Comparison of the two fully automated anti-HCMV IgG assays: Abbott Architect CMV IgG assay and Biotest anti-HCMV recombinant IgG ELISA.

OBJECTIVE AND BACKGROUND: To assess the performance characteristics of two fully automated human cytomegalovirus (HCMV) antibody tests. MATERIALS AND METHODS: Samples from negatively or not pre-screened blood donors were tested by the Biotest anti-HCMV recombinant IgG enzyme-linked immunosorbent assay (ELISA) in comparison to the Abbott Architect CMV IgG assay [chemiluminescent microparticle immunoassay (CMIA)]. For clarification, samples with discordant results between both assays were subjected to supplemental testing for anti-HCMV IgG, IgM and HCMV DNA in plasma. RESULTS: From 4938 samples tested, 362 delivered positive results in both assays (7.3%). 91 (1.8%) samples were discordant. Of 43 (two not further tested) samples positive only by ELISA, 41 were false positive, one true positive and one indeterminate. Of 45 (one not further tested) samples positive only by CMIA, 20 were false positive, 9 indeterminate and 16 true positive. Anti-HCMV IgM and HCMV DNA testing from the plasma were negative in indeterminate samples. Considering the results of supplemental testing, the CMIA achieved altogether better results concerning resolved sensitivity, resolved specificity as well as negative predictive value. Both assays had an inferior positive predictive value, with a better result for CMIA. CONCLUSION: Overall, the performance characteristics of the CMIA were better than those of the ELISA. Owing to the inferior positive predictive value, positive test results require confirmation if blood products from donors with remote HCMV infection should be administered.

Preanalytical stability of HIV-1 and HCV RNA: impact of storage and plasma separation from cells on blood donation testing by NAT.

BACKGROUND: The aim of this study was to evaluate the optimal preanalytical conditions prior to nucleic acid amplification technology (NAT) for human immunodeficiency virus-1 (HIV-1) or Hepatitis C virus (HCV) RNA in pools of 96 plasma specimens with regard to storage temperature, time and plasma separation in a blood donation environment. STUDY DESIGN AND METHODS: Changes in viral nucleic acid concentration of HIV-1 and HCV were observed for 5 days according to the Paul-Ehrlich-Institute's (PEI) guidelines that demand 95%-detection limit of at least 10 000 IU mL(-1) for HIV-1 RNA and 5000 IU mL(-1) for HCV RNA within a single donor blood specimen. Ninety-five per cent detection limits of HIV-1 RNA over 3 days after storage at either 5 or 21 °C were evaluated by using standardised HIV-1 RNA-positive plasma. RESULTS: HCV RNA in whole blood samples proved to be more stable than HIV-1 RNA. Whole blood storage at 21 °C was shown to decrease the detectability of HIV-1 RNA even after only 18 h. Plasma samples once used for NAT at time 18 h did not alter viral stability up to 48 h after donation. Ninety-five per cent detection limits of HIV-1 RNA were securely below 10 000 IU mL(-1) for 24 h after whole blood storage at 5 °C. CONCLUSIONS: These results may lead to a discussion around the most suitable preanalytical conditions in blood donation environments. Contrary to the current PEI guidelines that allow storage of whole blood specimens up to 18 h at 21 °C, these results suggest that immediate storage in a 5 °C container after blood donation is more suitable and would permit storage of whole blood up to 24 h prior to the separation of plasma from cells.

Autologous blood donor screening indicated a lower prevalence of viral hepatitis in East vs West Germany: epidemiological benefit from established health resources.

Prevalence data concerning viral hepatitis and human immunodeficiency virus (HIV) in the general population are usually scarce. We aimed for a large cohort representative of the general population that required little funding. Autologous blood donors are relatively representative of the general population, and are tested for viral hepatitis and HIV in many countries. However, frequently these data are not captured for epidemiologic purposes. We analysed data from well over 35,000 autologous blood donors as recorded in 21 different transfusion centres for anti-hepatitis C virus (HCV), HBsAg and anti-HIV, as well as TPHA if available. We found a lower prevalence of hepatitis B virus and HCV in East vs West Germany, 0.2%vs 0.32% and 0.16%vs 0.32% respectively, which confirms earlier data in smaller cohorts, thus supporting the value of our approach. HIV was too rare to disclose significant differences, 0.01%vs 0.02%. TPHA was higher in East (0.34%) vs West Germany (0.29%) without significant differences. HCV was more frequent in women vs men. Transfusion institutes managing autologous blood donations should be used as a resource for epidemiological data relating to viral hepatitis and HIV, if such testing is performed routinely. This approach generates data relating to the general population with special emphasis on undiagnosed cases.

One-way synergistic effect of low superantigen concentrations on lipopolysaccharide-induced cytokine production.

Lipopolysaccharides (LPS) of gram-negative bacteria and superantigens of gram-positive bacteria are among the main causes of sepsis and septic shock. Symptoms are initiated primarily by the release of endogenous mediators, especially cytokines. In the last few years, increasing evidence for the clinical relevance of mixed sepsis caused by coinfections with both types of bacteria has been found. Therefore, we developed an in vitro mixed sepsis model investigating the effect of different superantigen doses, in combination with different LPS concentrations, on cytokine production in human PBMCs using ELISA and RT-PCR. Low, in vivo relevant concentrations of the superantigen toxic shock syndrome toxin-1 (TSST-1) synergistically enhance LPS-induced production of interferon-gamma (IFN-gamma) interleukin-1 beta (IL-1 beta), IL-6, and IL-10, but low LPS has no comparable effect. Signal transduction studies with different inhibitors suggest that this one-way synergism is caused by an interaction between the cAMP and the PIP2 signaling pathway. Furthermore, our findings support the idea that this interaction is one important crossover point of signal transduction pathways by LPS and superantigens, which seems to be predominantly regulated by IFN-gamma and PGE-2. The identification of additional crossover points in the genesis of a mixed sepsis and their selective influence could lead to identical treatment of both gram-negative and gram-positive sepsis.

Induction of a cytokine network by superantigens with parallel TH1 and TH2 stimulation.

Superantigens cross-link the MHC class II molecule on accessory cells with the V beta region of the TCR outside the antigen binding sites. In this study, we compared the capacity of the staphylococcal entertoxins (SE) A, B, C1, C2, C3, D, and E, the toxic shock syndrome toxin (TSST) 1, the exfoliative toxin (ExFT) A, and the Streptococcus pyogenes erythrogenic exotoxins (SPE) B and C to induce cytokine release in human peripheral blood mononuclear cells. We showed that all toxins tested induced IL-1 alpha and beta, IL-2, IL-4, IL-6, IFN-gamma, and TNF-alpha, but not IFN-alpha. However, we found that SPEB differed from all other toxins tested, because its cytokine induction was significantly lower than that of the other toxins. This was not true of IL-6 and IL-10 induction, in which SPEB showed similar amounts of IL-6 compared with all other toxins and of IL-10 in comparison to SEC2. SPEB showed a specificity for TH2 cells, whereas the other toxins stimulated TH1 as well as TH2 cells very strongly. As a result, superantigens appear to be able to uncouple the TH1/TH2 antagonism. Collectively, our results indicate that SPEB seems not to be a superantigen or represents a different group of microbial superantigens. Furthermore, superantigens stimulate TH1 as well as TH2 cells without any preference and therefore they are able to induce humoral as well as cellular immunity. This could be one reason for the existence of autoantibodies and autoreactive T cells in autoimmune diseases and one major step in the beginning of the induction of autoreactivity.

Induction of a proinflammatory cytokine network by Mycoplasma arthritidis-derived superantigen (MAS).

Mycoplasma arthritidis is an arthritogenic organism for rodents, producing a superantigen (MAS). It has been postulated that mycoplasmas or superantigens thereof might play a role in human rheumatoid arthritis. Since M. arthritidis fulfills both, the present study was performed to investigate MAS-specific cytokine induction. Human or murine leukocytes were stimulated with MAS, staphylococcal enterotoxin E (SEE), or lipopolysaccharide (LPS). Cytokines were measured by ELISA, Bioassay, and RT-PCR. The response to MAS in humans was individually restricted, in contrast to the response to SEE or LPS. Furthermore, MAS showed the same capacity for inducing proinflammatory cytokines as interleukin (IL)-1 IL-6, and IL-8 as SEE and LPS. However, MAS showed a significantly decreased capacity to induce the anti-inflammatory cytokine IL-10 and IL-1RA. In mice, the reactivity to MAS was strictly MHC-II restricted, in contrast to that of SEE or LPS. The individual response to MAS in humans might be explained by the difference of the HLA-DR haplotype because H-2-differing mouse strains showed the same discrepancies. MAS induced an overproduction of proinflammatory cytokines, when its ability to induce proinflammatory and anti-inflammatory cytokines was compared with those of SEE and LPS. The individual response may explain an MHC linkage, and the failure to induce anti-inflammatory cytokines may be the reason for a chronic disease in contrast to acute inflammation.

Kinetics and organ distribution of allogeneic natural killer lymphocytes transfused into patients suffering from renal cell carcinoma.

The transfusion of natural killer (NK) lymphocytes into patients suffering from malignant diseases is an approach of current interest in the field of immunotherapy. Little is known about the organ distribution, survival, and clearance of donor immune effector cells in cellular therapy, and no reports exist on these important parameters considering NK cells in particular or any other type of allogeneic lymphocytes in humans. In the context of a clinical Phase I/II study we examined the distribution of transfused allogeneic NK cells in patients suffering from renal cell carcinoma. The NK cells were ex vivo cultivated and activated before transfusion. To assess the circulation of the transfused cells in the peripheral blood, we used a nested PCR technique to detect HLA DRB1 alleles of the NK cell donors. Post-transfusion, all patients showed evidence of circulating donor cells for up to 3 days. After 7 days, all donor cells were cleared from the blood to undetectable levels. To assess organ distribution, (111)In-labeled NK cells were injected and monitored by whole-body scintiscans. A distribution to the whole body, with preference for liver, spleen, and bone marrow, was observed after a short initial uptake in the lungs. No activity was observed in lymphatic nodes. A total of 2/4 evaluable metastases showed a clear accumulation of transfused NK cells. The half-life corrected activity in all body compartments remained almost constant over the 6-day observation period in concordance with the absence of any excretion of radioactivity. This may indicate an extended survival of the transfused cells, despite their foreign nature, in the host organism.

A novel RT-PCR for reliable and rapid HCV RNA screening of blood donations.

BACKGROUND: The objective of this work was to develop a novel and highly sensitive RT-PCR method that is suitable for HCV RNA screening of blood donations according to the criteria released by the Paul Ehrlich Institute, the federal licensing agency of Germany, for routine HCV NAT. STUDY DESIGN AND METHODS: RNA was prepared from plasma pools of up to 20 single blood donations using an automated nucleic acid isolation system (NucliSens Extractor, Organon Teknika). For reverse transcription, amplification, and simultaneous detection of PCR products, a novel approach based on the TaqMan technology was developed. Glyceraldehyde-3-phosphate dehydrogenase messenger RNA, which is detectable in human plasma, was coamplified in each reaction as an internal positive control. RESULTS: The HCV genotypes and subtypes 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, and 5a were detected in parallel with comparable amplification efficiency. The 95-percent detection limit related to the WHO HCV RNA standard preparation was calculated to be 389 IU per mL of plasma of the single blood donation. Total CVs (%) were <4. The screening of up to 180 blood donations took 5 hours; as a rule, the blood components could be released on the day of donation. CONCLUSION: The TaqMan HCV RT-PCR is an almost completely automated, highly sensitive, specific, and rapid method that is reliable for HCV RNA screening of blood donations. It allows a closed-tube HCV RNA detection without risk of contamination by PCR products.

Zinc enhances lipopolysaccharide-induced monokine secretion by alteration of fluidity state of lipopolysaccharide.

Elevated zinc serum concentrations have been shown to restore impaired immune response. Therefore, pharmacologic zinc supplementation has been used to improve immune function, particularly in intensive care patients. In these patients, Gramnegative sepsis, the symptoms of which are predominantly caused by LPS-induced release of monokines, represents a serious problem. We have recently shown that zinc enhances induction of TNF-alpha and IL-1 beta in cultures of PBMC by LPS. By fluorescence polarization and infrared spectroscopic measurements we found that zinc addition leads to decreased fluidity of the hydrocarbon chains of LPS. Experiments at different temperatures showed that the less fluid gel (beta) phase of LPS is more effective in cytokine induction than the more fluid liquid-crystalline (alpha) phase. Our studies suggest that the synergistic effect of zinc on monokine induction by LPS is caused by direct interaction of zinc with LPS altering the fluidity of the hydrocarbon chains. Although this effect is zinc specific, other divalent ions, like cobalt and nickel, with a complex structure and size comparable to those of zinc also enhance LPS-induced monokine secretion but to a much lesser extent. Our data indicate that the zinc level represents a relevant clinical parameter in the treatment of Gram-negative infection. This reveals potential risks in the therapeutic application of zinc.

Hypothermia enhances the biological activity of lipopolysaccharide by altering its fluidity state.

Lipopolysaccharides (LPS, endotoxin) of gram-negative bacteria are among the main causes of sepsis and septic shock. In the present study, the influence of temperature on the biological activity of LPS was investigated. Lowering the temperature from 37 degrees C to 34.5 degrees C or to 30 degrees C significantly enhances in vitro tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-6 release induced by different LPS chemotypes and heat-inactivated Escherichia coli. This cytokine-increasing effect of lowering the temperature is highly mediated by serum proteins, particularly by LPS-binding protein (LBP) and low-density lipoproteins (LDL). In contrast, cytokine production induced by the superantigen toxic shock syndrome toxin-1 (TSST-1) from Gram-positive Staphyloccoccus aureus decreases by around 70% at 30 degrees C as compared with 37 degrees C, corresponding to the expected effect of change in temperature and regardless of the presence of serum proteins. In order to explain the unexpected biological hypothermia effect with regard to LPS, the fluidity state of the lipid A portion of LPS as one important physico-chemical property possibly involved was investigated. The fluidity, determined by fluorescence polarization measurements, was found to decrease with decreasing temperature. These data suggest that a low fluid LPS chemotype is biologically more active than a more fluid one (and vice versa). Statistical analysis of the results shows a strong correlation between cytokine secretion and fluidity state of a given LPS chemotype (0.71 < r < 0.89, all P<0.01). As a clinical consequence, these data may be one possible explanation for the higher mortality rate of hypothermic Gram-negative sepsis.