Quantification of IgM and IgA anti-pneumococcal capsular polysaccharides by a new ELISA assay: a valuable diagnostic and prognostic tool for common variable immunodeficiency.

PURPOSE: Existing ways of assessing CVID patients at risk of pulmonary infections are not universally accepted. The need to identify additional prognostic factors allowed us to evaluate the anti-polysaccharide IgA and IgM responses in 125 CVID patients immunized with the 23-valent pneumococcal polysaccharide (PS) vaccine (Pneumovax(®)). METHODS: We used a new anti-PS23 IgM and IgA ELISA assay, which evaluates a global response to all 23 polysaccharides contained in Pneumovax(®). RESULTS: Anti-PS23 IgM and/or IgA antibodies were detectable in a minority of CVID patients. Antibody responses were correlated to B cell subpopulations and serum immunoglobulin concentrations. The non responders had a higher incidence of pneumonia and bronchiectasis and responders had the lowest incidence of respiratory complications. CONCLUSIONS: This new ELISA assay allows for studying vaccine response in patients on Ig replacement therapy. This test also is an additional method of evaluation of specific antibody responses representing a valuable contribution to identify prognostic marker in CVID patients.

3'-Processed mRNA is preferentially translated in Chlamydomonas reinhardtii chloroplasts.

3'-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3' termini by processing yet are not polyadenylated, we asked whether 3' end maturation plays a role in chloroplast translation. A general characteristic of the 3' untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3'-end formation signals. Deletion of the Chlamydomonas chloroplast atpB 3' IR in strain Delta26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase beta-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in Delta26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3' end maturation. In this work, we have analyzed whether these unprocessed atpB transcripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase beta-subunit which accumulates in Delta26. Analysis of chloroplast rbcL mRNA revealed that transcripts extending beyond the mature 3' end were not polysome associated. These results suggest that 3'-end processing of chloroplast mRNA is required for or strongly stimulates its translation.

Autoreactive T cells in rheumatic disease. II. Function and specificity of an autoreactive T helper cell clone established from a HLA-B27+ reactive arthritis.

T cell lines were established by limiting dilution of peripheral blood (PBL) and synovial fluid lymphocytes (SFL) of a patient with HLA-B27+ reactive arthritis. Among these cell lines, the CD4 phenotype was dominant. Functionally, the majority of these cell lines exhibited helper activity for the immunoglobulin production by autologous B cells and proliferated in response to autologous mononuclear cells. In most cases, this autoreactive response was associated with alloreactivity. Only one cell line, the autoreactive CD4+ T cell clone, UA-S2, which was derived from the synovial fluid, proliferated in a highly specific manner in response to a determinant associated with MHC class II products present on autologous mononuclear cells. The restriction element was shown to be associated with DR molecules by inhibition experiments with monoclonal antibodies. Within the patient's family, the capacity of mononuclear cells to stimulate a proliferative response of UA-S2 segregated together with the HLA haplotype A2 or 32, B27, Cw1, DRw11 which was contributed by the patient's mother. UA-S2 proved to be a functional helper T cell clone. In the absence of additional antigen or mitogen, it induced IgG and IgM synthesis of autologous and family members' B cells. This helper activity of UA-S2 showed the same MHC restriction as the proliferative response. Although the patient's father also typed DRw11, this haplotype was not recognized by UA-S2. It is suggested that this autoreactive T cell clone detects a microheterogeneity of the serologically defined DRw11 haplotype. Indeed, typing of the patient's family members with cellular reagents established a difference between the two DRw11 haplotypes.

Monocyte differentiation and accessory function: different effects on the proliferative responses of an autoreactive T cell clone as compared to alloreactive or antigen-specific T cell lines and primary mixed lymphocyte cultures.

An autoreactive T cell clone derived from a patient with reactive arthritis, two alloreactive T cell lines, two antigen-specific T cell lines and allogeneic resting T cells were analyzed for their responses to monocytes and macrophages derived from monocytes by in vitro differentiation. The autoreactive T cell clone strongly proliferated in response to fresh monocytes and to macrophages derived from a 7 day culture, but only poorly to monocytes cultured for 2 days. In contrast, alloreactive and antigen-specific T cell lines proliferated to all stimulatory cells equally well. Finally, primary mixed lymphocyte reactions could be stimulated by both fresh and 2-day cultured monocytes, but not by in vitro derived macrophages. The impaired response of the autoreactive T cell clone to 2-day cultured monocytes could not be attributed to reduced expression of several well-defined surface molecules nor to induction of nonresponsiveness. Neither allogeneic monocytes nor cytokines (IL-1, IL-2, IL-4, IL-6) could correct the defective response of the autoreactive T cell clone. However, preculture of monocytes in the presence of interferon-gamma, IL-1, IL-4 or IL-6 retained their stimulatory capacity. Our interpretation of the selectively impaired response of the autoreactive T cell clone is that it most likely recognizes a differentiation-dependent monocyte/macrophage-specific peptide.

Dengue research and training supported through the World Health Organization.

The rapidly increasing burden of dengue, the varied and often poorly understood factors contributing to its global spread, and the challenges of preventing and controlling it have led to a renewed call for more research and training on the disease. The main aims are to improve vector control, case management, and primary prevention through vaccine development. The World Health Organization (WHO), through its inter-departmental working group on dengue, is actively engaged in supporting and co-ordinating the major research activities. The dengue research initiatives of the Special Programme for Research and Training in Tropical Diseases (TDR), other departments at the WHO's Geneva headquarters, the WHO's regional and country offices, and the organization's dengue-affected member states are summarized in this article. This intensified effort, in close collaboration with other stakeholders, is contributing towards the goals of reversing the current epidemiological trends and of reducing the global burden posed by dengue in all of its forms.

Retention mechanism of lactate dehydrogenase in anion-exchange chromatography.

The anion-exchange retention mechanism of lactate dehydrogenase (LDH) isoenzymes was examined under conditions where the muscle (M) subunit was not retained by the sorbent matrix. Using the stoichiometric displacement model of retention to quantitate the number (Zn) of ionic groups on the protein that interact with the sorbent matrix, it was determined that Zn for the LDH isoenzymes increases incrementally as the number of heart (H) subunits is increased up to a total of three H subunits. Both the MH3 and H4 isoenzymes have the same Zn. As the subunits in this tetrameric enzyme are arranged in a tetrahedral structure, these data indicate that steric limitations prevent any more than three subunits from interacting simultaneously with the sorbent surface. The reason why the MH3 and H4 isoenzymes could be separated is apparently that there are multiple equivalent faces in the H4 isoenzyme, and this both increases the probability and rate of adsorption and decreases the probability and rate of desorption for the H4 relative to the MH3 isoenzyme. It was concluded from these studies that the three-dimensional structure of a biopolymer will determine those surface residues which are in a position to interact with a sorbent surface and that multiple subunits or domains of a protein may act cooperatively over considerable distances in biopolymer-surface interactions.

Application of the stoichiometric displacement model of retention to anion-exchange chromatography of nucleic acids.

The stoichiometric displacement model has been refined in its application to anion-exchange chromatography. The revised stoichiometric displacement model has been shown to be valid for anion-exchange chromatography with respect to all particulars of the model tested. It has been shown that the use of displacing agent activity is not a necessary condition for valid application of the stoichiometric displacement model to anion-exchange chromatography. While the cation of the displacing salt can influence anion-exchange chromatography, the data indicate that the displacing anion is of primary importance. It has been shown that solutes with three-dimensional structure have Zn value to solute charge ratios less than unity, and that the stoichiometric displacement model may be useful as a probe of solute three-dimensional structure.

A novel Euglena gracilis chloroplast operon encoding four ATP synthase subunits and two ribosomal proteins contains 17 introns.

The structure of a Euglena gracilis chloroplast operon encoding four subunits of the chloroplast ATP synthase complex and two ribosomal proteins has been determined. These six genes contain 17 introns. This operon is transcribed as a hexacistronic primary transcript which is subsequently processed to monocistronic mRNAs. The linear order of these genes, 5'-rps2-atpI-atpH-atpF-atpA-rps18-3' , encoding ribosomal protein S2, chloroplast ATP synthase subunits CF0IV, CF0III, CF0I, CF1 alpha and ribosomal protein S18, respectively, is similar to the equivalent operons of prokaryotes, cyanelles and land-plant chloroplasts. This operon differs from those of these other organisms in the co-transcription of rps18 and in intron content.

A complex twintron is excised as four individual introns.

Twintrons are introns-within-introns excised by sequential splicing reactions. A new type of complex twintron comprised of four individual group III introns has been characterized. The external intron is interrupted by an internal intron containing two additional introns. This 434 nt complex twintron within a Euglena gracilis chloroplast ribosomal protein gene is excised by four sequential splicing reactions. Two of the splicing reactions utilize multiple 5'- and/or 3'-splice sites. These findings are evidence that introns with multiple active splice sites can be formed by the repeated insertion of introns into existing introns.

High-performance anion-exchange chromatography of oligonucleotides.

Several types of high-performance silica-based supports have been found to be effective in the separation of polynucleotides. The principal difference in these materials is the type of bonded phase and the method by which it is attached to the silica support. One approach is the coupling of stationary-phase groups to the surface through siloxane bonding. This technique is simple and produces a material of high capacity and resolution, but it suffers from poor bonded-phase stability. An alternative approach is the adsorption of low-molecular-weight polyethylene imines (PEI) that are crosslinked into a surface film. The stationary phase is held in place by adsorption of the film at multiple sites. A previous report on this material showed the resolution of oligonucleotides containing up to 30 bases. This paper reports further optimization of the PEI bonding chemistry in the preparation of HP-IEC columns for oligonucleotides and tRNA species. Quaternization of the ion-exchange matrix was found to increase resolution of oligonucleotides from 30 to 50 bases. The same support was found to be capable of resolving multiple tRNA species. Separations were achieved on small (0.42 X 5 cm) columns, using a 60- to 120-min ammonium sulfate gradient. The initial solvent was 15% acetonitrile in 0.05 M potassium phosphate (pH 5.9). The addition of 1 M ammonium sulfate to the initial solvent was used to prepare the final solvent.

Presence of IgE antibodies to bovine serum albumin in a patient developing anaphylaxis after vaccination with human peptide-pulsed dendritic cells.

Dendritic cells are professional antigen-presenting cells that can be generated in vitro either from monocytes or from CD34+ peripheral blood progenitor cells by using recombinant cytokines. These cells have potential implications for immunotherapeutic approaches in the treatment of cancer and other diseases. We have conducted a phase I study in melanoma patients using peptide-pulsed dendritic cells cultured in medium supplemented with 10% fetal calf serum (FCS) and a cocktail of cytokines. Peptide-pulsed dendritic cells were injected intravenously at 2-week intervals. Here we report on a case of type I hypersensitivity anaphylactic reaction after repetitive vaccination with autologous peptide-pulsed cells. Pre-vaccination and post-vaccination serum samples were evaluated for the presence of antibodies to FCS and bovine serum albumin (BSA). A retrospective study in 7 patients vaccinated with FCS-cultured dendritic cells demonstrated the presence of IgG and IgM antibodies to FCS and BSA after vaccination in 6 out of 7 patients. However, IgE antibodies were absent in all patients with the exception of the patient developing anaphylaxis. The patient's serum was demonstrated to contain a strong IgE response directed against BSA. In contrast, 2 patients vaccinated with dendritic cells cultured under serum-free conditions developed no antibodies to FCS and BSA after repetitive vaccination. We suggest that patients can be sensitized with an IgE response against BSA leading to anaphylactic reactions. On the basis of these data, dendritic cells cultured in autologous serum or under serum-free conditions are recommended for therapeutic applications in vivo.

Possible role of IL-2 deficiency for hypogammaglobulinaemia in patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) patients are unable to produce specific immunoglobulins after antigen contact in vivo. The aim of this study was to investigate whether in some cases of CVID a decreased de novo synthesis of IL-2 might be the cause of immunodeficiency and whether this deficiency can be corrected by IL-2 supplementation in vitro. Mononuclear cells from 17 CVID patients and from 10 healthy controls were cultured with monoclonal anti-CD3 antibody OKT3, pokeweed mitogen (PWM) or tetanus toxoid (TT) to stimulate IL-2 synthesis. In parallel, in vitro IgG and IgM synthesis was stimulated with Staphylococcus aureus Cowan I (SAC), PWM or TT in the presence or absence of IL-2. While lymphocytes of 11 out of 17 patients produced low to normal amounts of IL-2 upon stimulation with anti-CD3, only three patients showed low IL-2 production in response to PWM and five in response to TT. Regarding immunoglobulin synthesis in vitro, five patients completely failed to produce IgM or IgG upon stimulation with PWM, SAC or TT irrespective of the addition of IL-2. By contrast, four patients did not show any defect in vitro and synthesized normal amounts of IgM and IgG with any of the three stimuli. Finally, eight patients could be reconstituted for PWM-, SAC- and TT-induced IgM and/or IgG synthesis in vitro, by adding IL-2 to the culture system. This enhancing effect of IL-2 could be blocked by adding anti-IL-2 receptor antibodies to the cultures. Our findings indicate that a defective IL-2 synthesis after antigen stimulation may be one reason for the impaired immunoglobulin production in some cases of CVID.

The 3' untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators.

A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene. The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.

Transient CD80 expression defect in a patient with variable immunodeficiency and cyclic neutropenia.

CD80 expression on stimulated B cells of 9 common variable immunodeficiency (CVID) patients and 10 healthy control individuals was examined. Here we report on an exceptional CVID patient with recurrent episodes of cyclic neutropenia, skin vasculitis and recurrent infections associated with a transient, but reproducible CD80 expression defect on stimulated B cells. Concomitantly serum Ig levels were markedly reduced and B cell counts were low. Interestingly, after recovery from an episode of neutropenia and vasculitis we observed an improvement of the CD80 expression as well as an increase in the number of B cells and a recovery of IgM and IgG production in vivo and in vitro.

The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability.

A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract. To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3' UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase beta-subunit protein in all transformants was increased compared to a strain in which the atpB 3' UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3' UTR sequences was reduced to approximately one-half that of transformants containing the 3' UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3' UTRs are important for transcript 3'-end processing, and for accumulation of the mature mRNAs.

5' to 3' exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways.

Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5' and 3' untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5'-->3' and 3'-->5' exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions -20, +25, +165 or +25/+165 relative to the mature petD 5' end, mRNAs accumulate with 5' ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5' end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5'-->3' exoribonucleolytic activity. In the case of the -20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5' end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5' ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) -20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.

Altering the 3 UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3-end maturation in vitro but not in vivo.

The 3' ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3' ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3'-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3'-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3' end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3'-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3' end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3' UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3' end was generated. These results imply that Chlamydomonas atpB 3' processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.

[Terminal B-cell maturation and immunoglobulin synthesis in vitro in primary and secondary immune deficiencies]. / Terminale B-Zellreifung und Immunoglobulinsynthese in vitro bei primären und sekundären Immundefekten.

Peripheral blood lymphocytes (PBL) from patients with various forms of primary and secondary immunodeficiencies and from 30 healthy control persons were examined for their capacity to show terminal B-cell maturation and immunoglobulin (Ig) synthesis in vitro following pokeweed mitogen (PWM) stimulation. PBL were cultured for 9 days with or without PWM and the percentages of B-cells or plasma cells with strongly positive cytoplasmic Ig (CIg+) were determined by indirect immunofluorescence on methanol fixed cytocentrifuged lymphocyte smears. In addition, newly synthesized IgA, IgG and IgM was measured in the cell free culture supernatant by means of a sensitive microplate ELISA. In 29 out of 30 control cultures the proportion of CIg+-cells increased markedly following 9-day stimulation with PWM. CIg+-cells and newly synthesized Ig were significantly correlated. In a group of 18 patients with common variable immunodeficiency (CVID) maturation to CIg+-cells was greatly impaired and the in vitro production of IgA and IgG was reduced. Two infants with severe combined immunodeficiency (SCID) failed to produce Ig prior to and following haplo-identical bone marrow transplantation despite the presence of immature B-cells. A heterogeneous group of secondary immunodeficiencies showed different forms of an impaired terminal B-cell maturation and a reduced capacity to synthesize Ig in vitro. The described PWM culture techniques in combination with the evaluation of CIg+-cells and the measurement of newly synthesized Ig in the culture supernatant proved to be a reliable in vitro correlate for terminal B-cell maturation and represent an important tool for the classification of humoral immunodeficiencies.

In vivo evidence for 5'-->3' exoribonuclease degradation of an unstable chloroplast mRNA.

The acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5' untranslated region (UTR). However, when this 5' UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5' end of the petD 5' UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5' UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5'-->3' exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5' UTR protects the mRNA from 5'-->3' degradation in wild-type cells.