E. Stephen Buescher

The Effects of Intense Submicrosecond Electrical Pulses on Cells

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.

Clinical experience with linezolid for the treatment of Nocardia infection

Linezolid is an oxazolidinone that has activity against most gram-positive bacteria, including in vitro activity against all Nocardia species and strains. We describe 6 clinical cases of nocardiosis that were successfully treated with linezolid. Two patients had underlying X-linked chronic granulomatous disease, and 2 patients were receiving chronic corticosteroid therapy. Four of 6 patients had disseminated disease, and 2 of these 4 patients had multiple brain abscesses. Four patients primarily received monotherapy; for the fifth patient, linezolid was added to a failing multiple-drug regimen, and, for the sixth patient, it was used as part of combination therapy. All 6 patients were successfully treated, although 1 patient had a presumed relapse of central nervous system infection after premature discontinuation of the drug. Linezolid appears to be an effective alternative for the treatment of nocardiosis.

Should Central Venous Catheters Be Removed as Soon as Candidemia Is Detected in Neonates

Background. Controversy exists regarding the most appropriate acute management of central venous catheters (CVCs) in neonates with candidemia, with up to two thirds of neonatologists preferring to attempt antifungal therapy without removing CVCs. Objective. To determine whether CVCs should be removed as soon as candidemia is detected in neonates. Methods. A cohort study of candidemia and CVC was conducted in infants in a neonatal intensive care unit (NICU) over a 5-year period (1994–1998). Results. Fifty infants had early-removal CVC (ER-CVC) within 3 days and 54 infants had late-removal CVC (LR-CVC) >3 days after the first positive blood culture for Candidaspecies. All infants were treated with amphotericin B. There was no significant difference between infants in the ER-CVC and LR-CVC groups in terms of gender, ethnicity, birth weight, gestational age, age at candidemia, severity-of-illness scores, distribution of types of CVC, or in the distribution of Candida species causing candidemia. The ER-CVC group had significantly shorter duration of candidemia (median: 3 days; range: 1–14 days), compared with the LR-CVC group (median: 6 days; range: 1–24 days). The case fatality rate of Candida albicans candidemia was significantly affected by the timing of CVC removal: 0 of 21 (95% confidence interval [CI]: 0–14) infants died in the ER-CVC group in contrast to 9 of 23 (39%; 95% CI: 19–59) in the LR-CVC group. Conclusion. Failure to remove CVC as soon as candidemia was detected in neonates was associated with significantly increased mortality in C albicans candidemia and prolonged duration of candidemia regardless of Candidaspecies.

Distinctive Distribution of Pathogens Associated With Peritonitis in Neonates With Focal Intestinal Perforation Compared With Necrotizing Enterocolitis

Objective. Candida and coagulase-negative staphylococci are emerging pathogens associated with focal intestinal perforation (FIP) and necrotizing enterocolitis (NEC) in neonates. The objective of this study was to determine whether there are significant differences in the predominant pathogens in culture-positive cases of peritonitis associated with FIP compared with NEC in neonates. Methods. A retrospective cross-sectional study was conducted of neonates with peritoneal culture-positive peritonitis associated with FIP or NEC over a 12-year study period (1989–2000). Cases with peritonitis were identified from a microbiology database. NEC was defined by radiologic evidence of pneumatosis intestinalis or portal venous gas or by pathology reports or surgical operative notes describing large areas of transmural bowel necrosis. FIP was defined as a <1-cm intestinal perforation surrounded by otherwise normal tissue in the absence of NEC. Results. Thirty-six cases of FIP were compared with 80 cases of NEC. Birth weight and gestational age were significantly lower in infants with FIP compared with NEC. Age at intestinal perforation and case fatality rates were similar between FIP and NEC. There were striking differences in the distribution of predominant pathogens associated with peritonitis in NEC and FIP cases. Enterobacteriaceae were present in 60 (75%) of 80 NEC cases compared with 9 (25%) of 36 FIP cases. In contrast, Candida species were found in 16 (44%) of 36 FIP cases compared with 12 (15%) of 80 NEC cases, and coagulase-negative staphylococci were present in 18 (50%) of 36 FIP cases versus 11 (14%) of 80 NEC cases. There were no significant differences between FIP and NEC cases for the presence of Enterococcus species (28% vs 23%) or anaerobes (3% vs 6%). Stratified analysis for birth weight <1200 g found similar significant differences in the predominant pathogens for FIP (n = 29) and NEC (n = 38). Results from peritoneal fluid cultures resulted in changes in antimicrobial therapy in 46 (40%) of 116 cases. Conclusions. Candida species and coagulase-negative staphylococci were the predominant pathogens in FIP peritonitis in contrast to Enterobacteriaceae in NEC peritonitis. A peritoneal fluid culture should be obtained in all neonates with intestinal perforation, regardless of cause, because it may help to direct the choice of the most effective antimicrobial.

Central venous catheter removal versus in situ treatment in neonates with coagulase-negative staphylococcal bacteremia.

Objective. To determine how often neonates with coagulase-negative staphylococcal (CONS) bacteremia can be treated successfully without removing the central venous catheter (CVC). Methods. A cohort study of CONS bacteremia and CVCs was conducted in infants in a neonatal intensive care unit in a 5-year period (1994 through 1998). CONS bacteremia was defined as at least two positive blood cultures within 3 days of each other. Results. Fifty-six infants had early removal CVC (ER-CVC) within 3 days, and 63 infants had late removal CVC (LR-CVC) >3 days after the first positive blood culture. All cases of CONS bacteremia were treated with vancomycin. There was no significant difference between infants in the ER-CVC and LR-CVC groups in terms of recurrence of bacteremia or case fatalities. CONS bacteremia of >3 days duration was more frequent in LR-CVC patients than ER-CVC patients: 43%vs. 13% (relative risk, 3.4; 95% confidence interval, 1.6 to 7.2). CONS bacteremia was successfully treated without CVC removal in 46% of LR-CVC cases. Seventy-nine percent of LR-CVC cases with CONS bacteremia lasting 1 or 2 days were treated successfully without CVC removal. The success rate decreased to 44% with a 3- to 4-day duration of bacteremia. None of 19 infants with CONS bacteremia lasting >4 days was treated successfully until CVCs were removed. Conclusions. Prolonged CONS bacteremia was avoided by early removal of CVCs. Retention of CVCs was successful in 46% of neonates with CONS bacteremia in whom it was attempted, but it was never successful if bacteremia lasted >4 days.

Colostral antioxidants : separation and characterization of two activities in human colostrum

Human colostrum contains several antioxidants which prevent the detection of human polymorpho-nuclear leukocyte (PMN) respiratory burst products. Using column chromatography to fractionate colostrum, two peaks of antioxidant activity were resolved away from colostral proteins and further characterized. One peak contained both cytochrome c-reducing activity and H2O2-depleting activity. This peak had the chromatographic, spectral, and antioxidant characteristics of ascorbate, and by high performance liquid chromatography (HPLC) methods, was shown to contain ascorbate as well as at least four other materials. The antioxidant activity in this peak was totally ascorbate oxidase sensitive and partially uricase sensitive. The other peak contained only H2O2-depleting activity and had the chromatographic, spectral, and antioxidant characteristics of uric acid. By HPLC, uric acid was the only component in this peak and its antioxidant activity was completely uricase sensitive and ascorbate oxidase resistant. Colostral uric acid levels were measured in eight postpartum women and found to be approximately one-third of simultaneously determined serum uric acid levels. Colostrum contains at least two separate antioxidants, one of which is ascorbate-like and the other is uric acid. We speculate that these antioxidants may function in human colostrum as traps for neutrophil-generated reactive oxygen metabolites.

Anti-Inflammatory Characteristics of Human Milk

When first proposed, the hypothesis that human milk was anti-inflammatory was supported by 2 observations: poor function of milk leukocytes and the presence in milk of components that could modify inflammatory processes. This hypothesis is now supported by studies documenting anti-inflammatory effects in animal models and suppression of humoral and cellular components of inflammation in vitro. To date, two mechanisms have been demonstrated: alteration of leukocyte function and modification of cytokine biology. It is not clear whether these mechanisms are only topical effects in the digestive tract, or whether absorption of milk components results in systemic effects. While inflammation benefits the host as a defense mechanism and precursor to immune responses, it also contributes to the clinical manifestations of illness and is an important early component of wound-healing responses that result in scar. The biological effects of milk’s anti-inflammatory character may be to minimize clinical symptomatology without losing immunoresponsiveness for the breast-fed infant, and to minimize scar formation during healing responses.

Human Colostrum Has Anti-inflammatory Activity in a Rat Subcutaneous Air Pouch Model of Inflammation

ABSTRACT: An animal model was used to examine the effect of human colostrum on an acute inflammatory process in vivo. Subcutaneous air pouches on the backs of outbred rats were injected with carrageenan as an inflammatory challenge, normal saline, pooled aqueous human colostrum, or carrageenan plus colostrum concurrently. Oral dexamethasone or indomethacin was administered to some animals before and during challenge as anti-inflammatory agents. Polymorphonuclear leukocyte (PMN) counts in pouch fluid were determined 6 h postchallenge. Carrageenan challenge resulted in a significant acute inflammatory response [48.8 ± 4.9 × 106 PMN/pouch (mean ± SEM, n = 46)] compared with normal saline controls [0.9 ± 0.2 × 106 (n = 31, p < 0.001 versus carrageenan)] or with colostrum [4.3 ± 0.8 × 106 PMN/pouch (n = 11, p < 0.001 versus carrageenan)]. The concurrent injection of colostrum plus carrageenan challenge significantly reduced the PMN response compared with carrageenan alone [18.8 ± 2.9 versus 48.8 ± 4.9 × 106 PMN/pouch (carrageenan plus colostrum versus carrageenan, n = 41 versus 46, p < 0.001)]. This degree of suppression of PMN influx was not significantly different from that seen with indomethacin treatment but was significantly more than that seen with dexamethasone treatment. The decreases in PMN counts observed most likely reflect suppression of the acute inflammatory response because a significant amount of PMN lysis in colostrum was not observed in vitro and the accumulation of PMN granule contents was not seen in pouch fluid from colostrum-treated animals in vivo. These data directly demonstrate for the first time that human colostrum has a biologically significant effect on the inflammatory process in vivo.

Studies of the function and structure of in vitro propagated human granulocytes

ABSTRACT: In our study, hematopoietic progenitor cells isolated from human umbilical cord blood were grown in an in vitro liquid culture system using the recombinant colony stimulating factors IL-3 plus granulocyte-macrophage colony-stimulatory factor (GM-CSF) or IL-3 plus granulocyte colony stimulating factor (G-CSF). The morphology and function of the cells produced were then studied, and it was demonstrated that continuous exposure to IL-3 plus GM-CSF produced predominantly eosinophilic granulocytes, whereas IL-3 plus G-CSF produced neutrophilic granulocytes. Cells from IL-3/GM-CSF cultures showed progressively increasing oxygen metabolism and locomotive capabilities over time, which became equivalent to peripheral blood neutrophils at wk 4 and 3, respectively. Phagocytic activity of these cells was poor. IL-3/G-CSF cultures produced cells with progressive increases in oxygen metabolism, locomotion, and phagocytosis. These functions never became equivalent to those of peripheral blood neutrophils. Flow cytometric analysis of IL-3/G grown cells showed that they expressed CD11b on their surfaces and that surface expression increased 2-fold after secondary granule secretagogue exposure. Ultrastructurally, the eosinophilic granulocyte nature of the IL-3/GM grown cells was confirmed by immunogold-lectin staining and IL-3/G grown cells were shown to contain antigenic myeloperoxidase. The data demonstrate that human umbilical cord blood mononuclear cells can be used to propagate granulocytes in vitro, that the types of granulocytes produced in this culture system depend on the growth factors used, that the cells produced in vitro develop several of the functional characteristics of peripheral blood granulocytes, and that ultrastructural details of developing human granulocytes can be carefully examined in this model system.

Risk factors and management of Nuss bar infections in 1717 patients over 25 years

PURPOSE An increase in postoperative infections after Nuss procedures led us to seek risks and review management. We report potential risk factors and make inferences for prevention of infections. METHODS An IRB-approved retrospective chart review was used to evaluate demographic, clinical, surgical, and postoperative variables of patients operated on between 10/1/2005 and 6/30/2013. Those with postoperative infection were evaluated for infection characteristics, management, and outcomes with univariate analyses. RESULTS Over this 8-year period (2005-2013), 3.5% (30) of 854 patients developed cellulitis or infection, significantly more than 1.5% (13) in our previous report of 863 patients, 1987-2005 (p=.007). The most frequent organism cultured was methicillin-sensitive Staphylococcus aureus. Patients who were given clindamycin preoperatively (5 of 26 patients) had higher infection rates than those who received cefazolin (25 of 828) (19% vs 3%, p<.001). Patients treated with a peri-incisional ON-Q (I-Flow, Kimberly-Clark, Irvine, CA) also had higher infection rates (8.3% vs 2.4%, p<.001). Of the 30 patients who developed an infection, eighteen (60%) with cellulitis or superficial infections did not require surgical treatment or early bar removal. The other twelve patients (40%) with deep hardware infections required an average of 2.2 operations (range 1-6), with 3 (25%) requiring removal of their stabilizer and 3 (25%) requiring early bar removal. None of these three patients experienced recurrence of pectus excavatum at 2 to 4 years of follow-up. CONCLUSION Preoperative antibiotic selection and use of ON-Qs may influence infection rates after Nuss repair. Nuss bars could be preserved in 90% of all patients with an infection and even 75% of those with a deep hardware infection. Attempts to retain the bar when an infection occurs may help prevent pectus excavatum recurrence. Level of Evidence=III.