Insertional hypermutation in mineral oil-induced plasmacytomas.

Unless stimulated by a chronic inflammatory agent, such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions: What do inflammatory tissues provide to promote mutagenesis? And what is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements--ecotropic and polytropic murine leukemia virus and intracisternal A particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to ten de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice.

Increased toxin expression in a Clostridium difficile mfd mutant.

BACKGROUND: The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY. RESULTS: During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations. CONCLUSIONS: This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.

A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies.

Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1-binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1-binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding-like ß3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum-exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1-specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration.

The role of sphingosine-1-phosphate transporter Spns2 in immune system function.

Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm1a(KOMP)Wtsi) allele (Spns2(tm1a)). The Spns2(tm1a/tm1a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm1a/tm1a) mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm1a/tm1a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.

Expression, purification, crystallization and preliminary crystallographic analysis of hepatitis B virus core protein dimerized via a peptide linker containing an EGFP insertion.

Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.

A novel 13 base pair insertion in the sonic hedgehog ZRS limb enhancer (ZRS/LMBR1) causes preaxial polydactyly with triphalangeal thumb.

Mutations in the Sonic hedgehog limb enhancer, the zone of polarizing activity regulatory sequence (ZRS, located within the gene LMBR1), commonly called the ZRS), cause limb malformations. In humans, three classes of mutations have been proposed based on the limb phenotype; single base changes throughout the region cause preaxial polydactyly (PPD), single base changes at one specific site cause Werner mesomelic syndrome, and large duplications cause polysyndactyly. This study presents a novel mutation-a small insertion. In a Swedish family with autosomal-dominant PPD, we found a 13 base pair insertion within the ZRS, NG_009240.1:g.106934_106935insTAAGGAAGTGATT (traditional nomenclature: ZRS603ins13). Computational transcription factor-binding site predictions suggest that this insertion creates new binding sites and a mouse enhancer assay shows that this insertion causes ectopic gene expression. This study is the first to discover a small insertion in an enhancer that causes a human limb malformation and suggests a potential mechanism that could explain the ectopic expression caused by this mutation.

Mast cells contribute to the mucosal adjuvant effect of CTA1-DD after IgG-complex formation.

Mast cell activation is one of the most dramatic immune-mediated responses the body can encounter. In the worst scenario (i.e., anaphylaxis), this response is fatal. However, the importance of mast cells as initiators and effectors of both innate and adaptive immunity in healthy individuals has recently been appreciated. It was reported that mast cell activation can be used as an adjuvant to promote Ag-specific humoral immune responses upon vaccination. In this study, we have used a clinically relevant mucosal adjuvant, cholera toxin A1 subunit (CTA1)-DD, which is a fusion protein composed of CTA1, the ADP-ribosylating part of cholera toxin, and DD, two Ig-binding domains derived from Staphylococcus aureus protein A. CTA1-DD in combination with polyclonal IgG induced degranulation and production of TNF-alpha from mouse mast cells. Furthermore, CTA1-DD and polyclonal IgG complex induced mast cell degranulation in mouse skin tissue and nasal mucosa. We also found that intranasal immunization with hapten (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled to chicken gammaglobulin admixed with CTA1-DD complexed with polyclonal IgG greatly enhanced serum IgG anti-NP Ab responses and stimulated higher numbers of NP-specific plasma cells in the bone marrow as compared with that observed in mice immunized with NP-chicken gammaglobulin with CTA1-DD alone. This CTA1-DD/IgG complex-mediated enhancement was mast cell dependent because it was absent in mast cell-deficient Kit(W-sh/W-sh) mice. In conclusion, our data suggest that a clinically relevant adjuvant, CTA1-DD, exerts additional augmenting effects through activation of mucosal mast cells, clearly demonstrating that mast cells could be further exploited for improving the efficacy of mucosal vaccines.

Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses.

The nfkb2 gene is a member of the Rel/NF-kappa B family of transcription factors. COOH-terminal deletions and rearrangements of this gene have been associated with the development of human cutaneous T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas. To further investigate the function of NF-kappa B2, we have generated mutant mice carrying a germline mutation of the nfkb2 gene by homologous recombination. NF-kappa B2-deficient mice showed a marked reduction in the B cell compartment in spleen, bone marrow, and lymph nodes. Moreover, spleen and lymph nodes of mutant mice presented an altered architecture, characterized by diffuse, irregular B cell areas and the absence of discrete perifollicular marginal and mantle zones; the formation of secondary germinal centers in spleen was also impaired. Proliferation of NF-kappa B2-deficient B cells was moderately reduced in response to lipopolysaccharide, anti-IgD-dextran, and CD40, but maturation and immunoglobulin switching were normal. However, nfkb2 (-/-) animals presented a deficient immunological response to T cell-dependent and -independent antigens. These findings indicate an important role of NF-kappa B2 in the maintenance of the peripheral B cell population, humoral responses, and normal spleen architecture.

Caenorhabditis elegans as a host for the study of host-pathogen interactions.

Recently, pathogenicity models that involve the killing of the genetically tractable nematode Caenorhabditis elegans by human pathogens have been developed. From the perspective of the pathogen, the advantage of these models is that thousands of mutagenized bacterial clones can be individually screened for avirulent mutants on separate petri plates seeded with C. elegans. The advantages of using C. elegans to study host responses to pathogen attack are the extensive genetic and genomic resources available and the relative ease of identifying C. elegans mutants that exhibit altered susceptibility to pathogen attack. The use of Caenorhabditis elegans as the host for a variety of human pathogens is discussed.

TB tools to tell the tale-molecular genetic methods for mycobacterial research.

In spite of the availability of drugs and a vaccine, tuberculosis--one of man's medical nemeses--remains a formidable public health problem, particularly in the developing world. The persistent nature of the tubercle bacillus, with one third of the world's population is estimated to be infected, combined with the emergence of multi drug-resistant strains and the exquisite susceptibility of HIV-positive individuals, has underscored the urgent need for in-depth study of the biology of Mycobacterium tuberculosis address the resurgence of TB. In aiming to understand the mechanisms by which mycobacteria react to their immediate environments, molecular genetic tools have been developed from naturally occurring genetic elements. These include protein expressing genes, and episomal and integrating elements, which have been derived mainly from prokaryotic but also from eukaryotic organisms. Molecular genetic tools that had been established as routine procedures in other prokaryotic genera were thus mimicked. Knowledge of the underlying mechanisms greatly expedited the harnessing of these elements for mycobacteriological research and has brought us to a point where these molecular genetic tools are now employed routinely in laboratories worldwide.

Use of a novel mutagenesis strategy, optimized residue substitution, to decrease the off-rate of an anti-gp120 antibody.

We have developed a novel strategy to decrease the antibody:antigen off-rate which we call optimized residue substitution. This strategy employs alanine substitution to first identify residues non-optimal for binding, as evidenced by a decrease in off-rate upon alanine replacement. These positions are then individually randomized to all amino acids, and the best replacement for each position determined. Finally, a construct which combines all optimized substitutions is generated and evaluated. We applied this strategy to the heavy chain CDR3 of P5Q, a scFv antibody which recognizes an epitope on the V3 loop of HIV gp120. We identified two amino acid substitutions that together decrease the off-rate by nearly ten-fold. The contributions by the two substitutions were near additive, indicative of independent affects on binding. We suggest that this strategy can be generalized to strengthen protein:ligand and protein:protein interactions in other systems.

[Expression of recombinant inserting mutants of HBsAg in vitro and its antigenic analysis].

OBJECTIVE: To study the immunological characteristics of two inserting variants of HBsAg as further to elucidate the role of inserting mutants in the development of HBsAg-negative chronic hepatitis B. METHODS: The inserting mutants of HBsAg were constructed by PCR-mediated mutagenesis in vitro. Epstein-Barr virus vector pCEP4 and retrovirus vector PXT1 carrying the mutant genes of HBsAg were transfected into mammalian cells. The affinity of polyclonal and monoclonal antibodies against HBsAg for mutant antigens were studied by ELISA and western blot. RESULTS: Two insertions were introduced into s gene, and a six nucleotide insertion introduced two aminoacids (Arg and Ala) between codons 122 and 123 of the s polypeptide, whereas a nine nucleotide insertion introduced three aminoacids(Arg, Gly and Ala) between codons 123 and 124. These s genes were subcloned into two eukaryotic expression vectors (pCEP4 and PXT1), and the recombinant eukaryotic expression plasmids had been constructed, which could express s polypeptide in mammalian cells. Cell lines expressing wild-type or mutant-type HBsAg stably were obtained after transfection of these vectors into mammalian cells. Two two inserting variants failed to react with polyclonal and monoclonal antibodies against HBsAg by ELISA and Western blot. CONCLUSION: The insertions may result in a conformational change of the s protein, and affect its antigenicities, suggesting that the inserting mutations may be critical for immunoescape of HBV and in part responsible for chronicity of HBsAg-negative patients.

Genetic modification of lymphocytes by retrovirus-based vectors.

The genetic modification of lymphocytes is an important topic in the emerging field of gene therapy. Many clinical trials targeting immunodeficiency syndromes or cancer have shown therapeutic benefit; further applications address inflammatory and infectious disorders. Retroviral vector development requires a detailed understanding of the interactions with the host. Most researchers have used simple gammaretroviral vectors to modify lymphocytes, either directly or via hematopoietic stem and progenitor cells. Lentiviral, spumaviral (foamyviral) and alpharetroviral vectors were designed to reduce the necessity for cell stimulation and to utilize potentially safer integration properties. Novel surface modifications (pseudotyping) and transgenes, built using synthetic components, expand the retroviral toolbox, altogether promising increased specificity and potency. Product consistency will be an important criterion for routine clinical use.

IS200 and multilocus sequence typing for the identification of Salmonella enterica serovar Typhi strains from Indonesia

In this work, IS200 and multi-locus sequence typing (MLST) were used to analyze 19 strains previously serotyped as Salmonella enterica serovar Typhi and isolated in Indonesia (16 strains), Mexico (2 strains), and Switzerland (1 strain). Most of the strains showed the most common Typhi sequence types, ST1 and ST2, and a new Typhi genotype (ST1856) was described. However, one isolate from Mexico and another from Indonesia were of the ST365 and ST426 sequence types, indicating that they belonged to serovars Weltevreden and Aberdeen, respectively. These results were supported by the amplification of IS200 fragments, which rapidly distinguish Typhi from other serovars. Our results demonstrate the utility of IS200 and MLST in the classification of Salmonella strains into serovars. These methods provide information on the clonal relatedness of strains isolated worldwide (AU)

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Direct random insertion of an influenza virus immunologic determinant into the NS1 glycoprotein of a vaccine flavivirus.

A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.

Lupus-prone MRL/faslpr/lpr mice display increased AID expression and extensive DNA lesions, comprising deletions and insertions, in the immunoglobulin locus: concurrent upregulation of somatic hypermutation and class switch DNA recombination.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/fas(lpr/lpr) mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas(lpr/lpr) mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas(lpr/lpr) B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas(lpr/lpr) greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas(lpr/lpr) mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) theta, pol eta and pol zeta, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/fas(lpr/lpr) mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.

Genetic polymorphisms of chitotriosidase in Caucasian children with bronchial asthma.

In humans, two types of chitinases have been identified: chitotriosidase I (CHIT1) and acid mammalian chitinase (AMCase). They are enzymes that cleave chitin, a polysaccharide contained in many different human parasites. So far, only little is known about their function in human and especially in human diseases. Recently we have described association of polymorphisms of AMCase with bronchial asthma in a pediatric population. In this study we were interested in whether CHIT1 is also involved in the genetics of asthma. The amino acid variants Gly102Ser and Ala442Gly, as well as a 24 bp duplication within CHIT1, were typed by means of restriction fragment length polymorphisms on 322 children with asthma and 270 randomly chosen adult controls. Statistical analyses made use of the Armitage's trend test; haplotypes were calculated by FAMHAP and FASTEHPLUS. The amino acid variants showed no association with bronchial asthma. The 24 bp duplication, previously shown to completely demolish CHIT1 activity, was also evenly distributed between asthmatics and controls. Finally, the haplotype showed no association with the disease. We conclude from our results that CHIT1 does not play a major role in the development of bronchial asthma in Caucasian children. The results might also imply that the two human chitinases that have been identified so far have quite distinct functions in human diseases even though they have the same substrate.

Missense mutations in SH2D1A identified in patients with X-linked lymphoproliferative disease differentially affect the expression and function of SAP.

X-linked lymphoproliferative disease (XLP) is an immunodeficiency resulting from mutations in SH2D1A, which encodes signalling lymphocytic activation molecule (SLAM)-associated protein (SAP). In addition to SLAM, SAP associates with several other cell-surface receptors including 2B4 (CD244), Ly9 (CD229), CD84 and NTB-A. SAP contains a single src-homology-2 domain and acts as an intracellular adaptor protein by recruiting the protein tyrosine kinase FynT to the cytoplasmic domains of some of these receptors, which results in the initiation of specific downstream signal transduction pathways. XLP is likely to result from perturbed signalling through one or more of these SAP-associating receptors. In this study, we identified missense (Y54C, I84T and F87S) and insertion (fs82 --> X103) mutations in four different kindreds affected by XLP. Each mutation dramatically reduced the half-life of SAP, thus diminishing its expression in primary lymphocytes as well as in transfected cell lines. Interestingly, although the Y54C and F87S mutations compromised the ability of SAP to associate with different receptors, the I84T mutation had no effect on the ability of SAP to bind SLAM, CD84 or 2B4. However, signalling downstream of SLAM was reduced in the presence of SAP bearing the I84T mutation. These findings indicate that, irrespective of the type of mutation, signalling through SAP-associating receptors in XLP can be impaired by reducing the expression of SAP, the ability of SAP to bind surface receptors and/or its ability to activate signal transduction downstream of the SLAM-SAP complex.

Arrested natural killer cell development associated with transgene insertion into the Atf2 locus.

Natural killer (NK) cell development in the bone marrow is not fully understood. Following lineage commitment, these cells appear to advance through a series of developmental stages that are beginning to be characterized. We previously reported a selective deficiency of NK cells in a C57BL/6 mouse with a transgenic construct consisting of the cDNA for the Ly49A major histocompatibility complex (MHC) class 1-specific inhibitory receptor driven by the granzyme A gene. This mouse has few NK cells in peripheral tissues with relative preservation of other immune cells, including T and B cells. Herein we demonstrate that these mice have an accumulation of NK cells with an immature phenotype in the bone marrow, consistent with a block at a previously proposed stage in normal NK-cell development. The phenotype is associated with transgenic insertion into Atf2, the gene for the basic leucine zipper (bZIP) transcription factor family member ATF-2. Although analysis of Atf2-null NK cells shows no defect, the transgenic mice express abnormal truncated Atf2 transcripts that may mediate a repressor effect because ATF2 can heterodimerize with other bZIP molecules. The defect is cell intrinsic, suggesting that certain bZIP molecules play significant roles in NK-cell development.

Rescue of the hairless phenotype in nude mice by transgenic insertion of the wild-type Hfh11 genomic locus.

Mice and rats homozygous for mutations at the nude (nu) locus exhibit the pleiotropic phenotypes of hairlessness and athymia. A recent positional cloning study identified, as a nude gene, a novel fork head transcription factor, Hfh11 (also called whn), that is expressed in skin and thymus, and is mutated in nude rodents. To obtain the direct biological proof that this gene is responsible for nude phenotype, we microinjected a cosmid clone containing the wild-type Hfh11 genomic locus into fertilized nude eggs. Two independent founder lines of transgenic mice were generated that corrected the hairless phenotype, but not the thymic defect. This partial rescue demonstrates that Hfh11 is the gene responsible for the hairless defect in the nude mouse. Taken together with previous genetic studies, this complementation result indicates that Hfh11 is indeed the nude gene and the Hfh11 locus is likely to be subject to complicated regulation.