Showing papers in "Clinical and Vaccine Immunology in 2003"
TL;DR: A multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen was developed and proved to be a powerful tool in the quantitation of cytokines.
Abstract: Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1α [IL-1α], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.
577 citations
TL;DR: Streptococcus pneumoniae is a major human pathogen causing pneumonia, sepsis, meningitis, and otitis media, and elderly adults because their immune systems are either unprepared or unable to respond effectively to it.
Abstract: Streptococcus pneumoniae is a major human pathogen causing pneumonia, sepsis, meningitis, and otitis media ([12][1]). It causes infections most often in young children ([12][1]) and elderly adults ([1][2]) because their immune systems are either unprepared or unable to respond effectively to
317 citations
TL;DR: Age-specific efficacy estimates for MCC vaccines obtained from postlicensure surveillance in England were compared with the efficacy predicted by the percentage of individuals in these age groups with rSBA titers above different cutoffs, suggesting that continuing protection is less dependent on the SBA level at the time of exposure but is more reliant on immunologic memory.
Abstract: Meningococcal C conjugate (MCC) vaccines were licensed on the basis of serological correlates of protection without efficacy data. The original correlate of protection was established by using a serum bactericidal antibody assay (SBA) with human complement (hSBA), with titers > or =4 predicting protection. However, the antibody data supporting licensure were largely generated by SBA with rabbit complement (rSBA), which gives higher titers than hSBA. While rSBA titers > or =128 reliably predict protection, as measured by hSBA, sera with rSBA titers in the range of 8 to 64 may not have hSBA titers > or =4. For rSBA titers in this equivocal range, a fourfold rise pre- to postvaccination with the MCC vaccine and/or a characteristic booster response to a polysaccharide challenge was proposed as a correlate of protection. To validate this proposed rSBA correlate, age-specific efficacy estimates for MCC vaccines obtained from postlicensure surveillance in England were compared with the efficacy predicted by the percentage of individuals in these age groups with rSBA titers above different cutoffs at 4 weeks and at 7 to 9 months after vaccination with the MCC vaccine. The average time since vaccination in the cohorts in whom efficacy was measured ranged from 8 to 10 months. The rSBA cutoff of > or =128 was shown to significantly underestimate efficacy, with rSBA cutoffs of > or =4 or > or =8 at 4 weeks postvaccination with the MCC vaccine being the most consistent with observed efficacy. When the levels obtained 7 to 9 months postvaccination with the MCC vaccine were used, all rSBA cutoffs significantly underestimated efficacy, suggesting that continuing protection is less dependent on the SBA level at the time of exposure but is more reliant on immunologic memory.
306 citations
TL;DR: The HPV-Luminex competitive immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.
Abstract: Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 μl of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.
300 citations
TL;DR: Data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2, and that these effects are stimulatory on mouse immune cells.
Abstract: Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-α) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-κB activation and TNF-α production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall ≫ polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-α secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2−/− mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-κB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.
272 citations
TL;DR: The release of a biological weapon (BW) agent by a terrorist group or military force would likely be silent and undetectable or nearly so.
Abstract: BW agents.The release of a biological weapon (BW) agent by a terrorist group or military force would likely be silent and undetectable or nearly so. As shown by anthrax attack during the fall of 2001 in the eastern United States, patients would begin appearing at hospitals and clinics within several
206 citations
TL;DR: It is found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections.
Abstract: We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.
168 citations
TL;DR: VLP-based EIAs for SV40, BKV, and JCV are likely to contribute to a better understanding of the biology of these viruses with their greater versatility and their suitability for large-scale testing.
Abstract: Enzyme immunoassays (EIAs) for detection of serum antibodies to simian virus 40 (SV40), BK virus (BKV), and JC virus (JCV) were developed by using virus-like-particles (VLPs) produced in insect cells from recombinant baculoviruses expressing the VP1 protein of the respective virus. Rhesus macaque sera with neutralizing antibodies to SV40 showed a high level of reactivity in the SV40 VLP-based EIA, and these sera also showed lower levels of reactivity in the BKV and JCV VLP-based EIAs. Rhesus macaque sera negative for neutralizing antibodies to SV40 were negative in all three EIAs. Competitive binding assays showed that SV40 VLPs inhibited BKV reactivity. In rhesus macaque sera, high optical density (OD) values for antibodies to SV40 VLPs were correlated with high OD values for antibodies to BKV but not with high OD values for antibodies to JCV VLPs. Human sera with neutralizing antibodies to SV40 were more reactive to SV40 VLPs than human sera without neutralizing antibodies to SV40. The greater SV40 reactivities of human sera were correlated with greater reactivities to BKV VLPs but not JCV VLPs. These data suggest that cross-reactivity with BKV antibodies may account for part of the low-level SV40 reactivity seen in human sera. With their greater versatility and their suitability for large-scale testing, the VLP-based EIAs for SV40, BKV, and JCV are likely to contribute to a better understanding of the biology of these viruses.
149 citations
TL;DR: Given that saliva is a readily obtainable, noninvasive specimen, detection of DEN-specific salivary IgM should be considered a useful, cheaper diagnostic modality with similar sensitivity and specificity to IgM detection in serum.
Abstract: To evaluate alternative approaches to the serological diagnosis of dengue virus (DEN) infection, the detection of DEN-specific immunoglobulin M (IgM) and IgA antibodies in serum and saliva specimens was assessed in 147 patients with symptoms of DEN infection seen at the Ministry of Health in Nicaragua. Seventy-two serum samples were determined to be positive for anti-DEN antibodies by IgM capture enzyme-linked immunosorbent assay, the routine diagnostic procedure. Serum and saliva specimens were obtained from 50 healthy adults as additional controls. IgM was detected in the saliva of 65 of the 72 serum IgM-positive cases, 6 of the 75 serum IgM-negative cases, and none of the control group, resulting in a sensitivity of 90.3% and a specificity of 92.0% and demonstrating that salivary IgM is a useful diagnostic marker for DEN infection. Detection of IgA in serum may be another feasible alternative for the diagnosis of DEN infection, with serum IgA found in 68 (94.4%) of the IgM-positive cases. In contrast, detection of IgA in saliva was not found to be a useful tool for DEN diagnosis in the present study. Further studies of the kinetics of antibody detection in another set of 151 paired acute- and convalescent-phase serum samples showed that DEN-specific IgA antibodies were detected in more acute-phase samples than were IgM antibodies. Thus, we conclude that DEN-specific IgA in serum is a potential diagnostic target. Furthermore, given that saliva is a readily obtainable, noninvasive specimen, detection of DEN-specific salivary IgM should be considered a useful, cheaper diagnostic modality with similar sensitivity and specificity to IgM detection in serum.
146 citations
TL;DR: Serum glucan levels in intensive care unit (ICU) patients did not show a correlation with the presence of fungal infections and do not appear to be specific forfungal infections, but the assay may be useful as a negative predictor of infection.
Abstract: Fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. A major obstacle to managing fungal infection is the lack of a reliable clinical assay that will rapidly identify patients with fungal sepsis. Glucans are polymers of glucose that are found in the cell wall of fungi and certain bacteria. Glucans are also released from the fungal cell wall into the extracellular milieu. Several studies have reported that detection of fungal glucan in serum or plasma is useful in the diagnosis of mycoses. However, recent studies have questioned the clinical utility of this assay. In this study, we examined serum glucan levels in intensive care unit (ICU) patients and attempt to correlate serum glucan levels with the presence of fungal infection. Following attainment of informed consent, serum was harvested from 46 ICU patients with confirmed fungal infections, confirmed bacterial infections, or no evidence of infection. Sera from eight healthy volunteers served as control. Serum glucan was assayed with a glucan-specific Limulus assay. Serum glucan levels were increased (69.6 ± 17 pg/ml; P < 0.001) in ICU patients versus the normal (11.5 ± 1.3 pg/ml) and noninfected ICU (27.4 ± 17 pg/ml) controls. However, serum glucan levels were not different in patients with confirmed fungal infections versus those with confirmed bacterial infections. Thus, serum glucan levels did not show a correlation with the presence of fungal infections and do not appear to be specific for fungal infections. However, the assay may be useful as a negative predictor of infection.
146 citations
TL;DR: It is suggested that certain cytokines may play an important role in the pathogenesis of and the protection against rotavirus disease in children and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.
Abstract: Rotavirus is the most common cause of severe gastroenteritis in young children, but the pathogenesis and immunity of this disease are not completely understood. To examine the host response to acute infection, we collected paired serum specimens from 30 children with rotavirus diarrhea and measured the levels of nine cytokines (interleukin-1beta [IL-1beta], IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) using a microsphere-based Luminex Flowmetrix system. Patients with acute rotavirus infection had elevated median levels of seven cytokines in serum, and of these, the levels of three (IL-6, IL-10, and IFN-gamma) were significantly (P < 0.05) higher than those in serum from control children without diarrhea. Patients with fever had significantly (P < 0.05) higher levels of IL-6 in serum than control children, and those with fever and more episodes of diarrhea had significantly (P < 0.05) higher levels of TNF-alpha than those without fever and with fewer episodes of diarrhea. We further demonstrated a negative association (P < 0.05) between the levels of IL-2 and the number of stools on the day on which the first blood sample was collected. Finally, patients with vomiting had significantly (P < 0.05) lower levels of IFN-gamma than those without vomiting. Our pilot study provides evidence that the types and magnitudes of cytokine responses to rotavirus infection in children influence or reflect the clinical outcome of disease. These findings suggest that certain cytokines may play an important role in the pathogenesis of and the protection against rotavirus disease in children and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.
TL;DR: Irrespective of the route of administration, the potential probiotic strain Lactobacillus plantarum NCIMB8826 was found to persist for up to 10 days in the digestive tracts of mice, making it an attractive candidate as a probiotic to be used in the prevention or treatment of chronic inflammation.
Abstract: Recent clinical and experimental observations showed that specific probiotic microorganisms may provide therapeutic benefits in inflammatory bowel disease. However, a rigorous screening for new candidate probiotic strains with optimized therapeutic properties necessitates also determining possible adverse interactions with the host, particularly in individuals who are not healthy. We have evaluated the persistence of strains of lactic acid bacteria (LAB) in the digestive tracts of mice, their immunomodulation capacity, and their safety in healthy animals and in a colitis model. Following daily administration of 10(9) CFU of viable LAB orally, intragastrically, or intrarectally, the animals' feces were examined for bacterial excretion and cytokines were quantified in intestinal samples by quantitative reverse transcription-PCR. The level of bacterial translocation was assessed in healthy mice and in mice suffering from colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Irrespective of the route of administration, the potential probiotic strain Lactobacillus plantarum NCIMB8826 was found to persist for up to 10 days in the digestive tracts of mice. This strain did not induce detrimental effects in healthy or in TNBS-treated animals, as was reflected by the absence of weight loss, intestinal inflammation, modification of cytokine levels in the ileum and colon (healthy mice), and bacterial dissemination (healthy and colitic animals). Moreover, the translocation of endogenous microflora to the mesenteric lymph nodes and spleen was greatly reduced in the TNBS-treated mice after administration of LAB. This property, together with the strain's persistence capacity and innocuousness renders L. plantarum NCIMB8826 an attractive candidate as a probiotic to be used in the prevention or treatment of chronic inflammation.
TL;DR: The analysis of cytokine concentrations with regard to the outcome—in terms of the need for intensive care unit admittance and/or mechanical ventilation as well as mortality—suggests that there is a direct relationship between the intensity of the inflammatory response measured in blood and the severity of the episode.
Abstract: In order to analyze the characteristics of the inflammatory response occurring in blood during pneumonia, we studied 38 patients with severe community-acquired pneumonia. Venous and arterial blood samples were collected at study entry and on days 1, 2, 3, 5, and 7 after inclusion. The concentrations of proinflammatory (tumor necrosis factor alpha [TNF-α], interleukin 1β [IL-1β], IL-6, and IL-8) and anti-inflammatory (IL-10) cytokines were determined in order to detect differences related to the origin of the sample, the causative organism, the clinical variables, and the final outcome of the episode. Legionella pneumonia infections showed higher concentrations of TNF-α, IL-6, IL-8, and IL-10. After 24 h, plasma IL-6, IL-8, and IL-10 concentrations in pneumococcal episodes increased, whereas in the same time interval, cytokine concentrations in Legionella episodes markedly decreased. The characteristics of the inflammatory response in bacteremic pneumococcal episodes were different from those in nonbacteremic episodes, as indicated by the higher plasma cytokine concentrations in the former group. Finally, our analysis of cytokine concentrations with regard to the outcome—in terms of the need for intensive care unit admittance and/or mechanical ventilation as well as mortality—suggests that there is a direct relationship between the intensity of the inflammatory response measured in blood and the severity of the episode.
TL;DR: C-reactive protein levels obtained with the enzyme immunoassay were highly correlated with those obtained with an automated immunonephelometric assay, and levels were unaffected by delays in sample processing and storage temperature.
Abstract: C-reactive protein (CRP) is an acute-phase reactant whose levels increase in response to a variety of inflammatory stimuli. Elevated levels in serum are observed after trauma, tissue necrosis, infection, surgery, and myocardial infarction and are associated with an increased risk of cardiovascular disease. CRP levels are also elevated in noninflammatory states, such as obesity, sleep disturbances, depression, chronic fatigue, aging, and physical inactivity. In this study, the performance of a highly sensitive CRP enzyme immunoassay was evaluated, along with common laboratory variables (specimen type, processing time, and storage conditions) that may influence measured blood concentrations of CRP. The measurement range of the assay was from 0.4 to 50 μg/liter. Total imprecision (coefficient of variation) ranged from 8.1 to 11.4%. CRP levels obtained with the enzyme immunoassay were highly correlated with those obtained with an automated immunonephelometric assay. Comparable results were obtained for plasma (heparin and EDTA treated) and serum samples, and levels were unaffected by delays in sample processing and storage temperature. CRP levels were also unaffected by up to seven freeze-thaw cycles. The median CRP concentration in healthy adults was determined to be 0.94 mg/liter, with a 95% working reference interval of 0 to 6.9 mg/liter. In view of these data, we recommend that serial serum or plasma samples for CRP should be stored at 4oC for short periods of time or at −70oC for longer periods and tested within the same run to minimize interassay variability.
TL;DR: Serum levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b and for tetanus toxoid and for PCP were measured in serum samples of 386 age-stratified subjects to provide reference ranges to aid in the interpretation of specific antibody determinations in the clinical setting.
Abstract: Antibody levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) and for tetanus toxoid were measured in serum samples of 386 age-stratified subjects. The study group consists of healthy adult blood donors and hospitalized children undergoing elective surgery, excluding individuals with a history of infection. In children, anti-tetanus toxoid antibody levels displayed two peaks of 1.20 IU/ml (20.4 mg/liter) and 1.65 IU/ml (28.1 mg/liter) related to the schedule of routine childhood immunization in the first year and at 8 years of age. Eighty percent of the antibodies are of the immunoglobulin G1 (IgG1) isotype. For pneumococcal capsular polysaccharide (PCP), the specific antibody levels represent the acquisition of natural immunity. The initial concentration of 9.2 mg/liter was low in infancy (0.5 to 1 years of age) and remained low until 3 to 4 years of age (14.6 mg/liter). During this period PCP antibodies were almost 100% of the IgG2 subclass. Thereafter, IgG anti-PCP antibody titers increased steadily to adult levels (59.5 mg/liter). The data are intended to provide reference ranges to aid in the interpretation of specific antibody determinations in the clinical setting.
TL;DR: The flow assay requires neither specialized training nor equipment, the assay is very easy to perform and to read, and the components are stable without a requirement for refrigeration and well standardized, indicating that the Brucella IgM and IgG flow assays are ideal for use in clinical settings in rural and suburban areas in which brucellosis is endemic.
Abstract: To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG antibodies. The diagnostic values of these tests were examined. The tests are shown to detect acute, persistent, and relapsing disease and can be used to monitor treatment. The sensitivity of Brucella IgM and IgG flow assays calculated for the combined assay results is 96%, and specificity amounts to 99%. The flow assay requires neither specialized training nor equipment, the assay is very easy to perform and to read, and the components are stable without a requirement for refrigeration and well standardized. Together these characteristics indicate that the Brucella IgM and IgG flow assays are ideal for use in clinical settings in rural and suburban areas in which brucellosis is endemic.
TL;DR: Investigation of immune responses to Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, and Candida albicans indicates the presence of an active antibody response in synovial tissue and illustrates a potential connection between periodontal and joint diseases.
Abstract: The objective of the present study was to investigate immunoglobulin G (IgG) and IgA antibody immune responses to Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, and Candida albicans in the sera of patients with rheumatoid arthritis (RA), the synovial fluid (SF) of patients with RA (RA-SF samples), and the SF of patients without RA (non-RA-SF samples). An enzyme-linked immunosorbent assay was used to determine IgG and IgA antibody levels in 116 serum samples from patients with RA, 52 RA-SF samples, and 43 non-RA-SF samples; and these were compared with those in SF samples from 9 patients with osteoarthritis (OA-SF samples) and the blood from 100 donors (the control [CTR] group). Higher levels of IgG antibodies against B. forsythus (P < 0.0001) and P. intermedia (P < 0.0001) were found in non-RA-SF samples than in OA-SF samples, and higher levels of IgG antibodies against B. forsythus (P = 0.003) and P. intermedia (P = 0.024) were found in RA-SF samples than in OA-SF samples. Significantly higher levels of IgA antibodies against B. forsythus were demonstrated in both RA-SF and non-RA-SF samples than in OA-SF samples. When corrected for total Ig levels, levels of IgG antibody against B. forsythus were elevated in RA-SF and non-RA-SF samples compared to those in OA-SF samples. Lower levels of Ig antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group for both IgG (P = 0.003) and IgA (P < 0.0001). When corrected for total Ig levels, the levels of IgG and IgA antibodies against B. forsythus were still found to be lower in the sera from patients with RA than in the plasma of the CTR group (P < 0.0001). The levels of antibodies against P. gingivalis and C. albicans in the sera and SF of RA and non-RA patients were comparable to those found in the respective controls. The levels of IgG and IgA antibodies against B. forsythus were elevated in SF from patients with RA and non-RA-SF samples compared to those in OA-SF samples. Significantly lower levels of IgG and IgA antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group. This indicates the presence of an active antibody response in synovial tissue and illustrates a potential connection between periodontal and joint diseases.
TL;DR: The WBC and hemoglobin values of healthy HIV-negative adults from the CAR are lower than the reference values currently used in the CAR and the absolute CD4 T-cell counts ofhealthy HIV- negative adults fromThe CAR are similar to values for Europeans but theabsolute CD8 T- cell counts are much higher.
Abstract: A survey was carried out on 150 healthy adults to establish hematological reference ranges for human immunodeficiency virus (HIV)-negative adults from the Central African Republic (CAR). Immunohematological mean values, medians, and 95th-percentile reference ranges were established. Mean values were as follows: leukocyte (WBC) counts, 5.28 × 109/liter (males) and 5.11 × 109/liter (females); erythrocyte counts, 5.20 × 1012/liter (males) and 4.50 × 1012/liter (females); hemoglobin, 15.1 g/dl (males) and 12.5 g/dl (females); hematocrit, 45% (males) and 37% (females); lymphocytes, 2,587/μl (males) and 2,466/μl (females); CD4 T cells, 927/μl (males) and 940/μl (females); CD8 T cells, 898/μl (males) and 716/μl (females); and CD4/CD8 T-cell ratio, 1.13 (males) and 1.41 (females). We concluded that (i) the WBC and hemoglobin values of healthy HIV-negative adults from the CAR are lower than the reference values currently used in the CAR and (ii) the absolute CD4 T-cell counts of healthy HIV-negative adults from the CAR are similar to values for Europeans but the absolute CD8 T-cell counts are much higher. Thus, the CD4/CD8 T-cell ratios for healthy adults from the CAR are significantly reduced compared to the ratios for healthy Europeans.
TL;DR: It is established that LukM/LukF′-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukOToxin produced by mastitis isolates.
Abstract: Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF′-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM -positive isolates were much more leukotoxic than the supernatants of lukM -negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM / lukF ′- PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin ( P lukM / lukF ′-PV positive or negative isolates. These results establish that LukM/LukF′-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF′-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.
TL;DR: In this paper, the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma.
Abstract: The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue factor-factor VIIa.
TL;DR: A rapid diagnostic test for Vibrio cholerae O1 and O139 based on lipopolysaccharide detection using gold particles is described, which can provide a simple tool for epidemiological surveys.
Abstract: We describe the development and evaluation of a rapid diagnostic test for Vibrio cholerae O1 and O139 based on lipopolysaccharide detection using gold particles. The specificity ranged between 84 and 100%. The sensitivity of the dipsticks ranged from 94.2 to 100% when evaluated with stool samples obtained in Madagascar and Bangladesh. The dipstick can provide a simple tool for epidemiological surveys.
TL;DR: The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range.
Abstract: We describe a fluorescent covalent microsphere immunoassay (FCMIA) method for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes present in the pneumococcal polysaccharide vaccine (PPV23) licensed by the Food and Drug Administration, i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In addition, the assay incorporates an internal control that allows for contemporaneous evaluation of the effectiveness of pneumococcal cell wall polysaccharide (C-PS) preadsorption and a second control of PnPS 25 (which is not present in any polysaccharide or conjugate vaccine), which can be used to evaluate interassay reproducibility (useful for pre- versus postvaccination studies). The FCMIA was standardized with U.S. reference antipneumococcal serotype standard serum 89S-2. Preadsorption of 89S-2 with each PnPS and C-PS yielded homologous inhibition for serotypes 1, 6B, 9N, 9V, 11A, 12F,14, 15B, 18C, 19A, 19F, 20, 22F, 25, and 33F; heterologous inhibition for serotypes 9V, 10A, 11A, 12F, 15B, 17F, 20, and 23F; and neither homologous nor heterologous inhibition for serotypes 2, 3, 4, and 5. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14. The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range.
TL;DR: The results indicate that TLR4 is able to undergo multiple glycosylations without MD-2 but that the specific Glycosylation essential for cell surface expression requires the presence ofMD-2.
Abstract: MD-2 has been reported to be required for the translocation of the Toll-like receptor 4 (TLR4) to the cell surface. However, the mechanism by which MD-2 promotes TLR4 translocation is unknown. We identified the presence of two forms of TLR4 with different molecular masses (approximately 110 and 130 kDa) when TLR4 was expressed together with MD-2. Expressing TLR4 alone produced only the 110-kDa form. Using a membrane-impermeable biotinylation reagent, we found that only the 130-kDa form of TLR4 was expressed on the cell surface. When a cellular extract prepared from cells expressing TLR4 and MD-2 was treated with N-glycosidase, the two forms of TLR4 converged into a single band whose size was smaller than the 110-kDa form of TLR4. Mutation of TLR4 at Asn526 or Asn575 resulted in the disappearance of the 130-kDa form and prevented TLR4 from being expressed on the cell surface without affecting the ability of TLR4 to associate with MD-2. These results indicate that TLR4 is able to undergo multiple glycosylations without MD-2 but that the specific glycosylation essential for cell surface expression requires the presence of MD-2.
TL;DR: The detection of measles virus-induced IFN-γ and IL-4 production by ELISPOT can be used to identify measlesirus-specific low-frequency memory T cells in subjects immunized with measles vaccine and differences agree in directionality with the observed antibody response phenotype.
Abstract: The protective effect of measles immunization is due to humoral and cell-mediated immune responses. Little is known about cell-mediated immunity (CMI) to measles vaccine virus, the relative contribution of CD4+ and CD8+ T cells to variability in such immune responses, and the immunologic longevity of the CMI after measles vaccination in humans. Our study characterizes cellular immune response in subjects seronegative or highly seropositive for measles vaccine immunoglobulin G-specific antibody, aged 15 to 25 years, previously immunized with two doses of measles-mumps-rubella II vaccine. We evaluated the ability of subjects to respond to measles vaccine virus by measuring measles virus-specific T-cell proliferation. We examined the frequencies of measles virus-specific memory Th1 and Th2 cells by an ELISPOT assay. Our results demonstrated that proliferation of T cells in seronegative subjects was significantly lower than that for highly seropositive subjects (P = 0.003). Gamma interferon (IFN-γ) secretion predominated over interleukin 4 (IL-4) secretion in response to measles virus in both groups. The median frequency of measles virus-reactive CD8+ T cells secreting IFN-γ was 0.09% in seronegative subjects and 0.43% in highly seropositive subjects (P = 0.04). The median frequency of CD4+ T cells secreting IL-4 in response to measles virus was 0.03% in seronegative subjects and 0.09% in highly seropositive subjects (P = 0.005). These data confirm the presence of measles virus-specific cellular immune responses post-measles vaccine immunization in humans. The detection of measles virus-induced IFN-γ and IL-4 production by ELISPOT can be used to identify measles virus-specific low-frequency memory T cells in subjects immunized with measles vaccine. These differences agree in directionality with the observed antibody response phenotype.
TL;DR: The data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells and may be useful in evaluating antibodies elicited in response to PSAA vaccination.
Abstract: The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 104 to 105 bacteria/well, the mean ± standard deviation count in controls was 163 ± 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 μg/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination.
TL;DR: Whether elicited by infection or intraperitoneal injection of i-antigen, the serum and mucus antibody responses of channel catfish immunized against I. multifiliis did not occur synchronously and induced a transient mucosal antibody response that coincided with the resolution of infection.
Abstract: Fish acquire protective immunity against the ciliated protozoan parasite Ichthyophthirius multifiliis following sublethal infection or inoculation with I. multifiliis immobilization antigens (i-antigens). In both cases, parasite-immobilizing antibodies have been identified in sera and mucosal secretions. To investigate the kinetics of this immune response, antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) in the sera and cutaneous mucus of channel catfish (Ictalurus punctatus) that were either infected with parasites or given a single injection of purified i-antigen (5.0 μg/fish) in Freund's incomplete adjuvant. At 5 weeks, infected and inoculated fish had a mean serum (1:80 dilution) antibody absorbance (A405) value of 0.54 ± 0.17 and 0.35 ± 0.03, respectively, which were significantly higher (α = 0.05) than the pretreatment serum (1:80 dilution) antibody absorbance value of 0.24 ± 0.05. At 14 weeks, mean serum (1:80 dilution) ELISA absorbance values in the teo groups of fish increased to 0.79 ± 0.30 and 0.71 ± 0.24, respectively. In both groups of fish, antibody levels in cutaneous mucus (undiluted) were much lower than those in sera. Infected fish had detectable mucus (undiluted) antibody levels from 3 to 9 weeks, with the highest mean value (0.30 ± 0.07) occurring at 7 weeks. Although individual inoculated fish produced serum antibody absorbance values comparable to those seen in infected fish, the mean mucus antibody values in this group did not rise above pretreatment levels. I. multifiliis infection induced a transient mucosal antibody response that coincided with the resolution of infection. Whether elicited by infection or intraperitoneal injection of i-antigen, the serum and mucus antibody responses of channel catfish immunized against I. multifiliis did not occur synchronously.
TL;DR: Evidence is provided that probiotics modulate the oral tolerance response to BLG in mice and the mono-colonization effect is strain-dependant, the best result having been obtained with L. paracasei.
Abstract: In this study, the effect of Lactobacillus paracasei (NCC 2461), Lactobacillus johnsonii (NCC 533) and Bifidobacterium lactis Bb12 (NCC 362) on the induction and maintenance of oral tolerance to bovine beta-lactoglobulin (BLG) was investigated in mice. Germfree mice were monocolonized with one of the three strains before oral administration of whey protein to induce tolerance. Mice were then injected with BLG and sacrificed 28 or 50 days after whey protein feeding for humoral and cellular response measurement. Conventional and germfree mice were used as controls. Both humoral and cellular responses were better suppressed in conventional mice than in germfree and monoassociated mice throughout the experiment and better suppressed in L. paracasei-associated mice than in mice colonized with B. lactis or L. johnsonii. The latter two mono-associations suppressed humoral responses only partially and cellular responses not at all. This study provides evidence that probiotics modulate the oral tolerance response to BLG in mice. The mono-colonization effect is strain-dependant, the best result having been obtained with L. paracasei.
TL;DR: The ability to induce h βD-2 expression in combination with sensitivity to its antimicrobial effects may contribute to the rarity of skin infections with the gram-negative bacterial organisms, whereas lack of stimulation of hβD- 2 expression by S. pyogenes may be important in its ability to evade innate defenses and cause skin disease.
Abstract: Human beta defensin 2 (hbetaD-2) is thought to play an important role in cutaneous immune defense. We hypothesized that (i) keratinocyte expression of hbetaD-2, measured by reverse transcription-PCR, would be upregulated in response to challenge with pathogenic bacteria, particularly highly adherent strains of Streptococcus pyogenes and Staphylococcus aureus, and (ii) hbetaD-2 would have potent antimicrobial activity against pathogenic but not commensal organisms. Expression of hbetaD-2 was induced consistently by S. aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa, whereas strains of S. pyogenes were poor and variable inducers of hbetaD-2. No correlation was found between levels of bacterial adherence and keratinocyte expression of hbetaD-2. S. pyogenes was significantly more sensitive to killing by hbetaD-2 than S. epidermidis. We conclude that the ability to induce hbetaD-2 expression in combination with sensitivity to its antimicrobial effects may contribute to the rarity of skin infections with the gram-negative bacterial organisms, whereas lack of stimulation of hbetaD-2 expression by S. pyogenes may be important in its ability to evade innate defenses and cause skin disease. Induction of expression of hbetaD-2 but relative tolerance to it may enable S. epidermidis to survive on the skin surface and modulate hbetaD-2 expression when the stratum corneum barrier is disrupted.
TL;DR: Complement, as a vital part of the body's immune system, provides a highly effective means for the destruction of invading microorganisms and for immune complex elimination through activation and interaction with its respective receptors on various receptors.
Abstract: Complement, as a vital part of the body's immune system, provides a highly effective means for the destruction of invading microorganisms and for immune complex elimination (for reviews, see references [77][1] and [78][2]). Through activation and interaction with its respective receptors on various
TL;DR: Results show that VSVΔG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantviruses.
Abstract: A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVΔG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVΔG*G. Pseudotype VSV with the Hantaan (VSVΔG*-HTN) or Seoul (VSVΔG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 105 to 106/ml. The infectivity of VSVΔG*-HTN and VSVΔG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVΔG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVΔG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4°C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.