Clinical significance of a test for slime production in ventriculoperitoneal shunt infections caused by coagulase-negative staphylococci

Coagulase-negative staphylococci (CNST) are the most-common cause of ventriculoperitoneal shunt infections. Some of these strains produce a slime-like substance. We reviewed 19 episodes of ventriculoperitoneal shunt infections due to CNST in 17 patients. Eleven episodes of infection were caused by slime-producing CNST and eight by non-slime-producing CNST. Shunt obstruction and abdominal pain occurred more frequently when infectious episodes were due to slime-producing CNST than to non-slime-producing CNST (P less than .05). Despite appropriate antimicrobial therapy, the mean duration of fever was longer and the failure to eradicate the infecting organisms was more frequent when the infectious episodes were due to slime-producing CNST than to non-slime-producing CNST (P less than .025). Discriminate function analysis found, however, that only failure to eradicate the infecting organism (by antimicrobial therapy) occurred more frequently in infectious episodes due to slime-producing CNST than to non-slime-producing CNST (P = .01).     

Growth of purified astrocytes in a chemically defined medium.

Astrocytes purified from primary cultures of neonatal rat cerebrum can now be grown in a synthetic medium supplemented with putrescine, prostaglandin F2 alpha, insulin, fibroblast growth factor, and hydrocortisone. These five supplements have a marked synergistic effect on growth when used in combination but have little effect when used individually. Astrocytes grown in the defined medium exhibit dramatic changes in morphological characteristics in comparison to cells grown in serum-free or serum-supplemented medium. In addition, these cells express the astrocyte-specific marker glial fibrillary acidic protein and are estimated by several criteria to be greater than 95% astrocytes.     

Usefulness of a test for slime production as a marker for clinically significant infections with coagulase-negative staphylococci

The usefulness of a test for slime production as a marker for clinically significant infections with coagulase-negative staphylococci and its implications for therapy were examined. Hospital records were reviewed for 59 patients from each of whom more than one isolate of coagulase-negative staphylococci was obtained. In patients with a prosthetic device, 81% of 59 infectious episodes were due to a slime-positive coagulase-negative staphylococci. In contrast, 22 noninfectious episodes (in which the organisms were contaminants) were equally distributed between episodes due to slime-positive or slime-negative isolates (P = .005). Only 32% of infections caused by slime-positive organisms, in contrast to 100% of infections caused by slime-negative organisms, were improved by treatment with antibiotics alone (P = .02). Prosthetic device removal in addition to antibiotic treatment significantly improved the outcome in patients with infections due to slime-positive organisms when compared with treatment with antibiotics alone (93% vs. 32% improvement; P = .00025).     

Differentiation of human adipocyte precursors in a chemically defined serum-free medium

Stromal-vascular cells from the inguinal fat tissue of human (age range 1.5 month-27 years), were able to undergo adipose conversion when cultured in a medium containing insulin, transferrin and triiodothyronine. Between 10 and 20 per cent of the cells changed their morphology and accumulated lipid droplets within 10 to 15 days. In most cultures, differentiated cells were present in clusters. These clustered cells were shown by indirect immunofluorescence to contain lipoprotein lipase (located in the Golgi region) and by histochemistry to contain glycerol-3-phosphate dehydrogenase. The occurrence of both enzymes was assessed directly by determining enzyme activities and the synthesis of triacylglycerol was demonstrated by incorporation of [U-14C]glucose into lipids. Foreskin fibroblasts did not display any of these phenotypes. The development of a serum-free, chemically defined medium for the differentiation of diploid adipocyte precursors from human should be of interest for the characterization of factors involved in the stimulation or inhibition of the differentiation process.     

Long-term cultivation and differentiation of human erythroleukemia cells in a protein-free chemically defined medium.

To examine whether a human erythroleukemia cell line, K-562, can proliferate and differentiate without protein supplements, long-term cultivation of the cells was carried out in a protein-free chemically defined medium. By the use of stepwise decreases in the fetal bovine serum concentration, continuous growth of K-562 cells was established in a protein-free F-10 medium. The cells have been propagated in this medium for 3 years. The population-doubling time of the cells is about 30 hr. Growth was not stimulated by the addition of insulin, epidermal growth factor, fibroblast growth factor, multiplication-stimulating activity, transferrin, platelet-derived growth factor, or dexamethasone. Addition of serum stimulated the cell growth slightly and increased the saturation density. The saturation density of the cells could be increased to that seen with serum-supplemented cultures by changing the serum-free medium daily. The cells synthesized significant amounts of hemoglobin in the presence of hemin without serum supplementation. The results suggest that the human erythroleukemia cells grown in a protein-free medium do not require serum components for their growth or hemin-induced hemoglobin synthesis and provide an excellent model for better understanding of the growth and differentiation of human leukemia cells.     

Characterization of variant gamma-glutamyl transpeptidase produced by pancreatocholangiocarcinoma cell lines in a protein-free, chemically defined medium

Four pancreatocholangiocarcinoma cell lines (HPC-Y1, HPC-YT, MIA PaCa-2, and HChol-Y1) were established to propagate in a protein-free, chemically defined medium. High gamma-glutamyl transpeptidase (GGTP) activities were showed in their spent media (designated as the secreted (GGTP). Their GGTP activities in the spent media were 125, 85, 110, and 153 IU/L/mg of lyophilized spent media, whereas GGTP activities extracted from their cancer cell lines with bromelain were 105, 37, 86, and 112 IU/L/1 x 10(6) cells, respectively. The chemical characteristics of the GGTPs in the spent media from these cell lines resembled one of the GGTPs, sialic acid-rich GGTP, extracted from normal human pancreas with bromelain treatment as follows: the GGTPs secreted from the cancer cell lines bound to an anion exchange column moved fast on electrophoresis and then showed decreased electrophoretic mobility with neuraminidase treatment, showed a high affinity for concanavalin A and lentil lectin columns, and had an acidic isoelectric point. However, the elution patterns of erythroagglutinating phytohemagglutinin (E-PHA) column chromatography and thermostability tests demonstrated clear differences between the carcinoma GGTPs both in the spent media and cell lines and the sialic acid-rich GGTP of normal pancreas, namely the carcinoma GGTPs treated with neuraminidase showed affinity to E-PHA columns, and, in addition, the GGTPs in the spent media showed an apparent heat resistance at 56 degrees C. These findings indicate that the carcinoma GGTPs have a different oligosaccharide structure from that in normal pancreatic GGTPs.     

Characterization of cultured rat oligodendrocytes proliferating in a serum-free, chemically defined medium.

A serumless, chemically defined medium has been developed for the culture of oligodendrocytes isolated from primary neonatal rat cerebral cultures. Combined together, insulin, transferrin, and fibroblast growth factor synergistically induced an essentially homogenous population (95-98%) of cells expressing glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) activity to undergo cell division. Proliferating cels were characterized by several criteria: (i) ultrastructural analysis by transmission electron microscopy identified the cell type as an oligodendrocyte; (ii) biochemical assays showed expression of three oligodendrocyte biochemical markers, induction of both glycerol phosphate dehydrogenase and lactate dehydrogenase (EC 1.1.1.27), and presence of 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37); and (iii) immunocytochemical staining showed cultures to be 95-98% positive for glycerol phosphate dehydrogenase, 90% for myelin basic protein, 60-70% for galactocerebroside, and 70% for A2B5. Few cells (less than 5%) stained positive for glial fibrillary acidic protein, and none were detected positive for fibronectin.     

An unusual presentation of sepsis caused by coagulase-negative staphylococci.

We describe a 25-year-old male presenting with fever during the non-neutropenic phase of chemotherapy. The presentation was that of a viral infection. The cause of the fever turned out to be a bacteremia with coagulase-negative staphylococci (CONS) originating from a totally implanted venous access port (VAP). We briefly discuss the different types of VAP-related infections and treatment modalities.     

Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes.

A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed. Examination of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [3H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte proliferation.     

Establishment of a human carcinoembryonic antigen (CEA)-producing cell line in a protein-free chemically defined medium

Summary To examine whether a human carcinoembryonic antigen (CEA)-producing cell can proliferate and sythesize CEA in vitro in culture without protein supplements, long-term cultivation of such cells was carried out in a protein-free chemically defined medium. Using stepwise decreases in fetal bovine serum concentration, continuous growth of the cells was established in a protein-free am's F-12 medium. The cells, designated as HLC-Yl, have been propagated in this medium for 3 years. The population doubling time of the cells is about 52 h. Addition of the serum stimulated the cell growth (population doubling time = 27 h). Saturation density was not increased by the addition of serum. The cells grown in a protein-free F-12 secrete large amounts of CEA (65.4±2.6 ng/10⁶ cells/24 h). Addition of serum did not stimulate the production of CEA. The cells produced tumours when inoculated into athymic nude mice. The mice bearing the tumour showed high serum CEA levels, and CEA was demonstrated in the tumour tissue by the immunoperoxidase method. The present study suggests that cells grown in a protein-free medium do not require serum components for their growth or CEA synthesis and provide an excellent model for better understanding the growth and production of CEA in human lung cancer cells.Abbreviations FBS fetal bovine serum - CEA carcinoembryonic antigen - PAP peroxidase anti-peroxidase - DAB Diaminobenzidine - PAS periodic acid-Schiff - PBS phosphate buffered saline - TEM transmission electron microscopy - SEM scanning electron microscopy     

Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release

Summary Microdissected pancreatic islets from non-inbredob/ob-mice, were cultured for 6 or 7 days in serum-free tissue culture medium 199. The insulin content of the islets decreased 60% during culture in 17 mM or 28 mM glucose and about 70% in the presence of 3.3 mM or 5.6 mM glucose. At the end of a culture period in high glucose, the sum of the insulin in the islet plus that in the culture medium was almost twice as high as the insulin content of fresh islets, indicating an active insulin biosynthesis. The maximal insulin response to glucose after culture in 17 mM or 28 mM glucose was about 40% of that in fresh islets; after culture in 3.3 mM glucose it was 10%. Half-maximal stimulation was observed at a glucose concentration of 5 mM for islets cultured with high glucose as compared to 9 mM for fresh islets. Like glucose, glibenclamide was a more effective insulin stimulator after culture with a high glucose concentration than with a low one. However, leucine-induced insulin release was not affected by the glucose concentration in the preceding culture medium. Whereas potentiation of glucose-stimulated release by arginine or dibutyryl-cAMP was independent of glucose concentration during the culture, theophylline released three times more insulin when the islets had been cultured with high glucose.     

Small intestinal and colonic changes induced by a chemically defined diet

Three groups of rats were fed, respectively, a chemically defined diet intragastrically (IG), an equivalent diet intravenously (IV) and solid food orally (CH) for 8 days, and their small intestines and colons compared. All received equal calories. The small intestine was divided into equal proximal (A), middle (B), and distal (C) segments for measurements. Mucosal weight per cm in segments A and B of IG were, respectively, 65 and 38% higher than in IV (P<0.01), but 27 and 33% lower than in CH (P<0.01). However, in the distal segment, C, mucosal weight in IG was similar to IV and CH was 79% higher (P<0.01). DNA and protein followed the same pattern. Segment A sucrase activities were similar in CH and IG and were much higher than in IV (P<0.01). Sucrase in IG dropped very rapidly distally so that it became much lower than in CH (P<0.05) and similar to IV. Mucosal weight, DNA, and protein in the colon were not significantly different in IG and IV, which were both signficantly lower than in CH (P<0.01). The results indicate that a chemically defined diet maintains intestinal mass well in the proximal small intestine, but the effect diminishes rapidly in a distal direction so that distal small intestine and colon become atrophied and similar to those in intravenous feeding.This study was supported by Grant MA-3320 from the Medical Research Council of Canada, and by the Foundation Justine-Lacoste-Beaubien.     

Specific glucose protection of pancreatic beta-cell function during culture in chemically defined medium.

Microdissected pancreatic islets from non-inbred ob/ob-mice were cultured for 7 days in modified tissue culture medium 199 lacking serum. When 3 mM glucose was present during culture little or no insulin response to glucose stimulation was observed during the following incubation. Culture with 18 mM glucose on the other hand resulted in good preservation of glucose-stimulated insulin release, especially if release into the culture medium had been inhibited by lack of Ca++. High concentrations of leucine or its non-metabolizable analogue, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), stimulated insulin release into the culture medium but did not preserve glucose-stimulated insulin release. When compared to previously published fresh-islet levels, culture with 3 mM glucose alone or in combination with high concentrations of leucine or BCH resulted in a substantial loss of beta-cell insulin. This loss was less marked after culture with 18 mM glucose, and completely abolished if no Ca++ was included in the high glucose medium. The data indicate that glucose has a specific effect in protecting some glucoreceptor mechanism of the beta-cells during culture.     

The measurement of C-reactive protein and immune complexes in endocarditis caused by coagulase-negative staphylococci

Coagulase-negative staphylococci are a major cause of early endocarditis following the insertion of prosthetic heart valves but rarely cause endocarditis in natural valves. From a 2-year prospective study we report seven cases: six with endocarditis related to prosthetic valves and one with endocarditis related to a natural valve. Each isolate of coagulase-negative staphylococcus was identified biochemically (six Staphylococcus epidermidis; one Staphylococcus hominis) and characterised by bacteriophage typing. Six isolates were also examined for slime production and for extracellular toxins and enzymes: all produced toxin but enzyme and slime production was variable. Concentrations of immune complexes and C-reactive protein in the serum were measured in six of the patients. Our results suggest that measuring C-reactive protein may be useful but measuring immune complexes is not helpful and takes more time.     

Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting

We describe a platform technology, termed the dock and lock method, which uses a natural binding between the regulatory subunits of cAMP-dependent protein kinase and the anchoring domains of A kinase anchor proteins for general application in constructing bioactive conjugates of different protein and nonprotein molecules from modular subunits on demand. This approach could allow quantitative and site-specific coupling of many different biological substances for diverse medical applications. The dock and lock method is validated herein by producing bispecific, trivalent-binding complexes composed of three stably linked Fab fragments capable of selective delivery of radiotracers to human cancer xenografts, resulting in rapid, significantly improved cancer targeting and imaging, providing tumor/blood ratios from 66 +/- 5 at 1 h to 395 +/- 26 at 24 h.     

Prevalence and significance of coagulase-negative staphylococci isolated from blood cultures in a tertiary hospital

Blood cultures (BC) are the most important tool in the diagnosis of bloodstream infections. However, false positive results are associated with increased laboratory costs and inappropriate antibiotic use. In order to determine the prevalence and location of blood cultures contaminated with coagulase-negative staphylococci (CNS), we performed a retrospective analysis of all blood cultures performed at St. Vincent's Hospital, Sydney during a 6-month period. From a total of 4234 patients with BC collected, CNS was isolated from 109 patients (2.6%). 94% of all CNS isolates (101/109) were contaminants. In the emergency department (ED), CNS isolates were significantly more likely to be contaminants (62/63, p<0.02) compared with the rest of the hospital, representing a 3.3% patient BC contamination rate. Treatment for a contaminant with vancomycin was significantly more likely to occur in ward patients (14/28, p<0.01) compared to the rest of the hospital. Duration of therapy did not differ across the hospital. Strategies to reduce the numbers of contaminants should be directed at medical staff in ED. Inappropriate vancomycin therapy could be curtailed by greater clinical microbiology liaison and vancomycin stewardship.