Sera from Volunteers Immunized by Planned Blood Transfusions as a Source of Dr Cytotoxic Typing Reagents

Sera from volunteers immunized with planned blood transfusions were tested for anti DR cytotoxic antibodies with a panel of HLA typed cultured human lymphoid cells using a variety of serological techniques. The majority of sera contained DR cytotoxic antibodies. The specificity of DR antibodies in seven sera was determined by testing them with a panel of B peripheral lymphocytes typed with DR alloantisera submitted to the 7th International Histocompatibility Workshop. The temporal evolution of DR and HLA-A and B cytotoxic antibodies was determined in two subjects by testing serial bleedings with B lymphoid cells, coated with Fab₂ fragments from anti β₂μ and anti DR xenoantisera. Results indicated a parallel evolution of DR and HLA-A and B cytotoxic antibodies.     

Measurement of Serum Antigen Concentration by Ultrasound-Enhanced Immunoassay and Correlation with Clinical Outcome in Meningococcal Disease

 The distribution of Neisseria meningitidis serogroup B and C polysaccharide antigen in blood and the prognostic significance of antigen concentration was examined by ultrasound-enhanced immunoagglutination of coated microparticles. Specimens (169 sera/plasma from 145 patients with confirmed meningococcal disease) were tested retrospectively. The ultrasonic immunoassay detected serum antigen in 136 samples from 112 patients. Titration of antigen-positive specimens allowed estimation of blood antigen concentration. The modal blood antigen titre was 1/16, corresponding to an estimated polysaccharide concentration of 0.85 μg/ml. The lowest mean blood antigen concentration found ultrasonically was 0.05 μg/ml; compared to the 1.98 μg/ml found by conventional latex agglutination, this represents an approximately 30-fold improvement in sensitivity. Three grades of outcome were correlated with the presenting antigen titre in 83 patients: (i) <2 weeks hospitalisation, (ii) ≥2 weeks hospitalisation and (iii) mortality. High polysaccharide concentrations correlated with mortality. Nine of 15 patients with a serum antigen titre of 1/64 or greater (≥3.4 μg/ml polysaccharide) died, whereas no patient with titres equal to or less than 1/4 (≤0.21 μg/ml) died, including those patients in whom antigen was undetectable by ultrasonic immunoassay. Increasing antigen concentration significantly correlated with severity of outcome (P<0.001). Ultrasound-enhanced agglutination provides a rapid prognostic indicator by sensitive measurement of serum antigen level.     

CAMP Factor of Group B Streptococci: Production, Assay, and Neutralization by Sera from Immunized Rabbits and Experimentally Infected Cows

Quantitative assay methods for CAMP factor activity of group B streptococci and for neutralization of this activity by animal sera are described. The course of the CAMP reaction is influenced by preincubation of red cells with staphylococcal beta-hemolysin and the rate of the reaction by the concentration of CAMP factor. The production of CAMP factor is enhanced by substitution of maltose for glucose in culture broth. Rising inhibitory titers to CAMP activity in sera of rabbits injected with sterile culture supernates of group B streptococci suggest that CAMP factor is an antigen in the rabbit. Cows with experimentally induced group B streptococcal mastitis also demonstrated significant rises in neutralizing titers for CAMP activity.     

Necessity of Molecular Techniques To Distinguish between Neisseria meningitidis Strains Isolated from Patients with Meningococcal Disease and from Their Healthy Contacts

Serogroup C strains of Neisseria meningitidis were isolated from a Germany patient with severe meningococcal disease after a trip to the Czech Republic. These strains (case isolates) were characterized by classical and molecular techniques, as were other strains (carrier isolates) isolated from healthy contacts. Five of 10 carrier isolates had switched off the expression of capsular polysaccharide, as demonstrated by a serogroup-specific PCR. The two case isolates were indistinguishable by multilocus sequence typing and belonged to the ET-37 complex. The carrier isolates belonged to four different sequence types, all unrelated to that of the case strains. Pulsed-field gel electrophoresis showed that the case isolates differed from reference ET-37 complex strains from the Czech Republic and Canada as well as from all the carrier isolates. The isolate from the patient’s nasopharynx was indistinguishable from the blood isolate except for a 40,000-bp chromosomal deletion that had occurred during systemic spread.Bacterial meningitis due to Neisseria meningitidis (the meningococcus) continues to be of global importance for public health authorities. While pandemics affecting China and Africa are usually caused by meningococci of the A capsular serogroup, sporadic meningitis, outbreaks, and hyperendemic disease in Central Europe and the United States are usually caused by serogroups B and C (2). For sporadic meningococcal meningitis, public health efforts often include bacteriological analysis of throat swabs obtained from close contacts of the patient and the treatment of healthy nasopharyngeal carriers with prophylactic antibiotics and/or vaccines (5).Meningococci isolated from the healthy contacts of a diseased patient (carrier isolates) are not necessarily related to the strain causing disease (case isolates), even if all these isolates express the same capsular serogroup. Various subtyping methods have been used to test relationships among the strains from a cohort, including serotyping and serosubtyping with monoclonal antibodies (MAbs) (11), pulsed-field gel electrophoresis (PFGE) (7), multilocus enzyme electrophoresis (MLEE) (2), ribotyping (22), the randomly amplified polymorphic DNA method (25), and PCR-restriction fragment length polymorphism analysis (16). Recently, a novel portable approach, multilocus sequence typing (MLST), which is based on the DNA sequences of six housekeeping gene fragments, has been developed (18). MLST allows assignment of meningococci to clonal groups within a globally accessible, continuously expanding central database.In outbreak situations, so many bacteria may be isolated that only some of the carrier strains, usually selected on the basis of their capsular serogroup by latex agglutination, are evaluated in detail. However, the expression of capsular polysaccharide by serogroup B meningococci can undergo phase variation, resulting in the isolation of strains from carriers which are not obviously related to the index strain because they are capsule negative and nonserogroupable (6, 14, 15). The siaD gene encodes a polysialyltransferase which is needed for the synthesis of capsular polysialic acid chains. siaD can be amplified from serogroup B and C meningococci by PCRs (6), enabling the determination of the potential serogroups of bacteria which have become phenotypically nonserogroupable. Here we describe a two-step PCR, an improved version of the siaD PCR (6), which distinguishes between serogroup B, C, W135, and Y meningococci.Antigenic variation through horizontal genetic exchange can also lead to capsule switching among highly related bacteria (23). Such capsule-switching variants would be classified as unrelated to the parent strain by classical serogrouping. Similarly, antigenic variation can also lead to the switching of other antigens (13), including those used for serological subtyping, such as PorA (21). Thus, serological methods cannot reliably recognize the relatedness of meningococci and epidemiological analyses should rely primarily on molecular techniques, particularly those based on multiple loci scattered around the chromosome (3).We report here on the molecular investigation of meningococci isolated from individuals who had been in contact with a patient with severe serogroup C meningococcal disease.     

Avidity of IgG Antibodies Against Meningococcal Serogroup A Polysaccharide and Correlations with Bactericidal Activity in Sera From Meningitis Patients and Controls From Ethiopia

Meningococcal meningitis is a significant global health challenge, especially for sub‐Saharan area: the African meningitis belt. Neisseria meningitidis of serogroup A (MenA) is responsible for the large number of epidemics that have been recorded in these countries. To determine the level of antibodies against meningococcal A polysaccharide (APS) that correlates with protection against MenA disease in the African meningitis belt, it may be important to consider antibody avidity along with quantity. In this study, two ELISA methods using the chaotropic agent ammonium thiocyanate were compared and employed to measure avidity indexes (AI) of IgG antibodies against APS in controls and in acute and convalescent sera from Ethiopian meningococcal patients. High statistical correlations between the AIs determined by the two methods were observed. The geometric mean AI (GMAI) increased with time from acute to convalescent sera indicating affinity maturation. GMAI was significantly higher in convalescent sera from the MenA patients and in sera from the controls than in acute sera from patients with meningococcal disease. A significant correlation between serum bactericidal activity titres (SBA) and concentration of IgG antibodies against APS was observed; however, our results did not indicate that determination of antibody avidities by the thiocyanate elution method gave a better correlation with SBA than anti‐APS IgG concentrations determined by the standard ELISA method.     

Isolation of IgG3 from Normal Human Sera and from a Patient with Multiple Myeloma by Using Protein A-Sepharose 4B

Affinity chromatography of IgG on protein A-Sepharose was used to isolate the human subclass IgG3 from normal serum and from a patient with multiple myeloma. The isolated material was purified by chromatography mi Sephadex G-150 and characterized immunochemically Ultracentrifugation studies gave s°₂₀ values of about 6.30 for both normal and myeloma IgG3 Approximately 54 halfcystine residues per molecule of IgG3 were obtained as judged from amino acid analysts after performic acid oxidation of the proteins. Polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate of the isolated and reduced material resulted in two bands correspoding to molecular weights of approximately 23.000 and 56.000. respectively. The yield of normal human IgG3 represented 1%-2% of the tout IgG.     

Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis A Antigen in Stool and Antibody to Hepatitis A Antigen in Sera: Comparison with Solid-Phase Radioimmunoassay, Immune Electron Microscopy, and Immune Adherence Hemagglutination Assay

Previously described techniques for detection of hepatitis A antigen (HA Ag) and antibody (anti-HA) have required purified HA Ag and expensive equipment. Herein is described an enzyme-linked immunosorbent assay (ELISA) for specific detection of HA Ag in human stool filtrates and of anti-HA in sera by using selected HA Ag-containing human stool filtrates as the antigen source. Because human stools often react nonspecifically in serological tests for HA Ag, blocking with preexposure and hyperimmune anti-HA sera from a chimpanzee inoculated with hepatitis A virus was used to confirm specific detection of HA Ag. The sensitivity of ELISA was found to be comparable to that of solid-phase radioimmunoassay (SPRIA) and immune electron microscopy (IEM). Of 37 acute-phase stools collected from nine patients, 16 were positive for HA Ag by ELISA. In 13 of these, HA Ag particles were found by IEM, and an additional 3 stools negative by ELISA contained HA Ag particles by IEM. Eight control stools were negative by both ELISA and IEM. Anti-HA was measured in sera by demonstrating its ability to block binding of the enzyme conjugate to HA Ag in a stool without detectable nonspecificity. This test (blocking ELISA) was as sensitive and specific as blocking SPIRA, IEM, and immune adherence hemagglutination and, like SPRIA and IEM, detected early-developing antibody. The ELISA is simple to perform and requires only a minimum of equipment. It is useful for screening stools for HA Ag and for monitoring HA Ag during purification, as well as for detecting early and late anti-HA in sera.     

The Repertoire of Anaplasma marginale Antigens Recognized by CD4+ T-Lymphocyte Clones from Protectively Immunized Cattle Is Diverse and Includes Major Surface Protein 2 (MSP-2) and MSP-3

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4⁺ T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406–5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.     

Identification of a 35 kDa Glycoprotein from Entamoeba histolytica by Sera from Patients with Amoebic Liver Abscess and with Mouse Monoclonal Antibody

In this work, we explored the relevance of a 35 kDa glycoprotein (Gm) of the outer membrane from E. histolytica in the diagnosis of the amoebic liver abscess (ALA) through ELISA and immunoblotting. We were interested in defining the relevance of this antigen in the immune response in patients with amoebic liver abscess and in exploring whether the mouse monoclonal antibody against this 35 kDa glycoprotein recognises the same epitope. We found that 87% of ALA patients had raised antibody levels to Gm antigen, whereas none of the healthy control subjects presented this same increase We also found 90% sensitivity, 100% specificity, 100% positive predictive value, 90% negative predictive value, and 90% prevalence value for this Gm antigen Nonetheless, we did not find any statistically significant differences in the levels of immunoglobulins against Gm, although IgG showed a tendency to increase, probably because we are dealing with a secondary immune response. Using electroimmunotransfer blot assay, we found that sera from ALA patients recognise the 35 kDa Gm protein in the same way as it is recognised by the mouse monoclonal antibody, suggesting that is a relevant molecule for the diagnosis of amebiasis, and eventually could lead to its use as protection against the disease.     

PSS病人血清中IL－2、IL－4、IL－6水平的测定

本文用生物测定法技术检测了３０例进行性系统性硬化症（ＰＳＳ）患者血清中白细胞介素２（ＩＬ－２）、白细胞介素４（ＩＬ－４）、白细胞介素６（ＩＬ－６）的水平。结果表明：在ＰＳＳ患者血清中，ＩＬ－２、ＩＬ－４的检出率显著高于对照组，ＩＬ－６的检出率虽然高于对照组，但是在统计学上无显著差异。结果提示：ＩＬ－２、ＩＬ－４可能在该病的发病机制中起一定作用。     

Reactivity of Sera from Systemic Lupus Erythematosus and Sjögren’s Syndrome Patients with Peptides Derived from Human Immunodeficiency Virus p24 Capsid Antigen

We have previously demonstrated that about one-third of patients with either Sjögren’s syndrome (SS) or systemic lupus erythematosus (SLE) react to human immunodeficiency virus (HIV) p24 core protein antigen without any evidence of exposure to, or infection with, HIV itself. Herein, we further characterize the specificity of this reaction using enzyme-linked immunosorbent assay to peptides representing fragments of p24. Characteristic epitope-specific profiles were seen for SS and SLE patients. SS patients had significantly increased responses to peptides F (p24 amino acids 69 to 86) and H (amino acids 101 to 111) and diminished reactivity to peptides A (amino acids 1 to 16) and P (amino acids 214 to 228). SLE patients had increased reactivity to peptides E (amino acids 61 to 76), H, and P. Utilization of peptide P hyporeactivity as the criterion to select for SS patients results in a screen that is moderately sensitive (64%) and specific (79.3%). Adding hyperreactivity to one other peptide (F or H) as an additional criterion yields an expected decrease in sensitivity (to 41%) while increasing specificity (to 93.1%). All sera-reactive peptides from regions of known structure of HIV p24 were located in the apex of the p24 molecule. Thus, the specificity of the peptide reactivities described here indicates a specific pattern of a nonrandom cross-reactivity between HIV type 1 p24 and autoimmune sera which may be partially syndrome specific. The future focus of our work will be to optimize assays of the peptide as diagnostic tools.     

Conversion of leukotriene A4 by neutrophils and platelets from patients with atopic dermatitis.

The generation of arachidonic acid-derived inflammatory mediators from unstimulated and stimulated neutrophils (PMN) and platelets in the presence of exogenous LTA4 has been studied in patients with atopic dermatitis (AD) as well as in healthy volunteers. PMN were stimulated with the interleukins IL-3, IL-8, C5a, and the Ca-ionophore A23187. In addition, NaF and thrombin were used to stimulate platelets. The release of leukotriene (LT)B4, 20-COOH- and 20-OH-LTB4, cysteinyl-leukotrienes and 12-HETE was measured. The proinflammatory mediator release from PMN and platelets of patients with AD was significantly higher as compared to the control group. The spontaneous conversion of LTA4 by PMN and platelets was markedly enhanced in patients with AD. Different results with receptor-specific and non-specific stimuli (Ca-ionophore A23187) in the presence of exogenous LTA4 were obtained. The results indicate a higher state of activation for enzymes involved in leukotriene formation. Furthermore, the production of 12-HETE by platelets from patients with AD was enhanced in unstimulated and stimulated cells. Our data emphasize that neutrophils and platelets may play an important role in the pathogenesis of AD by an increased responsiveness to receptor-specific stimuli and cell-cell interaction via LTA4.     

Large scale preparation of an immunoconjugate constructed with human recombinant CD4 and deglycosylated ricin A chain

A method for the preparation and purification of large amounts (grams) of a conjugate containing recombinant CD4 antigen (rCD4) and chemically deglycosylated ricin A chain (dgA) is described. The cross-linking of rCD4 and dgA molecules was accomplished with N-succinimidyl-oxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene (SMPT). The rCD4-dgA conjugate was purified by an automatic liquid chromatography system consisting of Blue-Sepharose CL-4B and Sephacryl S-200HR Pharmacia Bioprocess columns. The purified, endotoxin-free rCD4-dgA conjugate had a stable (hindered) disfulfide bond between rCD4 and dgA and was able to efficiently kill a human T cell line infected with HIV-1.     

Degradation of Amyloid A Precursor Protein SAA by Macrophage Cell Lines Obtained from Amyloid Resistant and Susceptible Strains of Mice

Reactive AA amyloidosis can be induced in mice in a model of sustained inflammation following daily casein subcutaneous injections. However, the development of AA amyloidosis is known to vary in different strains of mice. The C57BL/6 strain is susceptible to the development of amyloidosis while the A/J strain is resistant. The degradation of purified serum amyloid A (SAA) protein by human monocytes as well as by mouse macrophages has been shown. The resistance/susceptibility of different mouse strains to the development of systemic amyloidosis may therefore be related to a difference in the ability of macrophages to degrade SAA. The authors have used bone marrow-derived macrophage cell lines obtained from susceptible C57BL/6 (ANA-1) and resistant A/J (A/J 10) mouse strains to compare their ability to degrade HDL-SAA in vitro . Cells were incubated with HDL-SAA for up to 72 h and the culture medium was analysed by SDS-PAGE to determine the rate of SAA degradation by the macrophages. The A/J 10 cells (resistant) were found to initiate a constant HDL-SAA degradation promptly whereas ANA-1 cells (susceptible) showed an intermittent block in the degradation of the precursor. Activation of macrophages by lipopolysaccharide (LPS) or interferon-γ (IFN-γ) hampered the precursor degradation suggesting that the activation process may favour extracellular accumulation of the precursor leading to a partial degradation and fibril formation.     

Cytotoxic Effects of Klebsiella oxytoca Strains Isolated from Patients with Antibiotic-Associated Hemorrhagic Colitis or Other Diseases Caused by Infections and from Healthy Subjects

Antibiotic-associated hemorrhagic colitis (AAHC) is associated with Klebsiella oxytoca. This study analyzed whether cytotoxic properties are linked to specific subtypes of K. oxytoca. Klebsiella isolates from stools of AAHC patients, healthy carriers, and diarrhea patients as well as from infections of other organs were investigated. Cytotoxic effects on human epithelial cells were limited to the species K. oxytoca and were not detectable for any other Klebsiella species. Isolates from AAHC patients and from stools showed the highest proportion of cytotoxic strains. Urinary or respiratory tract isolates exhibited no cytotoxicity. Macrorestriction profiling of strains revealed no genetic relationships of AAHC isolates or the cytotoxic phenotype but identified that different K. oxytoca strains with different cytotoxic behaviors may be prevalent in the same AAHC patient. Under laboratory conditions, cytotoxicity was maximally effective after exponential bacterial growth and then declined despite the continued viability of K. oxytoca cells in culture. Given its capacity to induce AAHC and that a high proportion of stool isolates tested cytotoxin positive, we argue that K. oxytoca should be considered an opportunistic pathogen if detected in stools. The ability to induce disease after antibiotic treatment most likely represents an overgrowth of the toxin-producing bacterium due to an alteration of the normal colonic microflora.Antibiotic-associated colitis (AAC) is a frequent adverse effect observed when the normal bacterial flora is altered due to antibiotic therapy. Most cases of AAC are caused by infection by and extensive growth of Clostridium difficile, leading to pseudomembranous colitis. A special form of AAC is antibiotic-associated hemorrhagic colitis (AAHC), which was first described in 1978 (21) and has since been ascribed specific clinical, endoscopic, histopathological, and microbiological characteristics (9, 10). AAHC is not associated with C. difficile and was only recently shown to be caused by Klebsiella oxytoca (9, 10). AAHC is typically observed after a brief therapy with penicillins, with a sudden onset of bloody diarrhea often in combination with severe abdominal cramps, which often requires hospitalization. The key features of AAHC upon endoscopy are mucosal hemorrhage and mucosal edema, usually with segmental distribution, commonly affecting the ascending colon and the cecum (9). Histology typically resembles that of colitis induced by toxin-producing bacteria (10).For the majority of patients with AAHC, stool testing reveals K. oxytoca in significant amounts (>10⁶ CFU/ml) (9, 23). This Gram-negative rod is ubiquitous in the environment (e.g., soil and water) but can also be isolated from skin, mucous membranes, and the intestines of humans and animals (19). Human infections with K. oxytoca resemble those with Klebsiella pneumoniae; i.e., respiratory and urinary tracts are commonly affected (e.g., nosocomial pneumonia), in addition to soft tissue and hepatobiliary infections (6). Until recently, K. oxytoca was not considered to be an intestinal pathogen, and its presence in stool has placed the organism as a constituent of the normal gut microflora. For the healthy population, colonization of the intestine with K. oxytoca has been reported for 1.6% to 9% of subjects (1, 4, 10, 23). It is important that K. oxytoca constitutively produces β-lactamases conferring resistance to amino- and carboxypenicillins (13), agents typically given before the onset of AAHC. The association of K. oxytoca with AAHC was previously established by an animal model using a K. oxytoca strain isolated from a patient with AAHC in combination with an antibiotic to induce right-sided hemorrhagic colitis (10).In the 1990s, two independent groups reported that K. oxytoca strains isolated from patients with AAHC produce a cytotoxin, which caused cell death in cultured Hep2, Vero, CHO-K1, and HeLa cell lines as well as in an isolated intestinal-loop model (8, 15-17). In contrast, two laboratory strains of K. oxytoca exhibited no cytotoxicity, indicating that cytotoxin production might be strain specific (16, 17). The cytotoxin was reported to be heat labile, insensitive to proteinase digestion, and of a low molecular mass (8, 16, 17). A detailed analysis of the chemical nature and molecular structure of the cytotoxin is not yet available. Moreover, a causal link between toxin production in K. oxytoca and AAHC has not been established.The aim of the current study was to assess whether cytotoxin production is specific to certain subtypes of K. oxytoca and to test the hypothesis that AAHC uniquely correlates with those strains. A total of 121 Klebsiella isolates were investigated, including K. oxytoca strains isolated from stool samples from patients with AAHC, healthy carriers, and patients with colitis/diarrhea of other causes. K. oxytoca strains from infections involving other body sites and other Klebsiella spp. were analyzed in comparison. The characterization of all isolates was performed by using genotypic and biochemical methods, and the capacity of each isolate to induce cytotoxic effects on cultured eukaryotic cells was measured. A subset of strains was genotyped by macrorestriction profiling to assess their genetic relatedness.