Structure of bacterial luciferase.

The generation of light by living organisms such as fireflies, glow-worms, mushrooms, fish, or bacteria growing on decaying materials has been a subject of fascination throughout the ages, partly because it occurs without the need for high temperatures. The chemistry behind the numerous bioluminescent systems is quite varied, and the enzymes that catalyze the reactions, the luciferases, are a large and evolutionarily diverse group. The structure of the best understood of these intriguing enzymes, bacterial luciferase, has recently been determined, allowing discussion of features of the protein in structural terms for the first time.

Rational selection of adjuvant chemotherapy after cytoreduction surgery for murine neuroblastoma.

Improving the prognosis of advanced neuroblastoma remains an important yet unachieved goal of pediatric oncology, a fact which may be related to an insufficient analysis of the role played by cytoreductive surgery. Utilizing strain A mice bearing C-1300 syngeneic neuroblastoma, tumor biology and host immunocompetence were studied after cytoreduction surgery and adjuvant chemotherapy. Cell kinetic analysis in the residual tumor demonstrated an increase of the proliferative fraction 18 to 42 h after operation, but the same peak proliferation was delayed in bone marrow cells to 24 to 96 h. The potential for drug distribution to the tumor after cytoreduction surgery was assessed by injecting Na251CrO4 and measuring tumor uptake. There were two significant (P less than 0.05) peaks of activity at 6 h and 3 days, suggesting local edema and neovascularity, respectively. Injection of both cell cycle specific and nonspecific adjuvant chemotherapeutic agents in a dosage of one-fourth of their 50% lethal dose at 24 or 72 h following surgical cytoreduction did not induce any antitumor activity at either injection time. However, when cyclophosphamide was given in this dose, the C-1300 tumor growth was impaired, an effect which was largely abrogated by first subjecting the tumor bearer to thymectomy and irradiation. The transfer of spleen cells from adjuvant cyclophosphamide-treated mice to tumor-inoculated normal mice significantly delayed tumor appearance when comparison was made with animals treated by operation alone, and such recipients also exhibited a more prolonged survival. These data suggest that the antitumor activity of cyclophosphamide following cytoreduction surgery of C-1300 neuroblastoma is mediated by both pharmacological and immunological mechanisms.

Primary nonspecific ileal ulceration as a cause of massive rectal bleeding.

A case of primary ulceration of the ileum in the absence of a Meckel's diverticulum or ectopic gastric tissue is described. Although this condition is infrequently observed, it should be considered as a potential cause of massive rectal bleeding, iron deficiency anemia of unknown cause, perforation, or partial small bowel obstruction. The diagnosis is normally made at surgery. Segmental resection with end-to-end anastomosis is the treatment of choice. Recurrence following surgery is rare.

Tumor immunogenicity, nutritional repletion, and cancer.

Nutritional repletion in cancer reverses malnutrition and its associated immunodepression, but whether it benefits the host-tumor outcome has been variable. This study hypothesizes that such nutritional support will only favor that host generating a potent antitumor immune response. Murine neuroblastoma (NB) was characterized into immunizing C1300-NB and nonimmunizing TBJ-NB cell lines; and 100 6-week-old strain A mice were assigned on day -14 to isocaloric dietary groups (24% or 2.5% protein). At day 0, mice received either C1300-NB or TBJ-NB; on day 7 one half of the 2.5% group mice were repleted with 24% protein; on day 21, tumor weight/carcass weight (T/C) and mortality (M) were noted. Body weight increased 12.8% in the 24% group and fell 11.4% in the 2.5% group (p less than 0.01). The T/C ratios were smaller for immunogenic C1300-NB on 24% protein compared with 2.5% chow (0.0033 versus 0.0229; p less than 0.02). Repletion produced smaller tumors in the C1300-NB host; strikingly, repletion of TBJ-NB mice significantly increased tumor burden (T/C = 0.0186 versus 0.1657, p less than 0.01) and also increased animal mortality (M = 20% to 30%, p = NS). These data suggest that the influence of nutritional repletion on the tumor-bearing host varies specifically with the presence of an antitumor immune response.

Immunomodulatory mechanisms of arginine.

Dietary supplementation with the amino acid arginine augments T-lymphocyte activation in response to mitogens in clinical and experimental studies. In experimental models arginine enhances specific T cell antitumor immunity. The mechanism of these immunostimulatory effects of arginine on T cell function is unclear, and the scope and specificity of arginine's influence on other components of the effector immune response such as natural killer (NK) cells and macrophages are unknown. To evaluate the role of arginine in these responses, CBA/J mice (n = 100) were randomized to receive either 1% arginine supplementation or isonitrogenous (1.7%) glycine supplementation for at least 10 days. There was no significant differences in mean body weight between groups during feeding. Arginine supplementation significantly (p less than 0.05) enhanced cytotoxic T-lymphocyte development, poly IC-inducible NK activity, and the kinetics of interleukin-2 receptor expression on activated T cells. Basal- and endotoxin-induced macrophage interleukin-1 and superoxide production were not significantly influenced by supplemental arginine although macrophage cytotoxicity in response to gamma-interferon was augmented (p = 0.07). In subsequent in vitro studies we established that certain effects (NK, cytotoxic T-lymphocyte, and T cell activation) were reproducible after preincubation for 72 hours in pharmacologic levels of arginine. The data strongly suggest that arginine mediates a direct effect on components of the immune system, which results in enhanced production or use of lymphokines.

The influence of natural killer cells in neuroblastoma.

Human neuroblastoma (NRB) cell lines are markedly sensitive to natural killer (NK) cell lysis in vitro, but patients with NRBs have low or absent NK activity. This study evaluated the NK sensitivity of murine NRBs (C1300 and TBJ) in the regulation of NRB growth and determined the effects of recombinant (r) interferon gamma and recombinant interleukin 2 (rIL-2). Both basal (8% +/- 3% specific cytotoxicity) and induced (20% +/- 3%) NK lyses of C1300-NRB were observed. In vivo depletion of NK cells with anti-asialo GM-1 significantly enhanced growth of C1300-NRB and decreased survival. Treatment with r-interferon gamma or rIL-2 on days 1 through 3 after C1300-NRB inoculation significantly prolonged the mean tumor latency period, decreased the tumor growth rate, and enhanced in vitro NK killing of C1300-NRB and YAC-1. The effects of r-interferon gamma and IL-2 were abrogated by pretreatment with anti-asialo GM-1. These results demonstrated that NK cells form one important component of regulation of a murine NRB, but immunomodulation with potent lymphokines requires cooperation of more than one cell type.

Tumor immunization. Improved results after vaccine modified with recombinant interferon gamma.

The purpose of this study was to determine if a 3-day in vitro culture of the murine neuroblastoma C1300 with 500 U/mL of recombinant interferon gamma resulted in a protective crossreactivity to parent C1300. Twenty A/J mice received either a vaccine of 1 x 10(6) irradiated C1300 tumor cells intradermally or an equivalent inoculum of C1300 that had been incubated in recombinant interferon gamma (C1300*). One week later, all animals were rechallenged with 1 x 10(6) viable C1300. Animals immunized with C1300* had a significantly delayed early tumor incidence that translated into a survival advantage for the group. At the time of tumor rechallenge, a significantly increased level of nonspecific systemic immunity was conferred by the C1300* immunization. Thus, modification of tumor with recombinant interferon gamma before introduction as a vaccine may improve that vaccine's protective capability.

Evaluation of operative stress and peritoneal macrophage function in minimally invasive operations.

BACKGROUND: Laparoscopic operative procedures have decreased postoperative pain and the length of hospitalization. In addition, evidence supports a physiologic benefit from laparoscopic surgery. By analyzing several parameters of peritoneal macrophage function, we report a comparison of the magnitude of postoperative stress between two types of minimally invasive access techniques contrasted with an open laparotomy, in a murine model. STUDY DESIGN: Immature male A/J mice were exposed to pneumoperitoneum using carbon dioxide, gasless suspension, or laparotomy. Peritoneal macrophages were then harvested, and the number and viability of the macrophages from each group of mice were compared. Last, as a marker of postoperative stress, the in vitro production of nitric oxide and tumor necrosis factor alpha by these macrophages was determined. RESULTS: The number of peritoneal macrophages and the viability of the macrophages in the laparotomy group were significantly decreased 4 hours after operation compared with the minimally invasive and control groups. In addition, macrophage production of tumor necrosis factor alpha and nitric oxide, two markers of macrophage stress, 24 hours after operation was significantly increased in the laparotomy group compared with animals serving as controls. Gasless suspension and pneumoperitoneum decreased the number of macrophages to a lesser degree than did open laparotomy and did not affect macrophage viability. Moreover, gasless suspension and pneumoperitoneum did not lead to an increase in tumor necrosis factor alpha or nitric oxide production by peritoneal macrophages. CONCLUSIONS: Postoperative stress, assessed by a decrease in macrophage viability and an increase in cytotoxic cytokine production, is maximized after laparotomy compared with stress in murine hosts that underwent minimally invasive treatment. These data provide basic scientific evidence for the possible physiologic benefit of minimally invasive techniques.

Alternative techniques of feeding gastrostomy in children: a critical analysis.

BACKGROUND: In the era of managed care, the operative procedure applied to solve a given problem should vary with the status of the patient, the training and experience of the specialist, an analysis of morbidity and mortality rates, and a cost analysis of therapeutic alternatives. The purpose of this study was to critically analyze three different techniques for gastric feeding access in children. STUDY DESIGN: A retrospective analysis of patients who underwent primary feeding gastrostomy was performed at our institution. Patients who underwent gastrostomy placement concurrently with another major procedure were excluded. RESULTS: Over a 36-month period, 98 children underwent placement of a feeding gastrostomy by one of three alternative techniques: an open Stamm gastrostomy (Stamm, n=47), a pull-out percutaneous endoscopic gastrostomy (PEG, n=32), or an antegrade percutaneous fluoroscopically guided gastrostomy (PFGG, n=19). An open gastrostomy was performed more frequently in younger patients (average age, 49.7+/-11.9 months for PFGG). The sex distribution and indication for tube placement were similar in all groups (altered mental status: Stamm 43 percent, PEG 19 percent, and PFGG 38 percent; mechanical feeding difficulty: Stamm 66 percent, PEG 13 percent, and PFGG 21 percent; or failure to thrive Stamm 58 percent, PEG 17 percent, and PFGG 25 percent). Complications were most common in this high-risk patient population with PEG (19 percent), when compared with PFGG (16 percent) and Stamm (11 percent), although these were not statistically significant. Whereas reflux was frequent (Stamm 6 percent, PEG 9 percent, and PFGG 21 percent), only three patients in the entire series required a subsequent antireflux operation during the observation period. The three procedures were similar on hospital charge analysis (Stamm $1,316,29+/-63.33. PEG $1,130.04+/-94.88, and PFGG $1,079.83+/-109.12). When professional fees were included, the PFGG may be more economical than both the PEG and Stamm gastrostomy (Stamm $3,101.29+/-73/33. PEG $3,314.04+/-94.88, and PFGG $1,485.77+/-74.41, p<0.05). However, this may be misleading because the radiologist's fee was absorbed into the hospital charge is some cases, and therefore could not be fully accounted for in the total professional fee. CONCLUSIONS: The data from our institution demonstrate that there is no significant difference in these three feeding-access techniques when comparing procedural cost-effectiveness, indications for tube placement, or morbidity rates. The choice of procedure should be individualized giving consideration to the overall health of the child, the comfort of the specialist peforming the given procedure, and the institutional experience.

Intestinal ischemia: reduction of mortality utilizing intraluminal perfluorochemical.

This study reports an assessment of a method of intestinal protection by the intraluminal administration of an oxygenated perfluorochemical, perfluorotributylamine, or gaseous oxygen in an attempt to provide oxygen for mucosal cells rendered ischemia. A model of acute arterial or acute arteriovenous ischemia was produced in adult female rats. All control ischemic animals died within 12 hours. To assess intestinal protection, the mortality rates in the experimental groups were compared with the 100 percent control mortality. Gaseous oxygen and oxygenated perfluorochemical administered intraluminally reduced mortality significantly in the acute arterial model but not in rats with arteriovenous occlusion. Electron microscopy demonstrated preservation of villi after 1 and 2 hours of occlusion in the oxygen-treated groups. Light microscopy revealed massive destruction in control animals with preserved architecture in both the gaseous oxygen and oxygenated perfluorochemical groups. These results demonstrate that the intraluminal delivery of oxygen to bowel rendered ischemic by arterial occlusion may significantly decrease anatomic bowel disruption and improve animal survival. Such techniques have a potential clinical application to facilitate salvage of ischemic intestine and to augment intestinal preservation.

Central venous catheter access.

Central venous access for the purpose of supplying parenteral nutrition to the pediatric age group requires a careful definition of the patient's caloric need, estimated duration of therapy, and an assessment of available sites. Peripheral vein parenteral nutrition is limited by caloric density of the nutrient fluids, while peripherally inserted central catheters (PICC) offers a new technology for accessing central veins while obviating the risk of central vein access. Routes of central vein access are several and there are also a variety of catheters available for placement. Tunneled percutaneous placement of silicone rubber cuffed catheters via the subclavian vein approach is that technique we use most commonly. The risks of such access catheters include mechanical risks of placement, venous thrombosis of the access sites, and most importantly catheter related infections, either at the exit site, the subcutaneous tunnel or pouch, or even generalized sepsis. With a full knowledge of the spectrum of access techniques, access materials, and risks, safe total parenteral nutrition can be safely delivered to the children in need.

Tumor immunogenicity--the prime determinant of the nutritional influence on the host-tumor relationship.

The influence which malnutrition plays on the host-tumor relationship is controversial because of the disparity of human and rodent tumors, a critical difference being the minimal immunogenicity of human tumors and the variable antigenicity of rodent tumors. The hypothesis we tested is that the influence of malnutrition on tumor growth is a result of the immunogenicity of the host's tumor. C-1300 neuroblastoma (NB) is an immunogenic tumor by in vivo and in vitro assessment while the histologically identical TBJ-NB clone is non-immunizing. Isogeneic A/J mice were malnourished with 2.5% protein chow and were inoculated with C-1300-NB or TBJ-NB; either serial tumor volumes were assessed by three-dimensional measurement or animals were serially killed and tumor weight/carcass weight ratios (TW/CW) were calculated. Non-immunogenic TBJ-NB grew more rapidly than C-1300-NB in both control and malnourished groups, but there was no difference in either tumor size or TW/CW ratios between the two TBJ-NB nutritional groups. Contrasting with these data were immunogenic C-1300-NB in that the tumor grew significantly better in malnourished mice (tumor volume p less than 0.05 day 12 and 14; TW/CW p less than 0.026 by day 21). Prior whole-body irradiation abrogated this difference. These data demonstrate that for tumors differing only in antigenicity the influence of malnutrition is on that tumor which induces an immunologic antitumor response.

Simultaneous bilateral central catheters: a safe technique.

Optimal central catheter care includes restriction usage for blood sampling and blood product administration on enhance continued sterility, but our experience with 25 children receiving bone marrow transplants after cytoreduction challenges this concept. Prior to transplantation, bilateral percutaneous subclavian vein silastic catheters were inserted without incident, one utilized for continuous nutritional support in caloric quantity to assure body weight maintenance, and the contralateral catheter utilized for daily venous sampling plus administration of medications including blood products. Patients subsequently entered a protective environment and bi-weekly surveillance cultures were monitored. Nutritional therapy was given for 876 days through 53 catheters. One patient developed culture-proven sepsis, an organism first cultured from the skin. The patient complication rate of 4% and the per diem rate of 0.11% in this immunocompromised population compares favorably to the 10.5 and 0.32% incidence we previously reported for 200 children with unilateral catheters. These data demonstrate that bilateral central catheters can be safely utilized in children for nutrition and sampling.

Management of the short bowel syndrome in the pediatric population.

The short bowel syndrome in the pediatric population most commonly results from neonatal necrotizing enterocolitis. Multiple remedial surgical procedures have been developed to manage the rapid intestinal transit, decreased mucosal surface area, ineffective peristalsis, and short intestinal length in these patients. Despite significant morbidity, the overall outcome is favorable and warrants aggressive nutritional support, medical management, and surgical intervention in selected patients.

Short bowel syndrome in infancy: etiology and management.

Total parenteral nutrition permits normal growth and development of the infant during the adaptive phase of the short bowel syndrome. Skilled nutritional support combined with a variety of medical and surgical manipulations facilitates survival of infants with as little as 15 to 20 cm of intestinal length.

Nutritional care of the surgical neonate.

The surgical neonate is at great risk of developing nutritional deficiencies. However, recent advances in nutritional support, including the development of special elemental diets, infant formulas, nutritional supplements and more suitable additives for parenteral nutrition, have allowed health professionals effectively to prevent the occurrence of malnutrition in sick neonates. Strict monitoring of dietary intake, growth velocity, and biochemical parameters are essential while providing nutritional support. The complication of parenteral nutrition can be reduced by following standard recommendations for preparing solutions, caring for the central and the peripheral catheters, and biochemically monitoring the infant. The organization of a hospital nutrition support service composed of physicians, nurses, nutritionists, and pharmacists is essential in establishing guidelines for the safe delivery of nutritional therapy and in educating hospital personnel and parents on how to best meet the nutritional needs of the surgical neonate.

Serum hexosaminidase, host age, and nutritional status.

Hexosaminidase (HEX), an intestinal lysosomal acid hydrolase, has been suggested to be a useful early marker for both ischemic intestinal injury and recovery. The experimental results analyzing HEX levels in animals of varied ages, nutritional status, and stress levels reported here challenge that principle. In an experiment in which fetal malnutrition was induced by maternal restriction, normal and malnourished, stressed and unstressed 7-day-old pups were analyzed. In no circumstance was intestinal injury documented by either light microscopy or by proton nuclear magnetic resonance relaxation times, but in the 25 animals, serum HEX levels were significantly elevated in the malnourished group (2,108.7 v 1,724.6, P less than .001). In an additional experiment, 117,23-day-old weanling rats were randomized to receive protein restricted 2.5% or 8% chow whereas controls were fed standard 24% chow. Throughout 14 days rats were analyzed for growth (percentage of weight change) and serum HEX concentration. A variable level of serum HEX was seen during subsequent animal growth, but at each assessment values were uniformly higher in the 2.5% diet group, the group that was malnourished and grew less well (4% body weight gain with the 2.5% diet by day 14 v 76% weight gain with the 8% diet v 153% gain with the control 24% diet). (Table: see text). These data suggest marked variability in serum HEX levels with subject age and nutritional status, both factors likely to be highly variable in that patient with ischemic intestinal injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of primary tumor site on lymphocyte antitumor activity.

Clinical Stage IV-S neuroblastoma involving a primary, the liver, bone marrow, and/or skin has a favorable prognosis despite a considerable tumor burden. In a pilot experiment utilizing mouse C 1300 neuroblastoma, we demonstrated that animals receiving tumor to IV-S sites had a prolonged survival. To analyze the role which liver modulation of tumor might play in an immunologically mediated host-antitumor response, we applied an in vitro lymphocyte blastogenesis assay. Host-tumor response as reflected by blastogenesis and as quantified by tritiated thymidine incorporation into DNA demonstrated that normal lymphocytes were stimulated to a greater degree by subcutaneous site tumor than by tumor from the liver (260.7 +/- 71.9 vs. -54.2 +/- 194.3 CPM, p less than 0.05). Sensitized lymphocytes harvested from liver tumor bearers demonstrated a greater response when admixed with subcutaneous tumor in vitro (771.9 +/- 300.4 vs. 45.9 +/- 277.5 CPM, p less than 0.05) and sensitized lymphocytes harvested from subcutaneous tumor bearing animals also demonstrated more in vitro blastogenesis to subcutaneous tumor (577.9 +/- 200.2 vs. 98.9 +/- 154.1 CPM, p less than 0.05). However, host lymphocytes were themselves comparably reactive whether they were harvested from liver tumor or subcutaneous tumor donors (771.9 +/- 300.4 vs. 577.9 +/- 200.2, p = NS). These data suggest that lymphocytes in contact with murine liver neuroblastoma respond less well than when in contact with subcutaneous site tumor, a form of afferent blockade which may account for the propensity of the liver to serve as a site for metastatic disease.

Immunodepression secondary to malnutrition: assay by lymphocyte subset analysis using monoclonal antibodies and the cell sorter.

It has been well documented that the hospitalized child frequently is malnourished, and the considerably greater morbidity and mortality of such children, in large part, is due to the associated secondary immunodepression. To assess the mechanism of such immunodepression in acute and chronically malnourished subjects, we chose an experimental murine model of malnutrition. Immune function was assayed by lymphocyte subset population and mixed lymphocyte culture (MLC) assessment, determining whether an alteration of the T helper (TH) to T suppressor-cytotoxic (TS-C) lymphocyte ratio is the mechanism that produces immunoincompetence in malnutrition. Ten-week-old A/J mice were rendered acutely or chronically malnourished by graded protein restriction using a 2.5% protein diet. These animals were studied before and after protein depletion with the fluorescent activated cell sorter (FACS) and the MLC. FACS cell populations were defined by the monoclonal antibodies to THY 1.2, LYT 1 (TH), and LYT 2 (TS-C) antigens. MLC reactivity was assessed with A/J v C 57 BL/6 mouse lymphocytes and expressed as a stimulation index (S.I.). Results are expressed in Table 1, and suggest that these malnourished animals were significantly immunodepressed as measured in the MLC reaction. However, the mechanism of this depression is not mediated by an inversion of the TH/TS-C ratio. These data contrast with those previously reported for the immunodepression of the burned or traumatized patient in which the TH/TS-C ratios are reversed; these data suggest that a different, as yet to be elucidated, mechanism exists for the immunodepression of malnutrition.

Electrocoagulation induced immunity--an explanation for regression of neuroblastoma.

Electrocoagulation of tumor potentiates the host's antitumor immune response. Utilizing a murine neuroblastoma system and comparing surgical excision versus electrocoagulation we have demonstrated that electrocoagulation potentiates the immunogenicity of the neuroblastoma, potentiates the host antitumor immune response to residual primary tumor, and potentiates the host antitumor immune response to distant autochthonous neuroblastoma. These data suggest that such a heightened antitumor response might account for spontaneous regression of massive clinical neuroblastomas treated by electrocoagulation, and they suggest that electrococoagulation may be useful as a treatment modality for cytoreductive neuroblastoma surgery.