. RESULTS: RTs completed 99 surveys and ICU providers completed 96 surveys based on data from 10 patients. RTs and ICU providers found 63.9% and 65.3% of recommendations reasonable, respectively. There were 5 instances of disagreement between RTs and ICU providers. The percent of recommendations that RTs thought could be implemented was 29.9%, whereas this figure for ICU providers was 26.3%, with 4 instances of disagreement. Free-text responses indicated that many RTs and ICU providers were concerned about disrupting current patient stability and low tidal volumes. CONCLUSIONS: On initial evaluation, the decision support tool did not appear to be highly acceptable to RTs and ICU providers in our setting because recommendations were rarely implemented. In addition, acceptability did not increase over time as patients generally improved. Most respondents preferred to make no ventilator changes and felt the recommendations were too aggressive. The notable barrier to use was a perception of potential patient instability with weaning.

Short-Term Adverse Outcomes Associated With Hypoglycemia in Critically Ill Children.

OBJECTIVES: Previous studies report worse short-term outcomes with hypoglycemia in critically ill children. These studies relied on intermittent blood glucose measurements, which may have introduced detection bias. We analyzed data from the Heart And Lung Failure-Pediatric INsulin Titration trial to determine the association of hypoglycemia with adverse short-term outcomes in critically ill children. DESIGN: Nested case-control study. SETTING: Thirty-five PICUs. A computerized algorithm that guided the timing of blood glucose measurements and titration of insulin infusion, continuous glucose monitors, and standardized glucose infusion rates were used to minimize hypoglycemia. PATIENTS: Nondiabetic children with cardiovascular and/or respiratory failure and hyperglycemia. Cases were children with any hypoglycemia (blood glucose < 60 mg/dL), whereas controls were children without hypoglycemia. Each case was matched with up to four unique controls according to age group, study day, and severity of illness. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 112 (16.0%) of 698 children who received the Heart And Lung Failure-Pediatric INsulin Titration protocol developed hypoglycemia, including 25 (3.6%) who developed severe hypoglycemia (blood glucose < 40 mg/dL). Of these, 110 cases were matched to 427 controls. Hypoglycemia was associated with fewer ICU-free days (median, 15.3 vs 20.2 d; p = 0.04) and fewer hospital-free days (0 vs 7 d; p = 0.01) through day 28. Ventilator-free days through day 28 and mortality at 28 and 90 days did not differ between groups. More children with insulin-induced versus noninsulin-induced hypoglycemia had zero ICU-free days (35.8% vs 20.9%; p = 0.008). Outcomes did not differ between children with severe versus nonsevere hypoglycemia or those with recurrent versus isolated hypoglycemia. CONCLUSIONS: When a computerized algorithm, continuous glucose monitors and standardized glucose infusion rates were used to manage hyperglycemia in critically ill children with cardiovascular and/or respiratory failure, severe hypoglycemia (blood glucose < 40 mg/dL) was uncommon, but any hypoglycemia (blood glucose < 60 mg/dL) remained common and was associated with worse short-term outcomes.

An Elevated Low Cardiac Output Syndrome Score Is Associated With Morbidity in Infants After Congenital Heart Surgery.

OBJECTIVES: To evaluate an empirically derived Low Cardiac Output Syndrome Score as a clinical assessment tool for the presence and severity of Low Cardiac Output Syndrome and to examine its association with clinical outcomes in infants who underwent surgical repair or palliation of congenital heart defects. DESIGN: Prospective observational cohort study. SETTING: Cardiac ICU at Seattle Children's Hospital. PATIENTS: Infants undergoing surgical repair or palliation of congenital heart defects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Clinical and laboratory data were recorded hourly for the first 24 hours after surgery. A Low Cardiac Output Syndrome Score was calculated by assigning one point for each of the following: tachycardia, oliguria, toe temperature less than 30°C, need for volume administration in excess of 30 mL/kg/d, decreased near infrared spectrometry measurements, hyperlactatemia, and need for vasoactive/inotropes in excess of milrinone at 0.5 µg/kg/min. A cumulative Low Cardiac Output Syndrome Score was determined by summation of Low Cardiac Output Syndrome Score on arrival to cardiac ICU, and 8, 12, and 24 hours postoperatively. Scores were analyzed for association with composite morbidity (prolonged mechanical ventilation, new infection, cardiopulmonary arrest, neurologic event, renal dysfunction, necrotizing enterocolitis, and extracorporeal life support) and resource utilization. Fifty-four patients were included. Overall composite morbidity was 33.3%. Median peak Low Cardiac Output Syndrome Score and cumulative Low Cardiac Output Syndrome Score were higher in patients with composite morbidity (3 [2-5] vs 2 [1-3]; p = 0.003 and 8 [5-10] vs 2.5 [1-5]; p < 0.001)]. Area under the receiver operating characteristic curve for cumulative Low Cardiac Output Syndrome Score versus composite morbidity was 0.83, optimal cutoff of greater than 6. Patients with cumulative Low Cardiac Output Syndrome Score greater than or equal to 7 had higher morbidity, longer duration of mechanical ventilation, cardiac ICU, and hospital length of stay (all p ≤ 0.001). After adjusting for other relevant variables, peak Low Cardiac Output Syndrome Score and cumulative Low Cardiac Output Syndrome Score were independently associated with composite morbidity (odds ratio, 2.57; 95% CI, 1.12-5.9 and odds ratio, 1.35; 95% CI, 1.09-1.67, respectively). CONCLUSION: Higher peak Low Cardiac Output Syndrome Score and cumulative Low Cardiac Output Syndrome Score were associated with increased morbidity and resource utilization among infants following surgery for congenital heart defects and might be a useful tools in future cardiac intensive care research. Independent validation is required.

Effects of exendin-4, a glucagon like peptide-1 receptor agonist, on neutrophil count and inflammatory cytokines in a rat model of endotoxemia.

BACKGROUND: Sepsis remains a major cause of morbidity and mortality. A variety of strategies targeting modulation of the pro-inflammatory response associated with early sepsis have been reported without clinical success. GLP-1 enhances glucose-stimulated insulin secretion. In addition, it was shown to have anti-inflammatory effects. We hypothesized that treatment with exendin-4, a GLP-1 receptor agonist, would attenuate inflammation and improve glucose control in a lipopolysaccharide (LPS) rat model of inflammation. METHODS: Two-month-old male Wistar rats were randomly assigned to one of the following four groups: 1) treatment: intraperitoneal (IP) injection of LPS 10 mg/kg followed by exendin-4, 30 µg/kg, 10 minutes later; 2) control-1: IP injection of LPS 10 mg/kg, followed by normal saline (NS); 3) control-2: IP NS injection followed by exendin-4; 4) sham: IP injection of NS followed by another NS injection. Glucose concentration, total white blood count with absolute neutrophil count, and pro- and anti-inflammatory cytokine concentrations were measured at 0, 3, 6, and 10 hours following LPS injection. RESULTS: At 3 hours, rats injected with LPS developed neutropenia, elevated pro- and anti-inflammatory cytokines, and mild hypoglycemia. Treatment with exendin-4 significantly modulated neutropenia, and decreased pro-inflammatory cytokine concentrations (IL-1α, IL-1ß, IL-6, TNFα, and IFNγ). However, exendin-4 had no effect on IL-10 concentrations. LPS injection led to mild hypoglycemia, that was not observed in rats treated with exendin-4. Sham animals exhibited no significant change from baseline in all parameters. CONCLUSION: In this LPS model of acute early phase inflammation, treatment with exendin-4 decreased pro-inflammatory cytokine concentrations without changing IL-10 blood levels and improved neutropenia. Following LPS injection, rats developed a tendency toward hypoglycemia that improved with exendin-4. Overall our data suggest that exogenous exendin-4 mediates anti-inflammatory effects early in this rat model of endotoxin-induced inflammation.

Two pediatric cases of variant neurogenic stress cardiomyopathy after intracranial hemorrhage.

Takotsubo cardiomyopathy, also known as stress-induced cardiomyopathy, is an acquired form of left ventricular systolic dysfunction seen in the setting of physiologic stress and the absence of coronary artery disease. It is thought to be caused by excessive sympathetic stimulation. It is well described in the adult literature associated with subarachnoid hemorrhage where it is known as neurogenic stress cardiomyopathy (NSC), but few such pediatric cases have been reported. We describe our experience with 2 children (13- and 10-year-old girls) who presented with spontaneous intracranial hemorrhage followed by pulmonary edema and shock. Echocardiography revealed similar patterns of left ventricular wall motion abnormalities consistent with NSC, inverted Takotsubo variant. One child progressed to death, whereas the other made a remarkable recovery, including significant improvement in cardiac function over the course of 1 week. We argue that at least 1 of these cases represents true stress-induced cardiomyopathy. This report will alert pediatricians to this transient cardiomyopathy that is likely underdiagnosed in pediatric intensive care. We also highlight the challenges of managing both shock and elevated intracranial pressure in the setting of NSC.

Long-term dexmedetomidine use and safety profile among critically ill children and neonates.

OBJECTIVE: To determine whether long-term dexmedetomidine dosing is associated with lower opioid and benzodiazepine use without risk of significant hemodynamic changes and/or withdrawal. DESIGN: Retrospective, observational study. SETTING: PICU, cardiovascular ICU, and neonatal ICU in a single, tertiary care, academic children's hospital. SUBJECTS: We included all patients less than or equal to 21 years old, who received dexmedetomidine for greater than or equal to 72 hours from December 2008 to December 2010 resulting in a 98-subject cohort. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: The median duration of dexmedetomidine use was 141 hours. A decrease in systolic blood pressure and heart rate was seen after initiation of dexmedetomidine. After dexmedetomidine was discontinued, systolic blood pressure was statistically significantly higher than baseline. Similarly, heart rate showed a significant increase from baseline following discontinuation of dexmedetomidine. Starting dexmedetomidine was not associated with a significant difference in the dosing of opiates or benzodiazepines. Comfort scores were significantly lower at 2 and 72 hours of dexmedetomidine infusion. After stopping dexmedetomidine, the comfort score for patients at 1 hour was statistically higher than for patients at cessation of the infusion. Thirty percent of patients who were taken off dexmedetomidine, whether weaned or abruptly stopped, had withdrawal symptoms and scores recorded with agitation, tremor, and decreased sleep being most prominent. CONCLUSIONS: Hemodynamic effects of dexmedetomidine did not limit long-term use in this diverse population. After the addition of dexmedetomidine, opioid and benzodiazepine doses did not significantly escalate, and patients were more comfortable as evidenced by decreasing comfort scores. Withdrawal from dexmedetomidine may be an issue and manifests as agitation, tremors, and decreased sleep.

Common endocrine issues in the pediatric intensive care unit.

Thyroid hormone is central to normal development and metabolism. Abnormalities in thyroid function in North America often arise from autoimmune diseases, but they rarely present as critical illness. Severe deficiency or excess of thyroid hormone both represent life-threatening disease, which must be treated expeditiously and thoroughly. Such deficiencies must be considered, because presentation may be nonspecific.

Repeated lentivirus-mediated granulocyte colony-stimulating factor administration to treat canine cyclic neutropenia.

Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by repeated injections of recombinant granulocyte colony-stimulating factor (rG-CSF). As dose escalation of lentivirus may be clinically necessary, we monitored the outcome of four sequential intramuscular injections of G-CSF-lentivirus (3 × 10(7) IU/kg body weight) to a normal dog and a gray collie. In the normal dog absolute neutrophil counts were significantly increased after each dose of virus, with mean levels of 27.75 ± 3.00, 31.50 ± 1.40, 35.05 ± 1.68, and 43.88 ± 2.94 × 10(3) cells/µl, respectively (p<0.001), and elevated neutrophil counts of 31.18 ± 7.81 × 10(3) cells/µl were maintained for more than 6 years with no adverse effects. A gray collie dog with a mean count of 1.94 ± 1.48 × 10(3) cells/µl received G-CSF-lentivirus and we observed sustained elevations in neutrophil levels for more than 5 months with a mean of 26.00 ± 11.00 × 10(3) cells/µl, significantly increased over the pretreatment level (p<0.001). After the second and third virus administrations mean neutrophil counts of 15.80 ± 6.14 and 11.52 ± 4.90 × 10(3) cells/µl were significantly reduced compared with cell counts after the first virus administration (p<0.001). However, after the fourth virus administration mean neutrophil counts of 15.21 ± 4.50 × 10(3) cells/µl were significantly increased compared with the previous administration (p<0.05). Throughout the nearly 3 years of virus administrations the dog gained weight, was healthy, and showed neutrophil counts significantly higher than pretreatment levels (p<0.001). These studies suggest that patients with cyclic and other neutropenias may be treated with escalating doses of G-CSF-lentivirus to obtain a desired therapeutic neutrophil count.

Sustained glucagon-like peptide 1 expression from encapsulated transduced cells to treat obese diabetic rats.

Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major public health concern in industrialized countries. T2D is characterized by hyperglycemia and islet beta cell dysfunction. Glucagon-like peptide 1 (GLP-1) promotes ß cell proliferation and neogenesis and has a potent insulinotropic effect. Leptin receptor deficient male rats are obese and diabetic and provide a model of T2D. We hypothesized that their treatment by sustained expression of GLP-1 using encapsulated cells may prevent or delay diabetes onset. Vascular smooth muscle cells (VSMC) retrovirally transduced to secrete GLP-1 were seeded into TheraCyte(TM) encapsulation devices, implanted subcutaneously and rats were monitored for diabetes. Rats that received cell implants showed mean plasma GLP-1 level of 119.3 ± 10.2pM that was significantly elevated over control values of 32.4 ± 2.9pM (P<0.001). GLP-1 treated rats had mean insulin levels of 45.9 ± 2.3ng/ml that were significantly increased over control levels of 7.3±1.5ng/ml (P<0.001). In rats treated before diabetes onset elevations in blood glucose were delayed and rats treated after onset became normoglycemic and showed improved glucose tolerance tests. Untreated diabetic rats possess abnormal islet structures characterized by enlarged islets with α-cell infiltration and multifocal vacuolization. GLP-1 treatment induced normalization of islet structures including a mantle of α-cells and increased islet mass. These data suggest that encapsulated transduced cells may offer a potential long term treatment of patients.

Prolonged survival and improved glycemia in BioBreeding diabetic rats after early sustained exposure to glucagon-like peptide 1.

BACKGROUND: Type 1 diabetes (T1D) in both humans and BioBreeding (BB) rats is an autoimmune disease that results in complete destruction of islets and insulin dependency for life. Glucagon-like peptide 1 (GLP-1) promotes beta cell proliferation and neogenesis and has a potent insulinotropic effect. We hypothesized that the expression of GLP-1 before disease onset would increase islet mass, delay diabetes and prolong survival of BB rats. METHODS: Vascular smooth muscle cells retrovirally transduced to secrete GLP-1 were seeded into TheraCyte encapsulation devices, implanted subcutaneously, and rats were monitored for diabetes. RESULTS: In untreated control rats, plasma GLP-1 levels were 34.5-39.5 pmol/l, whereas, in treated rats, plasma levels were elevated, in the range 90-250.4 pmol/l. Hypoglycemia was not detected and this was anticipated from the glucose-regulated action of GLP-1. Diabetes onset (mean + or - SEM) in untreated rats occurred at 56.5 + or - 0.6 days (n = 6) and, in GLP-1-treated rats, was delayed until 76.4 + or - 3.3 days (n = 5) (p < 0.001). After disease onset, untreated control rats showed a rapid weight loss and elevated blood glucose (>650 mg/dl) and did not survive beyond 11 days. At 5 days after diabetes onset, insulin-secreting islets were absent in untreated rats. By contrast, treated rats maintained weight for up to 143 days of age and showed insulin-secreting beta cells. CONCLUSIONS: Sustained GLP-1 expression delivered by encapsulated cells before diabetes onset in BB rats showed an improved clinical outcome, suggesting the potential for treating patients using long lasting GLP-1 analogs.

Treatment of diabetic rats with encapsulated islets.

Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose>350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30-40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats.

G-CSF-lentivirus administration in rats provided sustained elevated neutrophil counts and subsequent EPO-lentivirus administration increased hematocrits.

BACKGROUND: Towards gene therapy treatment of patients with neutropenia, characterized by neutrophil counts < 500 cells/microl, we investigated the ability of lentivirus vectors to provide sustained granulocyte colony-stimulating factor (G-CSF) delivery and to permit transgene expression from a second virus administration in a preclinical rat model. METHODS: Rats were injected intramuscularly (IM) with 24 x 10(6) and 9 x 10(6) infectious units (IU) of a VSV-G-pseudotyped self-inactivating (SIN) lentivirus encoding rat G-CSF cDNA and containing cPPT and PRE elements. To determine the effectiveness of a second virus administration treated rats and a naïve rat received erythropoietin (EPO)-lentivirus IM. Rats were monitored for neutrophil and red blood cell production. Lentivirus antibodies were assayed by virus-neutralizing assay and ELISA. RESULTS: High and low dose virus administration increased neutrophil counts to 5660 +/- 930 cells/microl (mean +/- SD) and 4010 +/- 850 cells/microl, respectively, that were sustained for > 17 months and were significantly higher than controls counts of 1890 +/- 570 cells/microl (p< or =0.0002). Rats treated with EPO-virus produced significantly increased hematocrits (HCT) (63.1 +/- 4.3% vs. 46.0 +/- 3.2%, p < 0.0001) without effect on G-CSF-virus-mediated neutrophil production. Antivirus antibodies were not detectable at serum dilutions > or =1:10 by virus neutralization or ELISA. Lymphocytes and platelets were not significantly different between control and treated animals (p > 0.1). Only genomic DNA from muscle at injection sites was positive for provirus suggesting lack of virus spread. CONCLUSIONS: G-CSF-lentivirus administered IM provided elevated, sustained neutrophil counts that were unchanged by subsequent EPO-lentivirus administration. Significantly increased hematocrits were obtained following EPO-lentivirus delivery. These data support the treatment of patients with severe chronic neutropenia by dosed lentivirus delivery IM.

An adult dog with cyclic neutropenia treated by lentivirus- mediated delivery of granulocyte colony-stimulating factor.

Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by daily injections of recombinant granulocyte colony-stimulating factor (G-CSF). After showing that canine recombinant G-CSF increased neutrophil counts in an affected dog, we administered intramuscularly 2 x 10(9) infectious units (IU) of a lentiviral vector encoding canine G-CSF cDNA. Elevated, therapeutic neutrophil production was obtained for nearly 18 months. Lentiviral vector treatment provided a mean neutrophil count of 29,230 +/- 12,930 cells/microl, which was significantly increased over both the pretreatment value (5,240 +/- 4,800 cells/microl; p < 0.0001) and the neutrophil count during G-CSF administration (17,820 +/- 11,100 cells/microl; p < 0.0001). By systemic infusion of recombinant G-CSF to normal dogs we estimated that 2 x 10(9) IU of lentivirus delivered 3.5 microg of G-CSF per kilogram per day. After lentiviral vector treatment the gray collie gained weight, showed no clinical signs of infection and fever, and no longer needed housing in a pathogen-free environment. Genomic DNA harvested from muscle at the injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, and noninjected muscle samples were negative. These studies show that an adult animal is responsive long-term to lentivirus-mediated G-CSF delivery, suggesting this approach may be applied for treatment of adult patients with cyclic and other neutropenias.

Sustained elevation of neutrophils in rats induced by lentivirus-mediated G-CSF delivery.

BACKGROUND: Patients with severe chronic and cyclic neutropenia, characterized by neutrophil numbers <500 cells/microl, are treated daily with recombinant granulocyte colony-stimulating factor (G-CSF). As an alternative delivery approach we investigated the ability of lentivirus vectors to provide sustained G-CSF expression. METHODS: Fischer rats were injected intramuscularly (IM) with vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus pRRL-CMV-G-CSF-SIN that encoded rat G-CSF cDNA regulated by the human cytomegalovirus (CMV) promoter and incorporated a self-inactivating (SIN) construct in the 3' long terminal repeat (LTR). Control rats received normal saline or lentivirus encoding the enhanced green fluorescent protein (eGFP). Rats were serially monitored for blood cell production and tissues assayed for provirus distribution. RESULTS: Rats receiving a single IM injection of lentivirus exhibited elevated neutrophil counts for 14 months. Virus administration of 6 x 10(7) infectious units induced sustained levels of neutrophil production having a mean +/- standard deviation (SD) of 5650 +/- 900 cells/microl and rats that received a 10-fold lower dose of virus showed mean neutrophil counts of 3340 +/- 740 cells/microl. These were significantly higher than the mean of control animals receiving saline or control lentivirus (1,760 +/- 540 cells/microl, P < 0.0001). White blood cell (WBC) counts were significantly elevated in treated over control animals (P < 0.0001). Hematocrits (P > 0.3), lymphocytes (P > 0.2) and platelets (P > 0.1) were not significantly different between control and treated animals. Genomic DNA from muscle at the injection sites was positive for provirus, whereas lung, spleen, liver, kidney and non-injected muscle samples were all negative, suggesting lack of virus spread. CONCLUSIONS: These studies indicate that lentivirus vectors administered IM provide sustained, therapeutic levels of neutrophils and suggest this approach to treat patients with severe and cyclic neutropenia.

Continuous pentobarbital infusion in children is associated with high rates of complications.

PURPOSE: To evaluate the incidence and severity of complications related to continuous pentobarbital (PB) infusion for sedation in the Pediatric Intensive Care Unit (PICU). MATERIALS AND METHODS: We conducted a retrospective, chart review study. All patients admitted to the PICU from January 1997 through June 1998 who received continuous IV PB infusion for sedation (n = 8) were included. RESULTS: All patients were intubated and mechanically ventilated prior to PB infusion. PB was used only as a second line sedative after a combination of an opioid and benzodiazepine failed to achieve adequate sedation. After initiation of PB, we were able to decrease or discontinue benzodiazepines and/or opioid doses and discontinue neuromuscular blocking drugs in all patients. We observed a high incidence of complications (62.5%) related to PB or the phenobarbital treatment used for barbiturate weaning, including blood pressure instability (25%), oversedation (12.5%), drug reaction (12.5%) and neurologic sequelae (12.5%). Discontinuation of the drug was required in 25% of the cases. CONCLUSIONS: We found continuous PB infusion to be an effective sedative for children when other drugs fail. However, we observed a high rate of clinically significant complications requiring discontinuation of the drug.

Long-term erythropoietin gene expression from transduced cells in bioisolator devices.

Recombinant erythropoietin (EPO) is widely administered for long-term treatment of anemia associated with renal failure and other chronic diseases. The ability to deliver EPO by gene therapy would have clinical and economic benefit. We compared autologous and allogeneic transduced primary vascular smooth muscle cells for their ability to provide sustained EPO gene expression when encapsulated in TheraCyte devices implanted subcutaneously (SQ) or intraperitoneally (IP) in rats. Cells were transduced with retrovirus vector LrEpSN encoding rat EPO cDNA. Rats that received either autologous or allogeneic transduced cells showed elevated hematocrits (HCTs) ranging from 50 to 79% that were sustained for more than 12 months. The HCT of control rats remained at baseline (45.8%). Rats that received second SQ implants of either autologous or allogeneic cells showed elevations in hematocrit that were sustained for up to 12 months, suggesting the absence of immunological responses to transduced cells or implant material. All experimental groups had statistically significant elevated HCT (p < 0.001) when compared with controls. Both SQ and IP implantation were equally effective in delivering EPO long term. There were no significant differences in white blood cell (WBC) or platelet (PLT) values between treated and control animals. Implantation of TheraCyte devices was well tolerated and histological evaluation of the devices up to 12 months after surgery revealed a high degree of vascularization and no evidence of host immune response. TheraCyte devices offer a simple and safe gene delivery system that provides sustained therapeutic gene expression, permit removal and implantation of new devices, and do not require immunosuppression of the host.