William Browder

Serum Glucan Levels Are Not Specific for Presence of Fungal Infections in Intensive Care Unit Patients

ABSTRACT Fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. A major obstacle to managing fungal infection is the lack of a reliable clinical assay that will rapidly identify patients with fungal sepsis. Glucans are polymers of glucose that are found in the cell wall of fungi and certain bacteria. Glucans are also released from the fungal cell wall into the extracellular milieu. Several studies have reported that detection of fungal glucan in serum or plasma is useful in the diagnosis of mycoses. However, recent studies have questioned the clinical utility of this assay. In this study, we examined serum glucan levels in intensive care unit (ICU) patients and attempt to correlate serum glucan levels with the presence of fungal infection. Following attainment of informed consent, serum was harvested from 46 ICU patients with confirmed fungal infections, confirmed bacterial infections, or no evidence of infection. Sera from eight healthy volunteers served as control. Serum glucan was assayed with a glucan-specific Limulus assay. Serum glucan levels were increased (69.6 ± 17 pg/ml; P < 0.001) in ICU patients versus the normal (11.5 ± 1.3 pg/ml) and noninfected ICU (27.4 ± 17 pg/ml) controls. However, serum glucan levels were not different in patients with confirmed fungal infections versus those with confirmed bacterial infections. Thus, serum glucan levels did not show a correlation with the presence of fungal infections and do not appear to be specific for fungal infections. However, the assay may be useful as a negative predictor of infection.

Early Activation of Pulmonary Nuclear Factor kappa B and Nuclear Factor Interleukin-6 in Polymicrobial Sepsis

BACKGROUND Transcription factor activation may be a pivotal step in the pathophysiology of sepsis syndrome and adult respiratory distress syndrome. This study investigated the activation of lung nuclear factor kappaB (NFkappaB) and nuclear factor interleukin-6 (NF-IL6) and how they correlate to proinflammatory cytokine expression and mortality in a murine model of cecal ligation and puncture (CLP). METHODS Polymicrobial sepsis was induced by CLP. Transcription factor activation was assessed at 0, 1, 2, 3, 4, 5, 6, 8, and 24 hours after CLP by the electrophoretic mobility-shift assay. Lung cytokine mRNA levels were established by reverse transcriptase-polymerase chain reaction. RESULTS CLP induced pulmonary NFkappaB activation at 3, 4, and 8 hours (p < 0.05). Lung NFkappaB activation peaked at 3 hours (533% vs. no surgery, 2,900% vs. sham treatment) after CLP. Supershift analysis revealed a predominance of p50 subunits in the lung nuclear extracts of septic mice 3 hours after CLP, indicating the presence of p50 homodimer. In contrast, liver nuclear extracts from septic mice indicated the presence of both p65 and p50 subunits at 3 hours. Lung NF-IL6 activation (p < 0.05) was observed at 4 hours (649% vs. no surgery, 296% vs. sham treatment) and 6 hours after CLP. Lung tumor necrosis factor-alpha mRNA levels were increased (p < 0.05) at all time intervals after CLP. Lung IL-6 mRNA levels were increased at 3, 6, and 8 hours after CLP. CONCLUSION Early activation of lung NFkappaB and NF-IL6 and lung cytokine mRNA expression correlated with mortality in polymicrobial sepsis. Although IL-6 mRNA levels correlated with NFkappaB and NF-IL6 activation, tumor necrosis factor-alpha mRNA levels did not, in that they preceded transcription factor activation. These data suggest a potential role for NFkappaB and NF-IL6 activation in the initiation and propagation of acute lung injury.

INHIBITION OF LPS-INDUCED NFκB ACTIVATION BY A GLUCAN LIGAND INVOLVES DOWN-REGULATION OF IKKβ KINASE ACTIVITY AND ALTERED PHOSPHORYLATION AND DEGRADATION OF IκBα

Growing evidence supports the role of transcription factor activation in the pathophysiology of inflammatory disorders, sepsis, ARDS, SIRS, and shock. Kinase mediated phosphorylation of IκBα is a crucial step in the NFκB activation pathway. We investigated IκBα phosphorylation in murine liver and lung extracts after cecal ligation and puncture (CLP) in the presence and absence of a glucan ligand. ICR mice were subjected to CLP. Unoperated and sham-operated mice served as the controls. Glucan phosphate (50 mg/kg) was administered 1 h before or 15 min after CLP. CLP increased hepatic and pulmonary levels of phospho-IκBα by 48–192%. Pre- or post-treatment with glucan phosphate decreased (P < 0.05) tissue phospho-IκBα levels in CLP mice. Phospho-IκBα in the glucan-CLP group were not significantly different from the unoperated controls. To investigate mechanisms we examined IKKβ kinase activity, IκBα phosphorylation and degradation, and NFκB activity in a murine macrophage cell line, J774a.1, treated with LPS (1 μg/mL) and/or glucan phosphate (1 μg/mL) for up to 120 min. The glucan ligand blunted LPS-induced IKKβ kinase activity, phosphorylation and degradation of IκBα, and NFκB nuclear binding activity. The data indicate that one mechanism by which (1→3)-β-D-glucan may alter the response to endotoxin or polymicrobial sepsis involves modulation of IKKβ kinase activity with subsequent decreases in IκBα phosphorylation and NFκB activation.

Protective effect of nonspecific immunostimulation in postsplenectomy sepsis

The enhanced risk of severe sepsis following splenectomy is now well recognized in both adult and pediatric patients. Prophylactic antibiotics and bacterial vaccines have been utilized with limited success to inhibit the high morbidity and mortality. This study reports the use of glucan, a beta-1,3-polyglucose, as a nonspecific immunostimulant for postsplenectomy pneumococcal sepsis. ICR mice were treated with glucan or glucose (5% w/v) following splenectomy or sham operation. Mice were then challenged with 1 X 10(9) Streptococcus pneumoniae intranasally. Glucan significantly increased survival in the splenectomy group (75%) compared to controls (27%). Phagocytic function, as measured by the clearance of 131I-triolein-labeled reticuloendothelial test lipid emulsion, was increased in the glucan group when compared to control glucose animals, both in the presence and absence of pneumococcal infection. Splenectomy alone did not significantly decrease phagocytic function. An increased leukocytosis in response to pneumococcal infection was observed in splenectomized glucan-treated animals. Nonspecific immunostimulation appears to have significant potential as a treatment strategy against postsplenectomy infection.

Malignant cystosarcoma phylloides: Treatment and prognosis

Twenty-six cases of malignant cystosarcoma phylloides treated at The Charity Hospital of Louisiana were reviewed. No metastases to axillary lymph nodes was observed. The lesion appears to metastasize seldom, if ever, to lymphatics, and axillary dissection is seldom required. Local recurrence was common, however, and justifies wide resection of the primary. Mortality was related more to the size of the lesion than to other findings such as skin or muscle involvement. No lesion with less than three mitoses per ten high power fields and minimal stromal atypia proved fatal. Associated neoplasia was common, particularly in the opposite breast.

Primary hyperparathyroidism in the seventies: A decade of change?

In an effort to identify new trends in the presentation and treatment of primary hyperparathyroidism, 66 patients treated since 1975 were compared with 100 patients diagnosed and treated from 1948 to 1970. Despite widespread use of multichannel analyzers, the late patients had an insignificant increase in diagnosis while asymptomatic (18 percent versus 9 percent in the early group). Hypertension was the most common presenting complaint in patients seen since 1975, compared with renal disease in patients seen before 1970. Findings of diffuse hyperplasia were more common in the late patients (17 percent versus 3 percent in the early patients). There were no differences in rates of operative complications or persistent postoperative hypercalcemia. In the late series of patients persistent hypercalcemia after surgery for hyperplasia was due to inadequate resection of parathyroid tissue. In the adenoma patients, failure to locate the abnormal parathyroid gland was the cause of operative failure.

Glucan Immunomodulation in Experimental E. Coli Sepsis

In spite of the increased efficacy of antibiotic therapy, sepsis, particularly with endotoxin containing gram-negative bacilli, is a serious complication in patients whose defense mechanisms have been compromised by such events as trauma, burns, hematological malignancies, x-irradiation or therapy (1,2). While a number of defects in homeostatic mechanisms have been identified in such patients, attempts to significantly alter host defense mechanisms in order to modify gram-negative sepsis have been limited, since all currently employed experimental immunotherapeutic agents profoundly increase the sensitivity of the host to endotoxins. These agents include Bacillus Calmette-Guerin (3), zymosan (4), C. parvum (5), glucan (6) and muramyl dipeptide (7).

Adverse effect of splenectomy in experimental peritonitis

Patients undergoing splenectomy have increased operative morbidity and mortality, especially when associated with gastrointestinal surgery or injury. This present study was designed to assess the effect of splenectomy on mortality in a polymicrobial fecal peritonitis model and evaluate therapy with antibiotic (cefoxitin) or immunomodulation (glucan). Human stool-barium (0.15 cc) was placed in the peritoneum of Sprague-Dawley rats at the time of splenectomy or sham surgery. Splenectomy animals were then treated with 5% dextrose, cefoxitin (60 mg im q 6 hr), glucan (7.5 mg ip prior to surgery), or cefoxitin plus glucan. Splenectomy resulted in decreased survival (5% vs 30%, P less than 0.05). Treatment with cefoxitin (90%) or glucan (47%) significantly improved survival. Combined glucan-cefoxitin therapy had no improvement over cefoxitin alone. Peritoneal and blood cultures were performed 12 hr postoperatively. There were no significant differences in growth of bacteria between sham and splenectomy animals. Cefoxitin treatment resulted in lower growth of bacteria from both blood and peritoneum (P less than 0.05). Glucan treatment caused a significant decrease in the number of bloodborne bacteria (P less than 0.05). Intravascular colloidal carbon clearance and leucocyte counts were performed at 12 hr postoperatively. Presence of peritonitis significantly enhanced intravascular clearance, while splenectomy had no effect. Addition of glucan or cefoxitin therapy to splenectomy animals did not enhance intravascular clearance. Leucocyte counts were significantly lower (P less than 0.05) when splenectomy was added to peritonitis animals. Glucan and cefoxitin therapy did not increase leucocyte counts. Based on these studies we conclude that (1) splenectomy increases mortality in fecal peritonitis, (2) antibiotic and immunomodulator afford some protection, and (3) exact mechanism of protection remains unclear.