Whole Blood Cultures Research Articles

Study on blood cultures and bacteria counts in the blood of paratyphoid fever A patients

Paratyphoid fever A (PA) is a systemic infection caused by Salmonella enterica serotype Paratyphi A (SPA) [1]. Although blood cultures are considered to be the ‘gold standard’ for the detection of bloodstream pathogens, there is little information on the controlled clinical comparison of the paired aerobic and anaerobic bottles in the automated continuously monitoring instrument systems, and bacterial counts in the blood of patients with bloodstream infections [2, 3]. Yuxi City is the most severe endemic area of PA in China [4]. In order to determine the factors determining the yields from blood cultures, we have performed broth blood cultures (BBC) by the BacT/ALERT 3D automated blood culture system (bioMerieux, Inc., Durham, NC) and quantitative whole-blood culture (QWBC) for a large series of patients with suspected PA between 1999 and 2008. A total of 13,500 patients admitted with suspected PA to our institutions between June 1999 and June 2008 were studied. The available blood samples were obtained with vacuum system blood collection tubes. A blood culture set included one aerobic bottle (AEB) and one anaerobic bottle (ANB) (bioMerieux), and one QWBC plate (Columbia agar, bioMerieux). A 13-ml quantity of blood from each patient for one test set was considered to be the maximum amount to obtain from a single venipuncture and was distributed as follows: 5 ml to one AEB, 5 ml to one ANB, and 3 ml to one plate (only for 1,000 probable cases of PA). The culture bottles contained 5 ml of blood sample and 40 ml of culture media. Each bottle was monitored with the BacT/ALERT 3D system at 37°C for a standard 5-day protocol or until the instrument signaled a positive result [5]. Any bottle with a positive signal was immediately removed from the instrument, and an aliquot of the blood– broth mixture was taken from the bottle with a sterile needle and swab for subculture on sheep blood nutrient agar and XLD agar (Oxoid, England); the time-to-detection of bacteria causing PA was taken as the incubation time required before the instrument gave a positive signal. All bottles were treated independently until the end of the 7-day incubation and testing period. Both BBC and QWBC were performed for 1,000 probable cases of PA. QWBC were carried out by a pour plate method and the blood bacterial counts were estimated [3]. In brief, 3 ml of blood was drawn aseptically from each patient and injected into a sterile heparinized tube. Three milliliters of heparinized blood was mixed well with 19 ml of molten (50°C) Columbia agar in a sterile Petri dish, allowed to set for solidification, and then incubated at 37°C. After 2 to 4 days, colonies were identified, counted, and recorded as colony-forming units (CFU) per milliliter. The number of SPA bacteria per milliliter of blood was estimated from the number of CFU on each pour plate. The minimum volume of whole blood used for QWBC from any one patient was 3 ml. This gives a lower limit of Eur J Clin Microbiol Infect Dis (2009) 28:1259–1261 DOI 10.1007/s10096-009-0766-9

Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs

The impact of nonsteroidal anti-inflammatory drugs (NSAID) on prostaglandin E(2) (PGE(2)) production and cyclooxygenase 2 (COX-2) mRNA expression in bovine whole blood (WB) cultures stimulated by lipopolysaccharide (LPS) was determined, using the blood from six Holstein dairy cattle in various stages of lactation. Peak production of PGE(2) occurred 24 h after LPS stimulation but did not result in detectable concentrations of thromboxane B(2) (TXB(2)). The NSAID indomethacin, aspirin, flunixin meglumine, and 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide (PTPBS; celecoxib analogue), along with dexamethasone, were all equally effective in reducing the concentration of PGE(2) in the bovine WB culture supernatants. Bradykinin exhibited peak supernatant concentrations 1 h after LPS stimulation. Dexamethasone and the NSAID used in this study were equally effective at inhibiting bradykinin production. Peak induction of COX-2 mRNA occurred 3 h post-LPS stimulation. However, neither dexamethasone nor any of the NSAID used in this study altered COX-2 mRNA concentrations. In contrast, aspirin, flunixin meglumine, and PTPBS reduced tumor necrosis factor-alpha (TNFalpha) mRNA concentration. These results demonstrate that bovine blood cells respond to NSAID therapy like other mammalian cells with respect to inhibition of PGE(2) production and suppression of TNF mRNA induction, but do not inhibit induction of COX-2 mRNA.

C-Reactive Protein Elicits White Blood Cell Activation in Humans

Objective Consistent epidemiologic evidence suggests that acute infections increase the risk for acute cardiovascular events. We tested in humans whether activation of peripheral leukocytes in reaction to the administration of recombinant human C-reactive protein (rhCRP) may provide a mechanism for infectious diseases to promote atherosclerotic disease. Methods and Results By using quantitative real-time polymerase chain reaction analysis, whole-blood expression profiles were analyzed for 95 inflammatory markers before and after infusion of 1.25 mg/kg rhCRP in 5 male volunteers. Relevant transcript levels were measured at baseline and 4 and 8 hours after rhCRP-infusion. CRP caused significant up-regulation of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-1, plasminogen activator urokinase, macrophage inflammatory protein 1α, and nuclear factor of kappa B inhibitor mRNAs in peripheral leukocytes. mRNA up-regulation of MMP-9 and MCP-1 was 17- and 11-fold, respectively. The corresponding increase in plasma protein levels of MMP-9 (78 ± 32 ng/mL to 109 ± 41 ng/mL; P = .014) and MCP-1 (312 ± 92 pg/mL to 2590 ± 898 pg/mL; P = .007) closely mirrored mRNA findings. Also, in whole-blood culture stimulation assays, CRP induced proinflammatory changes. Notably, heat inactivation abolished the capacity of CRP to evoke these proinflammatory changes, excluding a role for contaminants within the purified CRP preparation. Conclusion CRP elicits activation of peripheral leukocytes with ensuing secretion of plaque-destabilizing mediators. These findings are consistent with the hypothesis that infectious diseases trigger manifestations of atherosclerosis, in which CRP elevation might contribute to the onset of cardiovascular events.

Changes in endocrine and immune responses of neonatal pigs exposed to a psychosocial stressor

The aim of the study was to determine the effects of a single social isolation (4 h) of piglets on immediate changes in stress hormones and immune responses at 7, 21 or 35 days of age. This social stressor caused an increase in plasma ACTH and cortisol concentrations and a decrease in plasma TNF-α. The percentage of CD8 + cells increased and the CD4 + cell percentage decreased, resulting in decreasing CD4 +/CD8 + ratio. The observed changes were consistent for all days studied. Further, the isolation treatment resulted in diminished LPS-stimulated IL-1β and IL-10 production in whole-blood cultures. In isolated piglets, positive correlations were estimated between changes in percentage of CD8 + cells and cortisol, and negative ones between changes in plasma TNF-α and culture supernatant IL-1β with ACTH and cortisol. The data suggest that psychosocial stress in neonatal pigs induced immune alterations to maintain adaptive stability, but reflecting also negative emotions experienced by this treatment.

Effect of aspirin treatment on TNFα production by women with a history of preterm birth

Lipopolysaccharide (LPS)-stimulated TNFα production is reported to be greater for whole blood (WB) cultures prepared from patients with a history of preterm birth than cultures obtained from women with a history of term birth. The objectives of this study were (1) to determine if there is a similar differential responsiveness for peripheral blood mononuclear leukocytes (PBML) and (2) to determine if treatment with aspirin influences LPS-stimulated TNFα production in these patients. WB and PBML were obtained from women with a history of preterm delivery before 32 weeks (cases; n = 5) and age- and race-matched controls ( n = 5) with a history of uncomplicated term delivery. WB and PBML were cultured and stimulated with LPS. All participants then took aspirin daily for 1 week and responsiveness of PBML and WB cultures to LPS was retested. The history of preterm labor was found to have no effect on LPS-stimulated TNFα production in cultures of WB or PBML. Aspirin treatment enhanced LPS-stimulated TNFα production by PBML from controls but not cases. We conclude that endotoxin responsiveness of women with a history of preterm birth is similar to that of women with a history of term birth in terms of in vitro TNFα production. Aspirin increases TNFα production by PBML in control women but not in women with a history of preterm birth. The divergent responses to aspirin treatment in patients with and without prior preterm labor may reflect differential regulation of cytokine production by prostaglandins in women with preterm labor associated with infection or inflammation.

Markers of Innate Immune Function Are Associated with Vitamin A Stores in Men

Recommendations for vitamin A intake and liver stores are based on maintaining normal vision. We propose that higher levels may be required to maintain normal innate immune function. To test this hypothesis, we conducted an 8-wk residential study among 36 healthy Bangladeshi men with low vitamin A stores. Subjects were randomized to receive vitamin A (240 mg in 4 doses) or placebo during study wk 2 and 3. They received 2 vaccines during wk 5 and vitamin A stores were estimated by isotopic dilution at wk 8. The serum concentration of the chemokine interferon-gamma-induced protein 10, a component of T-helper 1 (Th1) response, increased significantly after supplementation and was positively and significantly associated with vitamin A stores. Blood concentrations of natural killer (NK) and NK T-cells, which have anticancer and antiviral activity, were positively associated with stores (P < 0.05), as was monocyte oxidative burst (P < 0.05), a marker of bacterial killing ability. However, serum interleukin (IL)-6 and IL-17, cytokines that regulate the antibacterial Th17 response, were significantly and negatively associated with stores, as was production of the regulatory cytokine IL-10 by whole-blood cultures stimulated with bacterial lipopolysaccharide. In summary, vitamin A stores were positively associated with several measures of innate immune activity across a broad range of stores, suggesting that vitamin A enhances protection against diverse pathogens even at concentrations above those needed to maintain normal vision. The negative association of stores with serum IL-6 and IL-17 suggests that not all protective responses are similarly enhanced by vitamin A.

Whole-blood culture is a valid low-cost method to measure monocytic cytokines — A comparison of cytokine production in cultures of human whole-blood, mononuclear cells and monocytes

Whole-blood and peripheral blood mononuclear cell (PBMC) cultures are used as non-validated surrogate measures of monocytic cytokine production. The aim of this investigation was to compare ex vivo cytokine production from human whole-blood and PBMC with that from isolated monocytes. We also assessed the intra- and inter-individual variation in cytokine production. In 64 healthy men (age 19–40 years) IL-6, TNF and IL-10 were measured by enzyme-linked immunosorbent assay in supernatants from whole-blood, PBMC and monocytes cultured 24 h with lipopolysaccharide (LPS) or UV-killed L. acidophilus. Cytokines produced from whole-blood was found to be more strongly correlated with monocytic cytokines than cytokines from PBMC, particularly after LPS-stimulation: r=0.57, P<0.001 versus r=0.33, P=0.01 for IL-6 and r=0.43, P<0.001 versus r=0.30, P=0.02 for TNF-α. Adjustment for a preceding 8-week dietary fatty acid-intervention did not change any of the associations. Based on measurements at three time-points 8 weeks apart the intra-individual variation was ≥50% smaller than the inter-individual variation (P<0.05) in most whole-blood cytokine responses and LPS-stimulated IL-6 from PBMC. We conclude that whole-blood cultures are well-suited low-cost proxy-measures of monocytic cytokine production. Moreover, large inter-individual variation in cytokine production was demonstrated whereas the individual responses in whole-blood were reproducible even over long time-periods.

Cytokine profile associated with chronic and acute human schistosomiasis mansoni

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma (IFN-gamma) was evaluated in 43 schistosomiasis patients with different clinical forms. Whole-blood cultures cytokine production in response to soluble egg antigen (SEA), soluble worm adult preparation (SWAP), mitogens, neutralizing antibodies or recombinant IL-13 were measured by ELISA. After SWAP stimulation, chronic patients, particularly hepatointestinals, produced higher levels of IL-4 in comparison with acute patients, suggesting the presence of a type 2 cytokine profile in these patients. Following SEA and SWAP stimulation, hepatosplenic (HS) patients showed increased levels of IFN-gamma when compared with acute patients, indicating that HS disease in humans is associated with a type 1 cytokine response. The mechanisms of immune regulation are apparently different between the clinical stages of the disease, some of which are antigen-specific.

Effects of anti-CD3 monoclonal antibodies on functional activity of lymphocytes: studies in vivo and in vitro.

Lymphocyte functional activity was tested in 38 renal transplant recipients receiving induction treatment with various anti-CD3 MoAbs, i.e. OKT3, T3.G2a (an IgG2a anti-CD3 MoAb) or T3.A (an IgA anti-CD3 MoAb of the same idiotype). During treatment with OKT3 and T3.G2a, lymphocyte response to phytohaemagglutinin-P (PHA), as determined with the use of a whole-blood lymphocyte culture technique, decreased significantly. However, during treatment with T3.A PHA response was not affected. Using a conventional lymphocyte culture technique, PHA response was unchanged during treatment with all three MoAbs, indicating that the immunosuppressive effect of OKT3 and T3.G2a is probably dependent upon the presence of MoAb in culture medium and is reversible. In addition, we tested in vitro inhibition of aspecific mitogen- or antigen-induced lymphocyte stimulation by OKT3, T3.A and T3.G2a. It appeared that at low concentrations (< 25 ng/ml) T3.G2a and OKT3 exerted a stronger immunosuppressive effect than T3.A. However, at higher concentrations T3.A, OKT3 and T3.G2a were equally immunosuppressive. We conclude that the immunosuppressive effect of T3.A is caused by blindfolding. At low concentrations T3.G2a exerts its immunosuppressive effect mainly through modulation of the CD3 and/or T cell receptor complex, as a result of interaction with Fc receptors on monocytes. At higher concentrations blindfolding of the CD3/T cell receptor complex may contribute to immunosuppression.

Effects of four Fusarium toxins (fumonisin B 1, α-zearalenol, nivalenol and deoxynivalenol) on porcine whole-blood cellular proliferation

The in vitro effects of four Fusarium toxins, fumonisin B 1 (FB 1), α-zearalenol (α-ZEA), nivalenol (NIV) and deoxynivalenol (DON), on mitogen-induced cell proliferation were determined in swine whole-blood cultures. Considering the lack of sufficient toxicological data both on single and in combination effects, in vitro studies may contribute to risk assessment of these toxins. Incubation with increasing concentrations of FB 1 did not produce any consequence on proliferation; in contrast α-ZEA, NIV and DON showed an inhibitory effect. Dose–response curves for each mycotoxin were generated. NIV was found to be the most potent toxin followed by DON and α-ZEA. The effects of both FB 1 + α-ZEA and NIV + DON mixtures were also analysed to investigate possible interactions. The results indicated that combination of FB 1 + α-ZEA produces a synergistic inhibition of porcine cell proliferation; whereas there is no interaction between DON and NIV on porcine whole-blood proliferation, at tested concentrations.

159C>T CD14 genotype—Functional effects on innate immune responses in term neonates

Given the susceptibility of newborns to infection and the potential harm of overwhelming proinflammatory immune responses, the impact of genetic variation in innate immune molecules is of increasing interest for risk stratification and prevention. We studied the functional relevance of the 159C>T CD14 single nucleotide polymorphism in cord blood samples of n=135 healthy term neonates by investigation of sCD14, interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha concentrations in whole-blood culture supernatants and intracellular assessment of IL-1beta, IL-6, and TNF-alpha expression by flow cytometry. The 159C>T CD14 genotype frequencies were n=42 (0.31) for homozygous CD14-159 CC, n=69 (0.51) for heterozygous CD14-159 CT, and n=24 (0.18) for homozygous CD14-159 TT. No genotype-associated differences were noted for ex vivo baseline expression of sCD14, IL-6, IL-10, and TNF-alpha. After in vitro stimulation of cord blood cultures with lipopolysaccharide, carriers of the CD14-159 T allele were determined to have higher levels of sCD14 compared with carriers of the CD14-159 C allele (p=0.04) and increased concentrations of IL-6 (p=0.009) in culture supernatants (one-way analysis of variance). The 159C>T CD14 polymorphism is associated with soluble CD14 expression and cytokine expression, which might influence the balance of pro- and anti-inflammatory immune responses in healthy term neonates. Further studies are needed to delineate whether the 159C>T CD14 genotype is a risk factor for severity of neonatal infection in the clinical setting and a potential target for prevention strategies.

Reduced Maximal Oxygen Consumption and Overproduction of Proinflammatory Cytokines in Athletes

Objective: It was the aim of this study to evaluate whether chronic pain in athletes is related to performance, measured by the maximum oxygen consumption and production of hormones and cytokines. Methods: Fifty-five athletes with a mean age of 31.9 ± 4.2 years engaged in regular competition and showing no symptoms of acute inflammation, particularly fever, were studied. They were divided into 2 subgroups according to the occurrence of pain. Plasma concentrations of adrenaline, noradrenaline, cortisol, prolactin, growth hormone and dopamine were measured by radioimmunoassay, and the production of the cytokines interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor-α, interferon-α and prostaglandin E₂ by whole-blood culture. Maximal oxygen consumption was determined during an incremental treadmill test. Results: There was no change in the concentration of stress hormones, but the athletes with chronic pain showed a reduction in maximum oxygen consumption (22%) and total consumption at the anaerobic threshold (25%), as well as increased cytokine production. Increases of 2.7-, 8.1-, 1.7- and 3.7-fold were observed for IL-1, IL-2, tumor necrosis factor-α and interferon-α, respectively. Conclusions: Our data show that athletes with chronic pain have enhanced production of proinflammatory cytokines and lipid mediators and reduced performance in the ergospirometric test.

Possible involvement of heparin-binding protein in transfusion-related acute lung injury

In antibody-mediated nonhemolytic transfusion reactions, transfusion-related acute lung injury (TRALI) tends to occur typically within 2 hours after a blood transfusion. White cell antibodies or immune complexes have been frequently shown to be associated with the syndrome, although the mechanisms by which they induce TRALI are poorly understood. The aim of this study was to characterize soluble mediators that are released from cells at an early stage after immune stimulation. To explore the mechanism of TRALI, an in vitro whole-blood cell culture assay was established in which cells were stimulated by human antibodies and the activation of neutrophils was monitored by a cell surface marker (Mac-1) with flow cytometry and by measurement of the release of soluble factors, including perforin, interleukin-6, tumor necrosis factor-alpha, and heparin-binding protein (HBP) with enzyme-linked immunosorbent assays. In addition, the involvement of two neutrophil FcgammaRs (FcgammaRIIIb and FcgammaRIIa, also known as CD16 and CD32, respectively) was examined during antibody-induced cell activation with anti-FcgammaR blocking antibodies. Substantial amounts of HBP were released within 30 minutes of stimulation by human antibodies, although other soluble mediators were not released within the same period. Furthermore, the release of HBP was mediated via signals through both FcgammaRIIIb and FcgammaRIIa. HBP appears to be one of the primary effector molecules of antibody-mediated nonhemolytic transfusion reactions including TRALI.

Hepatosplenomegaly Is Associated with Low Regulatory and Th2 Responses to Schistosome Antigens in Childhood Schistosomiasis and Malaria Coinfection

Hepatosplenomegaly among Kenyan schoolchildren has been shown to be exacerbated where there is transmission of both Schistosoma mansoni and Plasmodium falciparum. This highly prevalent and chronic morbidity often occurs in the absence of ultrasound-detectable periportal fibrosis and may be due to immunological inflammation. For a cohort of school-age children, whole-blood cultures were stimulated with S. mansoni soluble egg antigen (SEA) or soluble worm antigen (SWA). Responses to SWA were found to be predominantly Th2 cytokines; however, they were not significantly associated with either hepatosplenomegaly or infection with S. mansoni or P. falciparum. In comparison, SEA-specific Th2 cytokine responses were low, and the levels were negatively correlated with S. mansoni infection intensities and were lower among children who were coinfected with P. falciparum. Tumor necrosis factor alpha levels in response to stimulation with SEA were high, and a negative association between presentation with hepatomegaly and the levels of the regulatory cytokines interleukin-6 and transforming growth factor beta(1) suggests that a possible mechanism for childhood hepatomegaly in areas where both malaria and schistosomiasis are endemic is poor regulation of an inflammatory response to schistosome eggs.

Oral administration of the probiotic Lactobacillus fermentum VRI-003 and mucosal immunity in endurance athletes

ObjectiveTo evaluate the ability of a probiotic Lactobacillus fermentum VRI-003 (PCC) to enhance the mucosal immune system of elite athletes.Design and settingA double-blind, placebo-controlled, crossover trial was conducted over a...

Development and evaluation of a gamma-interferon assay for tuberculosis in badgers ( Meles meles)

In this paper we report the development of a sensitive and specific assay for the detection of tuberculosis (TB) in European badgers (Meles meles), based on the stimulation of lymphocytes in whole-blood culture and the subsequent detection of gamma-interferon (IFNgamma) by sandwich ELISA. The comparative levels of IFNgamma produced to bovine and avian tuberculin (B-A) was used as the basis of determining the TB status of badgers, resulting in a more sensitive test than that based on the defined Mycobacterium bovis antigens ESAT6 and CFP10. The assay was evaluated using 235 badgers. The IFNgamma EIA (enzyme immunoassay) based on a monoclonal pair (mEIA) was more sensitive than one using a rabbit polyclonal antiserum (pEIA). At a specificity of 93.6%, the mEIA was 80.9% sensitive, compared to a sensitivity of 74.5% for the pEIA. At the same specificity as the EIA, the current serological ELISA test for TB in badgers (Brock test) had a sensitivity of 48.9%. Only one of the culture positive badgers missed by the mEIA was correctly diagnosed by the Brock test, suggesting that the combination of both a T-cell and serological test has little diagnostic advantage.

T-helper 1 and 2 serum cytokine assay in chronic opioid addicts.

There are a few studies with conflicting results on the effects of opioids on the functioning of immune system. This study was performed to investigate the in vitro production of interferon-gamma and interleukin-10 after antigenic stimulation of cells using whole blood from opioid addicts. Blood samples were taken from 20 chronically opioid-addicted persons, who voluntarily enrolled for detoxification (10 opium and 10 heroin addicts). Blood samples were also taken from 10 healthy individuals with no history of drug abuse as the control. Cell culture was performed in a whole blood culture assay. Diluted blood samples were stimulated with phytohemagglutinin or with lipopolysaccharide and the supernatants were collected to measure cytokine production. The results demonstrated a significant decrease in interferon-gamma production and an increase in interleukin-10 secretion in heroin addicts, relative to the control group (35.9+/-26.3 versus 110.2+/-60.3 pg/mL, p<0.01 and 71.8+/-28.4 versus 17.1+/-13.5 pg/mL, p<0.01, respectively), however the changes in these values in opium addicts were not significant compared to healthy individuals. The results could suggest that opioid addiction leads to a shift in the Th1/Th2 cytokine balance of peripheral CD4+ cells towards the Th2 response, and opioid addicts demonstrate reduced mitogenic responsiveness of lymphocytes relative to healthy individuals.

In vitroresponse toCandida albicansin cultures of whole human blood from young and aged donors

Invasive infections with opportunistic fungi, such as Candida albicans, have become an increasing problem in aged adults in recent years. This work investigates the influence of human ageing on C. albicans recognition by toll-like receptors (TLRs), essential components of the innate immune system, using a cohort of 96 young (15-42 years) and aged (>70 years) human volunteers. No significant differences between aged and young donors were observed on (1) cell surface TLR2, TLR6 and TLR4 expression on lymphocytes, monocytes and granulocytes, (2) production of cytokines [IL-8, IL-1beta, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and IL-12p70] and prostaglandin E(2) (PGE(2)) by whole human blood in response to C. albicans and (3) fungicidal activity of whole blood. A statistically significant higher titre of natural anti-C. albicans antibodies was found in plasma of volunteers between 80 and 95 years old when compared with other age groups, probably as a consequence of the increased levels of serum Ig that has been described in elderly subjects. Therefore, the results indicate that the increased susceptibility to C. albicans infections in the elderly is not a consequence of defects in TLRs expression or signalling, nor of an impaired fungicidal activity of blood.

Cytokine levels in supernatants of whole blood and mononuclear cell cultures in adults and neonates reveal significant differences with respect to interleukin‐13 and interferon‐gamma

Findings regarding cytokine levels in neonates and their impact on the development of allergic diseases are controversial. This might be attributed to different laboratory approaches. To compare cytokine levels in supernatants of whole blood (WB) and mononuclear cell (MC) cultures in response to unspecific and allergen specific stimuli. A total population of n = 25 healthy full-term neonates and n = 25 adults was recruited. WB was diluted 1 in 5 and incubated with phytohaemagglutinine (PHA; 20 mug/ml) and the cows' milk protein betalactoglobulin (BLG) for 24 and 120 h. In parallel, cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cells (PBMC) were isolated, and cells were cultured with PHA and BLG in the same concentrations in a medium supplemented with fetal calf serum (FCS) and in a serum- free medium (only PBMC from adults). The cytokines interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and IL-13 in the cell culture supernatants were measured using the ELISA technique. IFN-gamma and IL-10 levels in response to PHA in supernatants of MC of neonates were significantly lower compared to that in adults (p < 0.05, Wilcoxon two-sample test). IL-13 levels were significantly higher in response to PHA in neonates. In adults, only levels of IL-10 were significantly correlated in WB and PBMC cultures (PHA: r(S) 0.6; p = 0.002; BLG: r(S) 0.54; p = 0.005). In neonates, IL-10 (PHA: r(S) 0.77; p < 0.001; BLG: r(S) 0.63; p < 0.001) and IFN-gamma (PHA: r(S) 0.48; p = 0.02; BLG: r(S) 0.4; p < 0.047) were significantly correlated. Supernatants of PBMC cultured with an FCS-supplemented medium showed a significant lower IFN-gamma release (PHA 1297 pg/ml; BLG 2762 pg/ml) compared to serum-free cell cultures (PHA 6592.5 pg/ml, p < 0.0001; BLG 14228 pg/ml, p = 0.04). IFN-gamma and IL-13 levels in WB and MC supernatants revealed significant differences. Our data indicate the need for thoroughly defined and standardized culture conditions for the detection of in vitro cytokine levels.

Increased micronucleus frequency in phytohaemagglutinin‐stimulated blood cells of patients with vitiligo

Vitiligo is a relatively common, acquired pigmentary disorder characterized by areas of depigmented skin resulting from loss of melanocytes in the epidermis. Although several hypotheses have been proposed for the aetiology and pathogenesis of vitiligo, the cause of vitiligo remains unclear. To evaluate spontaneous micronucleus (MN) frequency using the cytokinesis block MN assay to determine damages at the DNA or chromosome level in phytohaemagglutinin (PHA)-stimulated blood cells of patients with vitiligo and healthy control subjects. Peripheral blood samples were obtained and cultured from 21 patients with vitiligo (mean age: 21.48 +/- 9.78 years) and 21 age- and sex-matched healthy control subjects (mean age: 21.52 +/- 9.80 years). MN values were scored in binucleated cells obtained from whole-blood cultures of patients and control subjects. MN frequencies (mean +/- SD) in PHA-stimulated blood cells of patients with vitiligo and control subjects were 0.94 +/- 0.58 and 0.58 +/- 0.32, respectively. Compared with control subjects, MN frequencies of patients with vitiligo were found significantly higher than those of the control subjects (P = 0.012). Our results indicate unexpectedly some chromosomal/DNA damage in whole-blood cultures of patients with vitiligo. We do not know, however, if these chromosome/DNA instabilities observed in the cells of vitiligo patients resulted from the cause or from the consequences of the disorder.