Radiometric Method for Detection of Bacteremia

A study was performed with simulated blood cultures to evaluate the production of (14)CO(2) as an index of bacterial growth. With a range of inoculum sizes from 4 to 4,250 colony-forming units, it was not possible to detect (14)CO(2) within 6 hr after inoculation in 59 separate experiments. In a limited trial with patients' blood cultures, the radiometric method failed to provide any earlier evidence of bacteremia than did routine broth cultures.     

A high-rate perfusion bioreactor for plant cells

A perfusion bioreactor allowing continuous extraction of secondary metabolites was designed and challenged for Eschscholtzia californica plant cell suspensions. Four sedimentation columns mounted inside a 2.5-L bioreactor separated single cells and cell aggregates from the culture medium. Cells were elicited with chitin at day 4 and the liquid medium free of cells and debris was then continuously pumped to the extraction columns containing fluidized XAD-7 resins, and then recirculated back to the cell suspension. A medium upward velocity corresponding to cell sedimentation velocity maintained a stable cell/medium separation front in the columns for sedimented cell volume (SCV) of 90% (70% packed cell volume, PCV). Two perfusion bioreactor cultures of 10 and 14 days were performed. A maximum dilution rate of 20.4/day was reached from day 4 to day 6, and was then reduced to 5/day at day 9 for 55% SCV. Control cultures were performed without and with free extraction resins into the cell suspension. Perfusion cultures showed similar specific growth rates of 0.24 +/- 0.04/day before and after elicitation. However, production level in the perfusion cultures was similar to that from the culture without resins with a maximum of 2.06 micromole/gDW total alkaloids, with 1.54 micromole/gDW in the resins. Cultures with free resins resulted in 30.94 micromole/gDW with 28.4 +/- 8.8 micromole/gDW in the resins. Difference in the cells nutritional state from elicitation was identified as a major cause in the production reduction. However, pathway to chelilutine was favored in the continuous extraction culture.     

Defective Fc-mediated phagocytosis in gamma-irradiated mouse peritoneal macrophages

Ingestion of bovine red blood cells opsonized with IgG, by irradiated and control cultures of mouse peritoneal macrophages, was monitored at various times following exposure to 7.5-20 Gy of 60Co. Radiation produced decreases in the percentage of phagocytic cells and reduced the phagocytic index of the macrophages at 6-10 days post-irradiation. Only a small decrease in the phagocytic index of irradiated cultures was noted on day 3 post-irradiation. Cell survival as monitored by cell number and lactic dehydrogenase release as well as the levels of beta-glucuronidase and lysozyme were less sensitive to radiation exposure than was the phagocytic ability of the cultures. Addition of 8-bromo-3',5'-cyclic adenosine monophosphate and prostaglandin E2 to cultures increased the phagocytic ability of both irradiated and control cultures but did not abolish the deficit produced by radiation. The data indicate that in vitro radiation exposure produces time-dependent changes in the ability of mouse peritoneal cells to ingest IgG coated red blood cells.     

A model culture system with human gingival fibroblasts for evaluating the cytotoxicity of dental materials

Summary A model experimental culture system and protocol are described to screen polymerized dental materials for diffusible toxic products. The system employs cultures of human gingival fibroblasts grown in plates containing immobilized samples of polymerized resins. Comparative cytotoxicity is evaluated by counting viable cells with the aid of phase optics at several time periods up to 48 h. To achieve adequate statistical sampling, multiple counts are made in four different zones at 90° angles from each sample and at three distances from the centers of samples. The most significant data were generated during a 24 to 48 h test period in culture. This cytotoxicity test measured cell death as a function of time of exposure and distance from the sample (24 h, 0 to 3 mm; 48 h, 3 to 6 mm) and permitted a calculation of the relative cytotoxicity for each material, which is termed the viability index (VI). This can be expressed as a percentage related to the control, which is called the time-distance cytotoxicity index (TDCI). This method is simple to carry out because it uses basic laboratory equipment, is rapid, and has a sound scientific basis. It focuses on times and distances when or where, or both, the greatest cellular changes are taking place. Some data illustrated are based on the screening of eight different restorative resins. The literature of cell culture testing of dental materials is reviewed. It is concluded that biotoxicity studies ideally should employ diploid human target cells from the oral cavity because the cells retain specialized features. Secondary cultures or strains of human diploid gingival fibroblasts, which are relatively easy to obtain and maintain, are recommended as cells of choice for screening dental restorative materials in vitro. This project was supported in part by an intramural grant from the Louisiana State University School of Dentistry, derived from BRSG Grant S07-RR-05704-10 awarded by the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health, Bethesda, MD.     

彩色多普勒超声对乳腺癌新辅助化疗疗效的评价研究

目的:探讨彩色多普勒超声(CDFI)对乳腺癌新辅助化疗(NCT)疗效评价的临床应用价值。方法:选取2013年1月~2013年12月在我院接受新辅助化疗后行手术治疗的女性乳腺癌患者55例,以病理学评价为金标准,化疗后根据化疗效果分为有效组和无效组,利用CDFI观察患者NCT前后病灶超声指标、病灶内血流分级及阻力指数(RI)值变化。结果:55例患者中,临床触诊疗效评价符合率为36.4%,敏感度为60.7%;CDFI评价符合率为70.9%,敏感度为85.7%。CDFI检查显示,乳腺癌NCT后原发肿瘤病灶大小显著缩小,边界多清晰可见,内部回声及后方回声倾向正常。有效组NCT前后病灶内的血流类型和RI值变化有统计学意义(P0.05),无效组NCT前后病灶内的血流类型和RI值无明显变化(P0.05)。结论:CDFI技术可对乳腺癌NCT前后病灶大小及病灶内部血流动力学变化提供客观参数,是评价乳腺癌NCT疗效的有效方法。     

Effect of polymeric adsorbents on the production of sanguinarine by Papaver somniferum cell cultures

The suitability of adsorbent polymeric resins, Amberlite XAD-4 and XAD-7 (Rohm and Hass, Inc.), was investigated for the accumulation of sanguinarine from Papaver somniferum cell cultures. The adsorption and desorption of sanguinarine from aqueous solution was most effective with XAD-7. In addition to sanguinarine, the resins were found to absorb growth regulators and vitamins from the culture medium. Growth inhibition was overcome by delaying for approximately 4 days resin addition after cell inoculation in fresh medium. Resin addition (5% wt/vol) to actively growing uneclicited cultures led to increases in sanguinarine production and release of 30% to 40% and 60%, respectively. The addition of resins to elicited cultures led to increases in alkaloid production of up to 50% to 85% with similar increases in alkaloid release as observed for nonelicited cells. Overall yield of sanguinarine increased from 21 mg . g biomass dry weight(-1) (dw) for elicited cultures to more than 39 mg . gdw(-1) when elicitation was combined with resin addition. Higher quantities of resin (10% to 20% wt/vol) increased marginally the release of sanguinarine into the medium, and on the resin, up to 85% of total production. The use of resin appears promesing for the development of a bioprocess for sanguinarine production by cultured plant cells. (c) 1992 John Wiley & Sons, Inc.     

Human nonspecific killer cells

The NK and K-cell activity of human leukocytes was investigated as compared with those cells of the K 562 cell line and murine cells covered by xenoantibodies in Graffi erythroblast leukaemia by means of the 51Cr release test. NK and K-cells could be identified in the blood and bone-marrow. However, they could not be identified in the thymus, lymph-nodes, and tonsils. Attempts of cell fraction with the blood of healthy donors revealed that the K-cells must be attributed to non-T-lymphocytes. NK-cells may be found in the fraction of non-T-lymphocytes as well as in that of T-lymphocytes. Killer cell activity tests in children with acute leukaemia resulted in leukaemia cells having NK and K-cell activity only in very rare cases. ALL patients in remission had strongly lowered NK-cell values under chemotherapy. In comparison to that, chemotherapy had no influence on K-cell activity. On the one hand, NK-cell activities were induced in mixed cultures of allogenous lymphocytes of the blood and, on the other hand, in cells of lymph-nodes. Attempts of fractionation, investigations for determining the influence of chemotherapy and attempts of inducing killer cell activity in vitro lead to the conclusion that NK and K-cells may be regarded as similar cell populations, being, however, not identical.     

Cultivation of Bordetella pertussis in liquid semisynthetic nutrient media with ion-exchange resins

The possibility of optimizing the processes of B. pertussis cultivation carried out in liquid semisynthetic culture media with strong-base anion-exchange resins is shown. The use of strong-base ionites enhances the reproducibility of cultivation processes, increases the activity of microbial populations and the concentration of microorganisms. B. pertussis cultures grown in the media with anion-exchange resins are characterized by pronounced biological activity.     

Different maturation capabilities of chronic lymphocytic leukaemia lymphocytes in vitro

Peripheral blood lymphocytes from five patients with B-derived chronic lymphocytic leukaemia were stimulated by Staphylococcus aureus strain Cowan together with T cell mitogen phytohaemagglutinin in 5-9 days suspension cultures. The responses of B lymphocytes were studied on a T cell depleted subpopulation, obtained from harvested lymphocyte cultures using the sheep red blood cell rosette technique. Proliferation tests were performed using a 3H-TdR blast cell index. The maturation process of B-lymphocytes was examined with cytoplasmic Ig studied by FITC-conjugated antisera. Results analysed indicate various degrees of maturation of B cells in different patients.     

Formation of broad-spectrum antibiotics by cultures of the genus Micromonospora]

Data on the study of antibiotic production by the representatives of Micromonospora and the use of ion exchange resins for intensification of screening antibiotic-producing organisms among Micromonospora are presented. It was found that out of 172 strains of Micromonospora tested 92 (53.5 per cent) cultures produced antibiotics, 18 of which were active against gramnegative bacteria. The use of carboxylic ion exchange resins at early microbiological stages of the screening provided an increase in the frequency of finding broad spectrum antibiotics from 10.4 to 19.7 per cent.     

Bacteraemia caused by Leptotrichia trevisanii in a neutropenic patient

We describe an episode of Leptotrichia trevisanii bacteraemia in a neutropenic hemato-oncology patient receiving chemotherapy for Refractory Anemia with Excess Blasts-2 (RAEB-2). Although Leptotrichia spp. colonize the oral cavity and genitourinary tract, serious episodes of bacteraemia might occur in immunocompromised patients, particularly in those with severe neutropenia. Therefore, microbiologists should consider the possibility of Leptotrichia spp. septicemia in patients with blood cultures positive for gram negative bacilli, when routine microbiology tests fail to reveal a correct identification of the organism.     

Early stage-specific immune responses in primary experimental human hookworm infection

To determine the cellular immune response during different stages of hookworm infection, we infected two human volunteers with Necator americanus and followed their immune responses against stage-specific, crude antigen extracts through larval migration, pre-patency, and early patency. After chemotherapy, the volunteers were followed for an additional 6 months. Low-dose N. americanus infection resulted in mild clinical signs and peripheral blood eosinophilia occurred during the estimated time of arrival of juvenile worms in the intestine. After an initial rise in proliferative responses against larval and adult worm antigens, the cellular reactivity decreased until the end of pre-patency, rose again during patency, and dropped after chemotherapy. Before infection and during the course of infection, elevated concentrations of TNF-alpha were detected in supernatants of peripheral blood mononuclear cells (PBMC) stimulated in vitro with third-stage (L3) or adult worm excretory-secretory (ES) antigens, which dropped off after chemotherapy. After stimulation with L3 antigen, elevated concentrations of CCL17 were detected in supernatants during pre-patency and patency. Interestingly, a prominent rise in IL-10 secretion was detected in ES antigen-stimulated PBMC cultures during late pre-patency. During reinfection studies in endemic areas, such distinct cytokine and chemokine profiles might be additional markers to better classify egg-negative patients.     

Synergistic effect of TPA and T-cell mitogens in nonmammalian vertebrates

Summary The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was used as a comitogen with the plant lectins phytohemagglutinin (PHA) and concanavalin A (ConA) in short-term cultures of whole blood from nonmammalian vertebrates. Stimulation with TPA in addition to standard mitogens resulted in a synergistic effect, consistently yielding more metaphases than cultures stimulated with either PHA, ConA, or TPA alone and is successful with blood samples as small as 0.1 ml. The increased mitotic index makes it possible to use different banding procedures for systematic studies. Also, because the amount of blood needed is so small, this procedure, unlike other published techniques, does not require the destruction of smaller animals to do chromosome studies. This work was supported in part by National Institute of Environmental Health Science Grant 5-T32-ES07015-08 to the Environmental Toxicology Center at the University of Wisconsin-Madison.     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

Cytogenetic studies in leprosy patients before and after chemotherapy

Summary The frequencies of chromosome aberrations and sister chromatid exchanges (SCEs), cell proliferation kinetics and mitotic indices were studied in peripheral blood lymphocyte cultures of leprosy patients both before and after chemotherapy. The differences in the frequencies of chromosome aberrations and SCEs between controls, paucibacillary and multibacillary patients were found to be statistically highly significant (P < 0.001). The extent of cytogenetic damage seemed to depend on the severity of the disease. Lymphocytes of untreated leprosy patients showed a low mitotic index and a slow rate of cell proliferation. Following combined treatment with dapsone and rifampicin there was an increase, but to a lesser degree (P < 0.01), in the frequency of SCEs and chromosome aberrations while the drug combination of dapsone, rifampicin and clofazamine had a non-mutagenic effect on chromosomes of the patient. Furthermore, after drug treatment, the cell proliferation rate and mitotic indices in paucibacillary patients were comparable to that of controls. These results indicate the clastogenic potency of Mycobacterium leprae and the remedial effects that follow therapeutic drug treatment.     

Premature chromosome condensation : Conformational changes of chromatin associated with phytohemagglutinin stimulation of peripheral lymphocytes

Phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes were shown to be distinguishable from unstimulated lymphocytes by the technique of premature chromosome condensation (PCC). Greater than 70% of the PCC from lymphocytes stimulated by incubating with PHA for 18–22 h showed greatly extended PCC as compared with only 30% in the unstimulated cultures. This decondensation pattern of the PCC paralleled with the previously reported increase in the template activity of chromatin. The PCC method can be useful in determining the proliferative potential of bone marrows of leukemic patients during and after chemotherapy.     

Effects of anticoagulants upon sister-chromatid exchanges, cell-cycle kinetics, and mitotic index in human peripheral lymphocytes

The effects of 3 blood anticoagulants, heparin, acid citrate dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) were investigated using human peripheral lymphocytes. Three different endpoints were examined: sister-chromatid exchange (SCE), cell kinetics index (CKI), and mitotic index (MI). SCEs were significantly increased in cells treated with EDTA, while the CKI and MI were significantly decreased in cultures treated with either ACD or EDTA when compared to cultures treated with heparin. These results suggest that anticoagulants may produce undesired effects upon cultured cells and indicate that the type of anticoagulant should be considered carefully prior to commencing cytogenetic studies using human peripheral lymphocytes.     

Selection of microorganisms capable of degrading petroleum and petroleum products at decreased temperatures

Of 150 cultures capable of degrading petroleum at +6 degrees C, 40 strains growing in the liquid mineral nutrient medium containing petroleum (2%) as a sole source of carbon were selected. Of them, 13 cultures displaying a petroleum degradation rate exceeding 25% were selected. Abilities of these cultures and their associations to utilize fuel oil and its components--oils and benzene resins--were studied. The culture exhibiting degradation rates of fuel oil, its oils, benzene resins, and petroleum amounting to 17, 26, 10, and 51%, respectively, was selected. This culture can be used for cleanup of petroleum pollution under cold climatic conditions.     

Micronucleus assay in bovine lymphocytes after exposure to bisphenol A in vitro

Bisphenol A (BPA) [2,2-bis-(4-hydroxyphenyl) propane] is an important industrial agent, made by combining acetone and phenol, that is used extensively as a monomer in the production of polycarbonate plastics and as a precursor of epoxy resins. Micronucleus assays have served as an index of cytogenetic damage in in vivo and in vitro studies. We studied the genotoxic and cytotoxic effects of BPA on bovine peripheral lymphocytes in vitro. Lymphocyte cultures from two donors were exposed to four different concentrations of BPA (1?×?10^?4, 1?×?10^?5, 1?×?10^?6, and 1?×?10^?7 mol.L^?1) for 48 h. The highest concentration of BPA (1?×?10^?4 mol.L^?1) resulted in a significant increase in the number of micronuclei in comparison with the negative control (67.50?±?2.121/1,000 binucleated cells versus 36.0?±?5.657/1,000 binucleated cells in the DMSO control, P??=??0.018). BPA did not affect the nuclear division index at any treatment concentrations. The present results thus demonstrate a significant genotoxic effect by BPA on bovine peripheral lymphocytes in vitro, only at the highest concentration.