CD40-CD154 interactions between macrophages and natural killer cells during sepsis are critical for macrophage activation and are not interferon gamma dependent.

Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.

Mucopolysaccharidosis VII in a cat.

Mucopolysaccharidosis VII was diagnosed in a domestic shorthair cat from California. The cat was small and had multiple abnormalities, including a small body disproportionate to the size of the skull, angular deformities of the ribs, abnormally short forelimbs, luxating patellas, generalized epiphyseal dysplasia involving the vertebrae and long bones, cuboidal vertebrae, pectus excavatum, subluxation of both hips, osteosclerosis of the tentorium cerebelli and left petrous temporal bone, tracheal hypoplasia, and corneal clouding. Beta-glucuronidase activity was markedly decreased in peripheral blood leukocytes. The cat died at 21 months of age, and a complete necropsy was performed. Tissues were examined by light and transmission electron microscopy. Large clear, round vacuoles representing distended lysosomes were present in many epithelial and connective tissue cells, including fibrocytes, chondrocytes, smooth muscle cells, hepatocytes, astrocytes, and macrophages.

Quantification of phagocytosis in human neutrophils by flow cytometry.

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

Hemimegalencephaly and focal megalencephaly in tuberous sclerosis complex.

We describe two children with complex cortical malformations as well as the typical intracranial manifestations of tuberous sclerosis complex. One child had hemimegalencephaly and the other had extensive focal megalencephaly. These cases are discussed in terms of the current concepts of cortical malformations.

Treatment of pneumonia in mechanically ventilated trauma patients. Results of a prospective trial.

OBJECTIVES: To determine the efficacy and magnitude of associated adverse effects of 2 different antibiotic regimens for the treatment of pneumonia in intubated surgical patients and to assay and compare blood samples and bronchoalveolar lavage fluid with respect to some host-defense parameters, especially in patients with unilateral pneumonia. DESIGN: Randomized, prospective, unblinded clinical comparison of 2 treatment arms with respect to intent to treat and clinical and microbiologically evaluable patients. SETTING: Six university surgical services in teaching hospitals with modern and well-staffed intensive care units. INTERVENTIONS: The consistency and objectively of the diagnosis of pneumonia was improved by the use of a grid of diagnostic parameters. Aggressive mechanical approaches to pneumonia in intubated surgical patients were supplemented by therapeutic use of aztreonam and vancomycin hydrochloride or combined imipenem and cilastatin sodium. RESULTS: Patients randomized to the aztreonam-vancomycin group were somewhat more ill, fared slightly better, and had fewer serious drug-related side effects than did those treated with imipenem-cilastatin (all P > .05). Immunologic parameters assessed by evaluation of bronchoalveolar lavage fluid showed differences between infected pulmonary lobes and noninfected ones; some changes were also noted in patients who recovered compared with those whose pneumonia persisted or recurred. CONCLUSIONS: Clinical studies of pneumonia in surgical patients need to be stratified to assure comparability, to identify patients in whom treatment is likely to fail, and to display differences between more and less effective therapies. Studies of blood and bronchoalveolar lavage samples showed that certain local and systemic immunologic parameters correlate with clinical status and outcome.

Bacterial cell wall products increase monocyte HLA-DR and ICAM-1 without affecting lymphocyte CD18 expression.

Bacterial cell wall products such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP) have the capacity to enhance immune responses to antigens. The expression of surface class II major histocompatibility antigens and the costimulatory receptors CD18 and CD54/ICAM-1 (intercellular adhesion molecule) was used to evaluate the comparative influence of these immunostimulators. On monocytes, both LPS and MDP increased the expression of human leukocyte antigen (HLA)-DR (maximal at 6 hr), CD18 (maximal at 1-3 hr), and ICAM-1 (maximal at 18-24 hr for LPS and 12 hr for MDP) without increasing the production of superoxide. MDP-induced ICAM-1 expression on monocytes returned to baseline values after 12 hr. On lymphocytes, only LPS increased ICAM-1 (after 18 hr) without affecting CD18, and a differential analysis demonstrated a generalized ICAM-1 upregulation in lymphocyte subsets after 18 hr: the most pronounced effect was measured in natural killer cells, followed by CD8(+) T cells, B cells, and CD4(+) T cells. MDP did not alter ICAM-1 or CD18 expression on lymphocytes. These similar but smaller effects of MDP may, in part, explain the lesser toxicity of MDP when compared to LPS.

Constituent analysis may permit improved diagnosis of intra-abdominal abscess.

BACKGROUND: Intra-abdominal abscesses (IAA) often fail to resolve with intravenous antibiotics alone and frequently require drainage. Diagnosis of IAA in postoperative patients with other likely sources of infection is very difficult. PATIENTS AND METHODS: In order to characterize IAA and identify parameters that might facilitate diagnosis, we prospectively examined peripheral blood and pus of 15 consecutive patients with IAA and compared them to samples from 34 consecutive patients with soft-tissue abscesses (STA). RESULTS: Serum interleukin (IL)-10 was elevated in IAA patients, while abnormally detectable serum IL-4 was demonstrated in the pus of both IAA and STA patients. IL-10 in IAA pus was more than 11-fold higher than in STA, whereas IL-4 in pus was similar in both types of abscesses. Both IL-4 and IL-10 were 4- to 10-fold higher in IAA and STA pus than in corresponding patient serum. Serum lysozyme was, however, significantly elevated in all abscess patients. CONCLUSIONS: The presence of IL-4 and IL-10 may indicate a T-helper 2 lymphocyte response in the etiology of abscess formation and persistence, although precise determination of T-helper 1-related cytokines is needed to verify this. Serum lysozyme and IL-10 may be reliable and relatively inexpensive diagnostic aids.

Assessing the role of routine choledochoscopy during exploration of the bile duct.

Two hundred and five consecutive explorations of the common bile duct were evaluated to assess the value of routine choledoschoscopic examination in preventing retained stones. Conventional exploration was performed in 139 instances (group 1), while routine choledochoscopy was added in 66 procedures (group 2). Preoperative temperature, leukocyte count, total bilirubin, amylase and alkaline phosphatase levels were not significantly different between the groups. Nine retained stones were demonstrated by postoperative cholangiography among the patients in group 1 as compared with three retained stones noted in those in group 2. This difference was not significant. Postoperative mortality and complication rates were unaffected by the performance of an endoscopic study of the biliary tract during ductal exploration. Choledochoscopy added an average of 36 minutes to the duration of the operative procedure (p less than 0.001). Choledochoscopy may prove helpful in selected instances, but the current data do not support its routine use during exploration of the common bile duct.