A new method to measure air-borne pyrogens based on human whole blood cytokine response

Air-borne microorganisms, as well as their fragments and components, are increasingly recognized to be associated with pulmonary diseases, e.g. organic dust toxic syndrome, humidifier lung, building-related illness, "Monday sickness." We have previously described and validated a new method for the detection of pyrogenic (fever-inducing) microbial contaminations in injectable drugs, based on the inflammatory reaction of human blood to pyrogens. We have now adapted this test to evaluate the total inflammatory capacity of air samples. Air was drawn onto PTFE membrane filters, which were incubated with human whole blood from healthy volunteers inside the collection device. Cytokine release was measured by ELISA. The test detects endotoxins and non-endotoxins, such as fungal spores, Gram-positive bacteria and their lipoteichoic acid moiety and pyrogenic dust particles with high sensitivity, thus reflecting the total inflammatory capacity of a sample. When air from different surroundings such as working environments and animal housing was assayed, the method yielded reproducible data which correlated with other parameters of microbial burden tested. We further developed a standard material for quantification and showed that this assay can be performed with cryopreserved as well as fresh blood. The method offers a test to measure the integral inflammatory capacity of air-borne microbial contaminations relevant to humans. It could thus be employed to assess air quality in different living and work environments.     

Differentiation between endotoxin and non-endotoxin pyrogens in human albumin solutions using an ex vivo whole blood culture assay

Purified E.coli endotoxin, Gram negative bacteria and Gram positive bacteria induce IL-6 secretion by whole blood cultures (WBC's). Polymyxin B at concentrations greater than 2 U/ml completely inhibits IL-6 secretion caused by 10 EU/ml of endotoxin. Polymyxin B has no effect on IL-6 secretion by WBC's in the absence of endotoxin. The inhibition of endotoxin induced IL-6 secretion is Polymyxin B concentration dependent at concentrations less than 1 U/ml. IL-6 induction caused by E.coli is only partially inactivated by 8 U/ml Polymyxin B. Polymyxin B has no effect on IL-6 secretion caused by B.subtilis. Two pyrogenic batches of human serum albumin (HSA), as tested by the rabbit assay for pyrogens, were also investigated. Polymyxin B at 4 U/ml inhibits less than 40 % of IL-6 secretion caused by these pyrogenic HSA batches. All the endotoxin activity in HSA samples spiked with purified endotoxin is inhibited by Polymyxin B indicating that HSA does not protect endotoxin against Polymyxin B inhibition. These results indicate that the pyrogenicity of these HSA batches are caused by Polymyxin B inhibitable and non-inhibitable fractions. This study shows that pyrogenic substances other than endotoxin can contaminate batches of pharmaceutical products and that results obtained using the Limulus Amoebocyte Lysate (LAL) assay does not necessarily indicate the pyrogenic status of pharmaceutical products. The WBC assay for pyrogens, having a broader sensitivity range than the LAL assay, is a better indicator of the pyrogenic status of pharmaceutical products.     

Study of the lymphocytein vitro response to rubella antigen and phytohemagglutinin by a whole blood method

Summary A whole blood culture method was used to study lymphocytein vitro responses to rubella antigen and to phytohcmagglutinin (PHA) in rubella infection. The acute phase of infection in four cases was characterized by high spontaneous incorporation of¹⁴C-thymidine in the cultures, unresponsiveness of lymphocytes to rubella antigen, and absence of response, or relatively low response, to PHA. Cells showing vigorousin vitro response to rubella antigen appeared at about two weeks after the onset of rash. Lymphocyte PHA response returned to normal by day 31. Three rubella vaccinees exhibited a similar response. The use of whole blood lymphocyte cultures stimulated with multiple doses of mitogen and with antigen appears to be a promising technique for studies of general and specific cell-mediated immunity in viral infections.With 4 Figures     

Assessment of immunotoxicity of irinotecan determined by the novel method, by which productivity of TNF-alpha from whole blood is stimulated by lipopolysaccharide

Irinotecan hydrochloride shows much different responses in each patient, and it has severe adverse effects. Therefore, a sensitive marker for the side effect of irinotecan on immunotoxicity may be able to prevent the severe complications by the early detection. We have recently developed a method to assess the immunotoxicity by measuring the productivity of TNF-alpha from whole blood containing monocytes when stimulated by lipopolysaccharide. By using this method, the effects of continuous low-dose irinotecan therapy on immunotoxicity were assessed in 10 patients with advanced gastric or colon cancer. When compared this method with the others such as white blood cell count, lymphocyte blastoid transformation by phytohem agglutinin (PHA), and natural killer cell activity in terms of the sensitivity, immunotoxicity by this method was found earlier than the other methods. Because our original method is easy to perform and sensitive as compared to the conventional methods, it can be widely used as one of the laboratory tests useful for patients treated with immunosuppressive agents.     

IgG response to Mycoplasma pneumoniae in patients with community-acquired pneumonia determined by ELISA

In a prospective study of community-acquired, radiologically verified pneumonia, a solubilized mycoplasma antigen was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG-antibodies to Mycoplasma pneumoniae in paired sera from 60 patients. All of the 13 patients with a positive complement fixation (CF) test for M. pneumoniae were positive in the ELISA, and 46 out of 47 patients with a negative CF test were negative. The only false positive test was recorded from a patient with a positive CF test for Chlamydia. Specific IgG antibodies were also determined in paired sera from 50 pneumonia patients, all positive in the CF test for M. pneumoniae, and collected over a period of 10 years. Of these 50 patients, 45 were recorded as positive in the ELISA for IgG antibodies to M. pneumoniae. In the prospective as well as in the retrospective study, the time for admission to hospital after onset of disease showed considerable variation (1-14 days), with the consequence that high titers were recorded in the CF as well as in the ELISA in some of the first serum samples. A tendency to earlier detection of significant titers was noted in the CF test as compared to the ELISA.     

Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants

Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production. The sensitivity of WB IL-8 production for pyrogens was comparable with that of MNC IL-6 production, but higher than WB IL-6 production. MNC IL-8 production as readout for pyrogenic stimulation was not useful due to high background IL-8 production. Surprisingly, contaminated culture media potently stimulated WB IL-8 production, but not MNC IL-6 production. Finally, the value of TLR-transfected HEK cells in the detection of pyrogenic contamination as well as the role of IL-10 in interindividual differences in cytokine production, is discussed. To summarize, the results presented herein together with literature data indicate that the measurement of WB IL-8 production may represent an advantageous alternative to the measurement of MNC IL-6 production, for the detection of pyrogenic contamination of pharmaceuticals.     

Platelet adhesion determination in whole blood using a simple stagnation flow method

A simple stagnation flow method for the determination of platelet adhesivity has been developed. The sensitivity of the method to its main operational parameters was tested, resulting in recommended standard flow conditions. The method was shown to be dependent on the surface properties of the test slide, on which platelets are deposited, thus proving its suitability to elucidate variations in the surface interaction of the platelets. Normal values were established for males and females.     

The detection of pyrogens in sera from patients with symptoms of sepsis using an ex vivo whole blood culture assay

The aim of this study was to investigate whether the ex vivo whole blood culture (WBC) assay system can be used to detect pyrogens in blood from patients with symptoms of sepsis. Blood samples from 35 patients with symptoms of sepsis were assayed for bacterial contamination using the radiometric blood culture assay. Serum from the same patients were screened for IL-6, C-reactive protein (CRP) and pyrogens using the whole blood culture assay. Serum samples from 26 patients tested positive for pyrogens. Of the 26 patients with pyrogenic serum, 15 had elevated serum IL-6 levels and 19 had elevated CRP levels. Only two of the samples had positive blood cultures as detected by the routine radiometric assay. Both of these patients had high serum CRP and pyrogen levels, while only one of them had an elevated serum IL-6 level. These results show that the WBC is very sensitive in detecting pyrogens in serum of patients. This technique can be a useful tool to quantitate pyrogens in sera from patients with symptoms of sepsis and to determine whether their clinical symptoms are caused by pyretic substances in their circulatory system.     

The estimation of whole blood viscosity by a porous bed method.

A significant impediment in determining the relative contribution of whole blood viscosity to the pathogenesis of cardiovascular and cerebrovascular disease has been the lack of an uncomplicated method to measure whole blood viscosity. To address this problem, a simplified porous bed viscometer has been developed to measure whole blood viscosity. Whole blood is passed through a porous bed of branching channels with a mean pore diameter of 69.6 +/- 20.2 microns and an estimated mean shear rate of 19.6 seconds-1. The effects of sample collection, sample storage, and temperature are described. The mean whole blood viscosity of 242 healthy persons was 22.7 +/- 5.3 seconds, which, when corrected to centipoise using Darcy's equation, corresponds to an apparent viscosity of 5.7 +/- 1.3 cp. There was a significant difference in the whole blood viscosity of normal men and women related to their different packed cell volumes. Platelets and granulocytes influenced whole blood viscosity in proportion to their contribution to the total packed cell volume. Fibrinogen levels did not significantly influence measured whole blood viscosity, which is consistent with the disaggregating conditions and the mean shear rate of the instrument. The porous bed viscometer is a convenient means to measure whole blood viscosity and it should be useful as a screening test for clinical and epidemiologic studies.     

Detection of antibodies to human immunodeficiency virus type 1 in whole blood and saliva by using a passive hemagglutination test.

A passive hemagglutination test (PHA) for detecting human immunodeficiency virus type 1 antibodies in serum samples by using envelope glycoprotein (gp160)-coupled sheep erythrocytes was described earlier (M.B. Vasudevachari, K. U. Uffelman, T.C. Mast, R.L. Dewar, V. Natarajan, H.C. Lane, and N.P. Salzman, J. Clin. Microbiol. 27:179-181, 1989). In the study reported here, the applicability of the PHA test to the detection of antibodies in whole-blood and saliva samples has been investigated. We observed a 100% correlation between PHA and commercial enzyme-linked immunosorbent assay in 101 whole-blood samples and 98% correlation between PHA and reactivity to envelope proteins in Western blots (immunoblots) of 53 saliva samples. Furthermore, salivary antibodies could be detected in 19 of the 22 seropositive individuals. As in serum, antibodies to envelope proteins were widely prevalent in all the Western blot-reactive saliva samples.     

Inflammatory cytokine (interleukin 6 and tumour necrosis factor alpha) release in a human whole blood system in response to Streptococcus pneumoniae serotype 14 and its capsular polysaccharide

Gram-positive bacteria, which lack lipopolysaccharide (LPS), produce a septic-shock-like condition, accompanied by release of pro-inflammatory cytokines. Various components of the bacteria may be responsible for this. We stimulated a whole blood system with heat-inactivated Streptococcus pneumoniae serotype 14 (S14) bacteria, with pneumococcal S14 capsular polysaccharide (PPS S14) and with PPS S14 coated on to latex beads, to compare interleukin 6 (IL-6) and tumour necrosis factor alpha (TNFalpha) production over a six hour period, to ascertain the contribution of PPS to the inflammatory response. This was compared with the response to LPS. After sonication of the bacteria, their PPS content was estimated by an enzyme-linked immunoabsorbent assay, to compare this with the concentration of free PPS needed to generate cytokine release. The whole bacteria elicited a much larger cytokine response than the equivalent amount of PPS alone, whereas the PPS-coated beads gave minimal response. The different cytokine responses to PPS and LPS suggest that there are differences in the receptors and/or signalling pathways for Gram-negative and Gram-positive bacteria. We conclude that the estimated amount of PPS in the bacteria is not enough to account for the large cytokine response we observed. Since PPS could not be shown to contribute significantly to cytokine induction, specific antibodies to PPS would not play any significant role in combating cytokine release associated with pneumococcal infection and possible septic shock. This needs to be considered in production of future vaccines.     

Oxygen saturation determined using a novel wavelet ratio surface

A wavelet-based method is presented for oxygen saturation measurement using photoplethysmogram signals from a standard pulse oximeter device. The transform moduli of both red and infrared signals are used to derive a novel wavelet ratio surface. Projection of the pulse component onto this surface allows optimal derivation of oxygen saturation.     

Blastogenic response of whole blood cells of turkeys to a T-cell mitogen.

Cellular immune mechanisms in the turkey are not well understood because adequately standardized, reproducible in vitro assays to measure cellular immunity are not available. Our purpose was to optimize conditions for a whole blood mitogenic assay that would facilitate quantitative assessment of the ability of circulating T cells of turkeys to respond to Con A. Heparinized peripheral blood from normal turkeys was examined. Data indicated that diluting the blood 1:20 or 1:40 and incubating the test cultures at 39 degrees C or 41 degrees C gave the best mitogenic stimulation. Presence of 7.5% turkey serum but not chicken or fetal bovine serum in the culture medium substantially enhanced the blastogenic response. Ontogeny of the whole blood mitogenic response was examined by repeat observations on a group of turkeys at various age levels starting at 1 week of age. Whole blood cells from 1-week-old turkeys responded poorly to Con A, although by 2 weeks of age, the response was well developed. Tests at subsequent ages revealed variable levels of activity. There was a considerable individual variation in the level of blastogenic response of turkeys within the same age group.     

Use of whole sheep red blood cells in ELISA to assess immunosuppression in vivo

An enzyme-linked immunosorbent assay (ELISA) using whole sheep red blood cells (SRBC) has been reported as one of the methods for detecting a T-lymphocyte-dependent antibody response. However, it has not been widely used because of SRBC problems such as the weak attachment to ELISA plates, specificity and short-term stability. The objectives of this study were to address these issues and to validate the SRBC-specific antibody response assay. Male Sprague-Dawley rats were bled after 6 days of SRBC immunization. In our new procedure, glutaraldehyde was added before discarding the supernatant of inoculated SRBC suspension to attach SRBC firmly to the plate, while in the original method it was added after discarding. As a result, the attached SRBC was maintained throughout the ELISA procedures. No interference was observed in the titration curve of IgM and IgG antibodies in rats and IgM-antibody in mice when control sera were analyzed to evaluate specificity of this method. The short-term stability of SRBC was overcome by using the different lots of SRBC. They provided antibody titers, which were consistent with those measured using the same lot for immunization. In addition, cyclophosphamide, cyclosporine, prednisolone and methotrexate, well-known immunosuppressive agents, were tested to confirm the applicability of the improved ELISA method to detect the T-lymphocyte-dependent antibody response. All four compounds inhibited the IgM antibody responses dose-dependently. These results demonstrate that the improved whole SRBC-ELISA method provides reproducible and reliable results in the T-lymphocyte-dependent antibody response assay.     

人乳头状瘤假病毒中和抗体滴度和ELISA法测定的小鼠血清抗体滴度的相关性研究

目的 探讨两种不同的报告基因纽扣珊瑚绿色荧光蛋白(Zoanthus sp.green fluorescent protein,ZsGreen)和分泌性碱性磷酸酶(secreted alkaline phosphatase,SEAP)测量的人乳头状瘤假病毒中和滴度之间的相关性,以及中和滴度和抗体滴度的相关性.方法 将密码子优化的人乳头状瘤病毒(HPV)衣壳蛋白L1、L2基因表达质粒和报告基因质粒共转染293FT细胞,48 h后收集细胞裂解上清,柱层析纯化假病毒,对假病毒滴度进行测定.采集免疫过候选HPV疫苗和Gardasil疫苗的小鼠血清,测量血清的中和滴度和抗体滴度.结果 经统计分析,这两种报告基因系统的假病毒检测的中和滴度结果高度相关(Spearman相关系数r=0.760),而且中和滴度和抗体滴度的相关度很高(Spearman相关系数r=0.577和0.741).结论 两种不同的报告基因ZsGreen和SEAP测量的HPV假病毒中和滴度之间,以及中和滴度和ELISA测定的抗体滴度之间高度相关,揭示了部分HPV疫苗预防病毒入侵的机制,为快速准确鉴定HPV-16和HPV-18候选疫苗的免疫保护效果奠定了基础.     

Endotoxin removal from whole blood by a novel adsorption resin: efficiency and hemocompatibility

The structural component of Gram- bacteria, endotoxin (ET), induces the release of endogenous mediators of sepsis. Attempts to remove these downstream molecules in vivo, have not improved survival. However, extracorporeal strategies such as continuous renal replacement therapy or therapeutic plasmapheresis have shown benefit. We are presenting an affinity-based extracorporeal technology for the removal of ET from whole blood. The small-scale device contains an adsorbent that removed 75% of ET present in whole blood. This affinity resin displayed good hemocompatibility regarding the coagulation pathway. Minimal platelet, neutrophil and complement activation were observed. There was also no evidence of consumption of coagulation factors or cell loss. In as much as ET participates in both the inflammatory and coagulation abnormalities in sepsis, this method represents an efficient and hemocompatible way to remove ET from whole blood, which, in an extracorporeal setting, may improve the outcome of sepsis.