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.

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.

Impaired up-regulation of CD70 and CD86 in naive (CD27-) B cells from patients with common variable immunodeficiency (CVID).

CVID is characterized by reduced serum levels of all switched immunoglobulin isotypes (IgG, IgA, IgE) predisposing patients to recurrent infections of their respiratory and gastrointestinal tract. Correspondingly, most CVID patients exhibit a severely decreased proportion of class switched memory B cells (CD19+CD27+IgD-IgM-IgG+ or IgA+) in their peripheral blood (CVID type I). We previously identified a subgroup of CVID patients showing a significantly reduced expression of CD86 and CD137 following activation in vitro of PBMC or purified B cells (CD19+) with anti-IgM plus IL-2. Here we extend our previous studies by asking whether highly purified, cell-sorted naive B cells show already an expression defect of B cell surface molecules relevant in activation (CD39, CD69), differentiation (CD24, CD27, CD38) or T-B interaction (CD25, CD70, CD86). We stimulated cell-sorted, naive B cells (CD19+CD27-IgM+IgDhighIgG-IgA-) from 10 CVID patients and 10 healthy controls for 4 days with anti-IgM plus IL-2 in the absence or presence of autologous CD4+ T cells and measured the expression of the referred surface molecules. Based on reduced or normal numbers of switched memory B cells the CVID patients had previously been classified into eight type I patients and two type II patients, respectively. Interestingly, only the molecules CD25, CD70 and CD86, all relevant in cognate T-B interaction, showed a significantly lower expression in naive B cells from CVID patients compared to controls. While coculture with autologous CD4+ T cells normalized the CD25 expression, CD70 and CD86 expression remained subnormal, notably in the eight CVID patients of type I. These findings strongly suggest an intrinsic signalling or expression defect for CD70/CD86 at the level of naive B cells in type I CVID patients.

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii.

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

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.

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.

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.

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.

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.

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 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.

Severe combined immunodeficiency due to defective binding of the nuclear factor of activated T cells in T lymphocytes of two male siblings.

Peripheral blood lymphocytes (PBL) and alloreactive T cell lines of two male infants born to consanguinous parents and presenting with severe combined immunodeficiency (SCID) showed a pronounced deficiency in T cell activation. Although phenotypically normal, the proliferative response of the childrens' T cells was strongly reduced but could be improved by the addition of interleukin-2 (IL-2). Furthermore both childrens' T cells were unable to produce the cytokines IL-2, interferon-gamma (IFN-gamma), IL-4 and tumor necrosis factor-alpha (TNF-alpha). This multiple cytokine production deficiency could not be restored by IL-2 or co-stimulatory signals provided by antigen-presenting cells (APC). Moreover, mRNA for IL-2 and IFN-gamma could not be detected. In contrast, expression of the activation-dependent cell surface markers CD25 and CD69 was within normal limits. To determine whether the functional defect of the patients' T cells was due to the absence or abnormal binding of transcription factors involved in cytokine gene expression, electrophoretic mobility shift assays were used to examine the DNA binding of AP-1, Oct, CREB, SP1, NF-kappa B and the nuclear factor of activated T cells (NF-AT) to their respective response elements in the promoter of the IL-2 gene. Whereas AP-1, NF-kappa B, Oct, CREB and SP1 displayed normal binding activities in nuclear extracts, the binding of NF-AT to its IL-2 promoter response element was barely detectable both before and after T cell stimulation. Our results strongly suggest that this NF-AT/DNA binding defect is responsible for the multiple cytokine deficiency and the SCID phenotype observed in the two infant brothers.

A chloroplast transcript lacking the 3' inverted repeat is degraded by 3'-->5' exoribonuclease activity.

Chlamydomonas reinhardtii strains harboring deletions of the chloroplast atpB 3' inverted repeat (IR) are weakly phototrophic due to reduced accumulation of discrete atpB transcripts and the chloroplast ATPase beta-subunit protein. A sequence of 18 guanosine residues, which can impede a 3'-->5' exoribonuclease in vitro, is able to substitute for the atpB IR in vivo. Strains containing the poly-guanosine tract in place of the atpB 3' IR are phototrophic and accumulate near wild-type levels of discrete atpB transcripts and the ATPase beta-subunit protein. Because these atpB transcripts contain the 18 guanosine residues, and the poly-guanosine tract is not a terminator of transcription, the accumulation of discrete atpB transcripts is likely the result of impediment of 3'-->5' exoribonuclease activity. These findings support a model in which atpB transcripts lacking the 3' IR are degraded by 3'-->5' exoribonuclease activity, and demonstrate that the poly-guanosine tract can be used to study chloroplast RNA metabolism in vivo.

Two novel cationic staphylococcal proteins induce IL-2 secretion, proliferation and immunoglobulin synthesis in peripheral blood mononuclear cells (PBMC) of both healthy controls and patients with common variable immunodeficiency (CVID).

Two cationic proteins, a neutral phosphatase (NP-tase) and a 70-kD protein (p70) were isolated from Staphylococcus aureus by ion exchange chromatography. We compared their properties to those of the well established B cell mitogen of whole, fixed Staph. aureus strain Cowan I cells (SAC). Both purified proteins were able to induce immunoglobulin synthesis in PBMC cultures of healthy donors. NP-tase and p70 also induced immunoglobulin synthesis of PBMC from those patients with CVID who were also responsive to SAC plus IL-2 stimulation. Immunoglobulin synthesis in response to NP-tase and to p70 was time- and dose-dependent and could be inhibited by addition of specific antibodies against the proteins. In contrast to SAC, no addition of exogenous IL-2 was necessary to obtain maximal immunoglobulin synthesis induced by NP-tase or p70. However, neither protein was able to induce immunoglobulin synthesis in B cell-enriched cultures. High amounts of IL-2 were found in supernatants of PBMC from healthy donors following stimulation with low concentrations of NP-tase or p70, and this was associated with vigorous lymphocyte proliferation. Both proteins behave like typical antigens, and not like lectins or superantigens, since an NP-tase-stimulated T cell line showed an antigen-specific, MHC-restricted secondary response. In addition, no preferential T cell receptor V beta chain usage was found with eight V beta-specific MoAb. It is likely that the two proteins induce antigen-specific T cell activation, which is then followed by polyclonal activation of B cells via CD40 receptors and cytokine release.

The stabilization of a human IgM monoclonal antibody with poly(vinylpyrrolidone).

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50 degrees C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpyrrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 kDa. When the PVP concentration was greater than 1.0%, antibody aggregation was enhanced, and with the highest molecular weight PVP, antibody precipitation occurred. HPLC was used to show that more PVP was associated with the 4B9 at 50 degrees C than at 25 degrees C. Differential scanning calorimetry revealed that PVP concentrations greater than 2.0% decreased the antibody thermal transition temperature. Enzyme-linked immunosorbent assays were used to assess the effects of PVP on the antigen binding capacity of 4B9 and on 4B9 quantitation. At 4 degrees C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50 degrees C, however, less 4B9 was detected in the 5.0% PVP solution. The heat stabilization of the 4B9 antibody by low concentrations of PVP can be explained by a weak binding of PVP to the native protein. The PVP may sterically interfere with protein-protein interactions, thus reducing aggregation. Higher concentrations of PVP lead to protein aggregation and precipitation, probably by a volume-exclusion mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Complete sequence of Euglena gracilis chloroplast DNA.

We report the complete DNA sequence of the Euglena gracilis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns.

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.