Measurement of macrophage-mediated cytotoxicity against adherent and non-adherent target cells by release of 11 indium-oxine

This report describes the utilization of 111 indium-oxine chelate ([111In]Ox) for studies of macrophage-mediated cytotoxicity. [111In]Ox efficiently labeled both non-adherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intracellularly incorporated [111In]Ox was very slow (0.25-0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [111In]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [111In]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [111In]Ox released in response to cytolytic macrophages correlated well with those observed for the 51Cr and [3H]TdR radiolabels. Therefore, [111In]Ox can be utilized for relatively short-term (less than 20h) assays with lymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity.     

A sensitive method to measure PHA-induced cytotoxicity to adherent target cells using [3H]uridine as a terminal label

This paper describes a modification of the PHA-induced cytotoxicity test of human peripheral blood lymphocytes against a tumour-derived adherent cell line (HeLa), in which the surviving target cells are labelled with [3H]uridine at the end of the assay. There is a direct correlation between [3H]uridine incorporation and the number of adherent target cells. The test proves to be very sensitive at low effector; target cell ratios. Frozen stored cells can be used in this system, a particular advantage because of the possibility of increasing the reproducibility of the assay by using the same batch of cryopreserved lymphocytes as a reference standard in each experiment. PHA-induced cytotoxicity was mainly found in the T cell enriched fraction.     

A terminal-labelling microcytotoxicity assay with 125I-iododeoxyuridine as a label for target cells

The development of a terminal-labelling microcytotoxicity assay is described in which target cells (fetal fibroblasts) were labelled with 125I-iododeoxyuridine after effector (lymphoid) cells had been incubated with them for 24 h. The time-course for the development of cell-mediated cytotoxicity was assessed following allogeneic skin grafting. 'Non-specific' cytotoxicity detracts from the sensitivity of all microcytotoxicity assays and the terminal-labelling assay using 125I is no exception. The non-specific effects can be reduced but not eliminated by the removal of adherent cells. The optimum target cell/effector cell ratio would seem to be between 1:100 and 1:250. Residual lymph node cells did not appear to incorporate enough label to affect the test results. In vivo correlates of in vitro findings are still not easy to determine.     

Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay

A case of Morris' syndrome in which the diagnosis has been realized only in old age is reported. A 69 year-old patient, with female external genitalia and secondary sexual characteristics, was referred to us with a diagnosis of a mass in the right inguinal region. Her personal history was based on a primary amenorrhoea, which was unsuccessfully investigated since she was adolescent. At the age of 63, during surgery for a left inguinal hernia realized in another hospital, a testis-like mass with the spermatic cord was casually found. During our hospitalization, a surgical removal of the right inguinal mass was performed, and the histologic examination showed the presence of a dominant sclerohyalin testicular tissue without evidence of seminal epithelium and sparse focuses of Leydig cells hyperplasia. Besides, the determination of gonadotropins and sex hormones yielded an increased production of LH, FSH, estradiol, testosterone and androstenedione. A cytogenetic analysis showed a 46, XY karyotype. The diagnosis realized only in old age has compelled the patient to live all her life, from sexual maturity, with indecision and doubt, and without a clinical explanation of fundamental utility even from the psychological point of view. Finally, in our patient the absence of cytologic aspect of malignant transformation in the removed testes in a six years period, seem fortuitous. It is always necessary to consider Morris' syndrome among the possible diseases causing primary amenorrhoea in the clinical evaluation of young phenotypic female patients.     

Macrophage scavenger receptor confers an adherent phenotype to cells in culture

Chromosomal imprints in the broadest sense can arise in somatic as well as germline cells. They can be imposed through the modification of chromosomal proteins or by the modification of chromosomal DNA, and they typically effect the expression of nearby genes. Modification enzymes--such as histone deacetylases and cytosine methyltransferases, as well as chromatin components--are known to play this role in animals and many of these same enzymes and components have been found in plants. Transposable elements are subject to chromosomal imprinting and may play a fundamental role in this process in plant and other eukaryotic genomes.     

Malarial immunodepression in vitro: adherent spleen cells are functionally defective as accessory cells in the response to horse erythrocytes

The basis for the depressed response of malarial infected mice to horse red blood cells (HRBC) has been studied in vitro. Results presented show that the adherent spleen cells from infected mice (a) are defective in their ability to allow nonadherent spleen cells of both normal and infected mice to respond to HRBC whereas a response does occur with adherent spleen cells from normal mice (b) do not suppress the response of unfractionated spleen cells from normal mice to HRBC (c) contain phagocytic cells as measured by the uptake of neutral red in numbers which are of the same order of magnitude as in adherent spleen cells from normal mice, but which are unable to take up HRBC. We conclude that a splenic adherent cell, probably the macrophage is functionally defective as an accessory cell in the response to HRBC of mice infected with Plasmodium berghei yoelii.     

Microbiologic assay for detection of antimicrobial agents in urine

Antimicrobial therapy can be a confounding factor in the diagnosis of urinary tract infection and is not always reported to laboratories by physicians. We developed a microbiologic assay for screening urine specimens for antimicrobial agents. Bacillus stearothermophilus was used as the indicator bacteria. A total of 1,921 urine specimens from three hospitals in Taiwan were screened using this assay. Of the samples assayed, 1,293 were positive for antimicrobial agents. Agreement between information provided by physicians and laboratory findings was 68.5% (419/612). In the presence of antimicrobial agents in the urine samples, the isolation of yeasts and Pseudomonas aeruginosa increased dramatically, from 4.5 to 19.5% and 4.2 to 13.2%, respectively. Additionally, Escherichia coli was more resistant to gentamicin (75.3% vs 48.7%, p < 0.0001), norfloxacin (85.2% vs 64.6%, p = 0.0006) and co-trimoxazole (58.5% vs 35.5%, p = 0.0018). In view of the high rate of occurrence of antimicrobial agents in urine specimens and the lack of information provided by most physicians to laboratories, a screening method to detect the presence (or absence) of antimicrobials in urine specimens may be a useful tool particularly in areas such as Taiwan where antimicrobial agents are commonly abused.     

Comparison of monolayer and bilayer plates used in antibiotic assay

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium-1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non-blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.     

Detection and quantification of apoptosis in transiently transfected adherent cells

Apoptosis is a highly conserved form of cell death present in all eukaryotic cell types and controlled by multiple genes. Several methods have been developed to quantify apoptosis, but none is adapted for all cell types. It is particularly difficult to reliably assay apoptosis of adherent cells. We describe a new, rapid and reliable flow cytometric method which can be used for quantifiying apoptosis in a sub-population of transiently transfected adherent cells. This technique is based on the detection of transfected cells and the apoptotic sub-population by immunofluorescence and Annexin-V labelling, respectively.     

An immunodiffusion assay for the detection of canine leishmaniasis due to infection with Leishmania infantum

An immunodiffusion assay (IDA) with polyethylene glycol (PEG) was tested for usefulness as diagnostic test for canine leishmaniasis (CL). A comparative analysis of dog sera was made using IDA with PEG, immunofluorescence assay (IFA) and enzyme immunosorbent assay (ELISA) techniques. Fourty-four dogs from Italy with CL (endemic dogs) and eight Dutch dogs with CL contracted in South Europe (expatriate dogs) were tested together with 40 endemic and 35 expatriate controls. Specificity did not differ substantially among the serotests, ELISA in endemic dogs being the least specific (mean specificity given in IFA, IDA and ELISA, 100%, 98% and 93.5%, respectively). Sensitivity in expatriate dogs was 100% for all serotests but was highly variable in endemic dogs. In parasite-negative dogs, IFA had the most sensitivity, i.e., 80.5% compared to 69% for both ELISA and IDA. In contrast, ELISA in parasite-positive endemic dogs had a sensitivity of 100% whereas both IFA and IDA gave a sensitivity of 93%. Despite its slightly lesser sensitivity than IFA or ELISA (2-6% and 5% respectively) in endemically infected dogs, IDA with PEG method may help to bring the diagnosis of CL within reach of the veterinary practitioner.     

Nested PCR assay for detection of granulocytic ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

Immunoglobulin M-capture biotin-streptavidin enzyme-linked immunosorbent assay for detection of antibodies to dengue viruses

Defensins and other antimicrobial peptides act in the innate host defense of epithelial surfaces. Human beta defensin 1 (hBD-1) has recently been shown to be expressed in airway epithelial cells and so has been implicated as a primary component of antibacterial activity in human lung. We attempted to purify these molecules from bronchoalveolar lavage fluid (BALF). Extraction of BALF on SepPak C-18 cartridges, followed by continuous acid-urea polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography yielded one fraction with antibacterial activity associated with factors of < 6.5 kD. N-terminal amino acid sequencing identified these peptides as human neutrophil defensins (HD) 1 through 3. No hBD-1 was detected. Together with lysozyme, it appears that HD-1 through -3 are the most prominent antimicrobial factors in BALF. The contribution of epithelial defensins such as hBD-1 to antibacterial defense of human airway in vivo remains to be elucidated.     

Binding of the Helicobacter pylori vacuolating cytotoxin to target cells

The vacuolating cytotoxin of Helicobacter pylori, VacA, enters the cytoplasm of target cells and causes vacuolar degeneration by interfering with late stages of endocytosis. By using indirect immunofluorescence and flow cytometry, we have demonstrated that VacA binds to specific high-affinity cell surface receptors and that this interaction is necessary for cell intoxication.     

Standardization of the chromium-51 release, cell-mediated cytotoxicity assay: cryopreservation of mouse effector and target cells

Utilizing controlled cryopreservation techniques, we were able to standardize the 51Cr release cytotoxicity assay and thereby ensured reliable comparisons between results obtained on different days. Optimal conditions for freezing of both effector and target cells were quite similar. Dimethyl sulfoxide (DMSO) at a concentration of 7.5-10.0% was employed as the cryoprotective agent and cells were frozen at the rate of -1 degrees C/minute. The handling procedures for the cells before and after freezing were important. Factors affecting recovery of functional reactivity were related to toxicity of DMSO for the cells, the osmotic stress placed upon the cells as the DMSO was being removed after thawing, the handling temperature of the freshly thawed cells, and the susceptibility of cells to mechanical damage immediately after thawing. The recovery of lymphocytes after freezing was about 70%; the recovery of cytotoxicity was around 85%. Syngeneic cytotoxic reactivity induced by inoculation with the Moloney strain of murine sarcoma virus was cryopreserved, as were allogeneic cytotoxicity and natural cytotoxic reactivity. Multiple tests employing effector cells from the same frozen pool gave reproducible results; the standard error of the mean percent cytotoxicity was less than 1.5%. Cryopreserved target cells gave decreased day-to-day variability in susceptibility to lysis, since the same population of cells could be employed in each assay. These results demonstrated conclusively that we can now have a constant source of effector cells and target cells, which can be used from assay to assay as an internal standard.     

Branched-DNA assay for detection of the mecA gene in oxacillin-resistant and oxacillin-sensitive staphylococci

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA-. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA-. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA- but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.     

Fluorescence in situ hybridization of progenitor cells obtained by fluorescence-activated cell sorting for the detection of cells affected by chromosome abnormality trisomy 8 in patients with myelodysplastic syndromes

Myelodysplastic syndrome (MDS) is believed to be a stem-cell disorder involving cytopenia and dysplastic changes in three hematopoietic lineages. However, the involvement of pluripotent stem cells and progenitor cells has not been clarified conclusively. To address this issue, we used fluorescence in situ hybridization (FISH) of blood and bone marrow (BM) smears for mature cells and FISH of cells sorted by fluorescence-activated cell sorting for progenitor cells. Seven patients with MDS associated with trisomy 8 were studied. FISH showed +8 in granulocytes, monocytes, and erythroblasts, but not in lymphocytes. Sorted cells of T (CD3(+)), B (CD19(+)), and NK cells (CD3(-)CD56(+)) from peripheral blood did not contain +8, nor did CD34(+) subpopulations from BM including B (CD34(+)CD19(+)), T/NK (CD34(+)CD7(+)) progenitors, and pluripotent stem cells (CD34(+)Thy1(+)). The +8 chromosome abnormality was identified in stem cells only at the level of colony-forming unit of granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM; CD34(+)CD33(+)). It may thus be concluded that cells affected by trisomy 8 in the context of MDS are at the CFU-GEMM level and that cells of lymphoid lineage are not involved. These results provide new insights into the biology of MDS and suggest that intensive chemotherapy and autologous BM transplantation may become important therapeutic strategies.