Genetic variants of Orientia tsutsugamushi identified from scrub typhus cases in Malaysia.

We report herein the clinical presentation and diagnosis of scrub typhus in three patients attending a teaching hospital in Malaysia. Three genetic variants belonged to the Karp and Gilliam strains of O. tsutsugamushi were amplified from the acute blood samples of the patients by a nested polymerase chain reaction assay. The circulation of different genetic variants of O. tsutsugamushi strains might complicate the presentation and severity of scrub typhus. Loop-PCR is a promising diagnostic tool for rapid diagnosis of scrub typhus.

Evaluation of land cover and prevalence of dengue in Malaysia.

Serological confirmation of dengue in 1,410 school-going children aged 7-18 years provided prevalence data for 16 different sites in Malaysia. These sites ranged from highly urbanized cities to small towns. We found that at least ~7 % of children in the study group had been exposed to dengue by age 12 and ~16% by age 18. Here we report that the dengue seroprevalence correlates with i) increasing land development and decreased vegetation, and ii) the overall population growth. Water bodies did not significantly affect dengue prevalence. High prevalence of dengue was also recorded in few of the non-urban sites suggesting the expanding geographical locality of those who get dengue in Malaysia in tandem with increased land usage activities. These findings highlight the need to give closer consideration to future urban planning and development, taking into consideration the changing demography and the importance of built environment to mitigate the increasing incidence of dengue in the non-urban areas of Malaysia.

Evaluation of land cover and prevalence of dengue in Malaysia

Serological confirmation of dengue in 1,410 school-going children aged 7-18 years provided prevalence data for 16 different sites in Malaysia. These sites ranged from highly urbanized cities to small towns. We found that at least ~7 % of children in the study group had been exposed to dengue by age 12 and ~16% by age 18. Here we report that the dengue seroprevalence correlates with i) increasing land development and decreased vegetation, and ii) the overall population growth. Water bodies did not significantly affect dengue prevalence. High prevalence of dengue was also recorded in few of the non-urban sites suggesting the expanding geographical locality of those who get dengue in Malaysia in tandem with increased land usage activities. These findings highlight the need to give closer consideration to future urban planning and development, taking into consideration the changing demography and the importance of built environment to mitigate the increasing incidence of dengue in the non-urban areas of Malaysia.

The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation.

Ig somatic mutations would be introduced by a polymerase (pol) while repairing DNA outside main DNA replication. We show that human B cells constitutively express the translesion pol zeta, which effectively extends DNA past mismatched bases (mispair extender), and pol eta, which bypasses DNA lesions in an error-free fashion. Upon B cell receptor (BCR) engagement and coculture with activated CD4+ T cells, these lymphocytes upregulated pol zeta, downregulated pol eta, and mutated the Ig and bcl-6 genes. Inhibition of the pol zeta REV3 catalytic subunit by specific phosphorothioate-modified oligonucleotides impaired Ig and bcl-6 hypermutation and UV damage-induced DNA mutagenesis, without affecting cell cycle or viability. Thus, pol zeta plays a critical role in Ig and bcl-6 hypermutation, perhaps facilitated by the downregulation of pol eta.

Dysregulation of CD30+ T cells by leukemia impairs isotype switching in normal B cells.

Chronic lymphocytic leukemia (CLL) is associated with impaired immunoglobulin (Ig) class-switching from IgM to IgG and IgA, a defect that leads to recurrent infections. When activated in the presence of leukemic CLL B cells, T cells rapidly up-regulate CD30 through an OX40 ligand and interleukin 4 (IL-4)-dependent mechanism. These leukemia-induced CD30+ T cells inhibit CD40 ligand (CD40L)-mediated S mu-->S gamma and S mu-->S alpha class-switch DNA recombination (CSR) by engaging CD30 ligand (CD30L), a molecule that interferes with the assembly of the CD40-tumor necrosis factor receptor-associated factor (TRAF) complex in nonmalignant IgD+ B cells. In addition, engagement of T cell CD30 by CD30L on neoplastic CLL B cells down-regulates the CD3-induced expression of CD40L. These findings indicate that, in CLL, abnormal CD30-CD30L interaction impairs IgG and IgA production by interfering with the CD40-mediated differentiation of nonmalignant B cells.

Characterization of electrolytic ZrO2 coating on Co-Cr-Mo implant alloys of hip prosthesis.

An electrolytic Zr(OH)4 gel has been coated on ASTM F-75 Co-Cr-Mo alloy specimens in 0.0625 M ZrO(NO3)2 solution with pH = 2.2 at a current density of 2 mA/cm2. After annealing at 623-973 K for 120 min in air, the ZrO2-coated specimen was evaluated by electrochemical polarization in Hank's solution, wear tests with UHMWPE (Ultra-high molecular-weight polyethylene) under a load stress of 50 MPa, scratch tests, surface morphology observations, and XRD analysis. The ZrO2-coated specimen annealed at 773 K for 120 min revealed a good adhesion of 610 MPa on Co-Cr-Mo substrate, a lower wear loss of UHMWPE and a higher protection potential than the uncoated specimen in Hank's solution. A monoclinic structure with (1 1 1) preferred orientation parallel to the sheet plane was detected at 623 K < or = T < or = 673 K and a tetragonal structure of ZrO2 was detected at T > or = 773 K. Then a monoclinic structure with random orientation and a tetragonal structure were mixed at T > or = 973 K.

Engagement of CD153 (CD30 ligand) by CD30+ T cells inhibits class switch DNA recombination and antibody production in human IgD+ IgM+ B cells.

CD153 (CD30 ligand) is a member of the TNF ligand/cytokine family expressed on the surface of human B cells. Upon exposure to IL-4, a critical Ig class switch-inducing cytokine, Ag-activated T cells express CD30, the CD153 receptor. The observation that dysregulated IgG, IgA, and/or IgE production is often associated with up-regulation of T cell CD30 prompted us to test the hypothesis that engagement of B cell CD153 by T cell CD30 modulates Ig class switching. In this study, we show that IgD+ IgM+ B cells up-regulate CD153 in the presence of CD154 (CD40 ligand), IL-4, and B cell Ag receptor engagement. In these cells, CD153 engagement by an agonistic anti-CD153 mAb or T cell CD30 inhibits S mu-->Sgamma, Smu-->Salpha, and S mu-->Sepsilon class switch DNA recombination (CSR). This inhibition is associated with decreased TNFR-associated factor-2 binding to CD40, decreased NF-kappaB binding to the CD40-responsive element of the Cgamma3 promoter, decreased Igamma3-Cgamma3 germline gene transcription, and decreased expression of Ku70, Ku80, DNA protein kinase, switch-associated protein-70, and Msh2 CSR-associated transcripts. In addition, CD153 engagement inhibits IgG, IgA, and IgE production, and this effect is associated with reduced levels of B lymphocyte maturation protein-1 transcripts, and increased binding of B cell-specific activation protein to the Ig 3' enhancer. These findings suggest that CD30+ T cells modulate CSR as well as IgG, IgA, and IgE production by inducing reverse signaling through B cell CD153.

B cell receptor engagement and T cell contact induce Bcl-6 somatic hypermutation in human B cells: identity with Ig hypermutation.

The human bcl-6 proto-oncogene has been found to be mutated in both neoplastic and normal B cells. We used CL-01 cells, our monoclonal model of germinal center differentiation, and normal human B cells to explore the induction requirements and the modalities of bcl-6 hypermutation. As we have previously shown, CL-01 cells are IgM+ IgD+ and effectively mutate the expressed Ig VHDJH and V lambda J lambda genes and switch to IgG, IgA, and IgE upon B cell receptor engagement and contact with CD4+ T cells through CD40:CD154 and CD80:CD28 coengagement. In this paper we showed that the same stimuli induce somatic hypermutation of bcl-6 in CL-01 and normal IgM+ IgD+ B cells. bcl-6 hypermutation was not accompanied by translocation of this proto-oncogene or hypermutation of the beta-actin gene, and it did mimic Ig hypermutation. It was associated with transcription initiation, in that it targeted the first exon and a 696-bp sequence immediately downstream (approximately 0.6 kb) of the transcription initiation site while sparing further downstream (approximately 2.5 kb) and upstream (approximately 0.1 kb) areas. bcl-6 hypermutation displayed an overall rate of 2.2 x 10-4 changes/base/cell division with characteristic nucleotide preferences and showed strand polarity. These findings show that B cell receptor engagement promotes hypermutation in genes other than Ig, and suggest that cis-regulating elements similar to those of the Ig locus exist in bcl-6.

Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution.

To investigate the role of somatic Ig hypermutation in the evolution of AIDS-associated B cell lymphomas, we analyzed the Ig V(D)J and c-myc genes expressed by neoplastic B cells in two extranodal sites, testis and orbit, and clonally related cells in the bone marrow. Testis and orbit B cells expressed differentially mutated but collinear V(H)DJ(H), V kappa J kappa and c-myc gene sequences. Shared mutations accounted for 10.2%, 8.4%, and 4.3% of the overall V(H)DJ(H), V kappa J kappa, and c-myc gene sequences. Tumor-site specific V(H)DJ(H), V kappa J kappa, and c-myc mutations were comparable in frequency, and a single point-mutation gave rise to an EcoRI site in the testis c-myc DNA. Both shared and tumor site-specific V(H)DJ(H), V kappa J kappa, and c-myc mutations displayed predominance of transitions over transversions. The "neoplastic" V(H)DJ(H) sequence was expressed by about 10(-5) cells in the bone marrow, and contained two of the three orbital, but none of the testicular V(H)DJ(H) mutations. The nature and distribution of the Ig V(D)J mutations found in the kappa chain suggested a selection by antigen in testis and orbit. Our data suggest that, in AIDS-associated B cell lymphomas, the Ig hypermutation machinery targets V(H)DJ(H), V kappa J kappa, and c-myc genes with comparable efficiency and modalities.

The evolutionarily conserved sequence upstream of the human Ig heavy chain S gamma 3 region is an inducible promoter: synergistic activation by CD40 ligand and IL-4 via cooperative NF-kappa B and STAT-6 binding sites.

Germline C gamma gene transcription is a crucial event in the process that leads to switch DNA recombination to IgG, but its regulation in the human is poorly understood. We took advantage of our monoclonal model of germinal center B cell differentiation, IgM+ IgD+ CL-01 cells, to define the role of the I gamma 3 evolutionarily conserved sequence (ECS) in the germline transcriptional activation of the human C gamma 3 gene. The I gamma 3 ECS lies upstream of the major I gamma 3 transcription initiation site and displays more than 90% identity with the corresponding human I gamma 1, I gamma 2, and I gamma 4 regions. Reporter luciferase gene vectors containing the human gamma 3 ECS were used to transfect CL-01 cells, which have been shown to undergo Smu-->S gamma 3 DNA recombination, upon engagement of CD40 by CD40 ligand (CD40L) and exposure to IL-4. In these transfected CL-01 cells, CD40:CD40L engagement and exposure to IL-4 synergistically induced gamma 3 ECS-dependent luciferase reporter gene activation. Targeted mutational analysis demonstrated that a tandem NF-kappa B/Rel binding motif is critical for the gamma 3 ECS responsiveness to both CD40L and IL-4, while a STAT-6-binding site is additionally required for IL-4 inducibility. Electrophoretic mobility shift assays showed that p50/p65/c-Rel and STAT-6 are effectively induced by CD40L and IL-4, respectively, and bind to specific DNA motifs within the ECS. These partially overlapping CD40L and IL-4 responsive elements are functionally cooperative as the disruption of one of them prevents synergistic promoter activation. Thus, the gamma 3 ECS is an inducible promoter containing cis elements that critically mediate CD40L and IL-4-triggered transcriptional activation of the human C gamma 3 gene.

Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation.

Partly because of the lack of a suitable in vitro model, the trigger(s) and the mechanism(s) of somatic hypermutation in Ig genes are largely unknown. We have analyzed the hypermutation potential of human CL-01 lymphocytes, our monoclonal model of germinal center B cell differentiation. These cells are surface IgM+ IgD+ and, in the absence of T cells, switch to IgG, IgA, and IgE in response to CD40:CD40 ligand engagement and exposure to appropriate cytokines. We show here that CL-01 cells can be induced to effectively mutate the expressed VHDJH-C mu, VHDJH-C delta, VHDJH-C gamma, VHDJH-C alpha, VHDJH-C epsilon, and V lambda J lambda-C lambda transcripts before and after Ig class switching in a stepwise fashion. In these cells, induction of somatic mutations required cross-linking of the surface receptor for Ag and T cell contact through CD40:CD40 ligand and CD80: CD28 coengagement. The induced mutations showed intrinsic features of Ig V(D)J hypermutation in that they comprised 110 base substitutions (97 in the heavy chain and 13 in the lambda-chain) and only 2 deletions and targeted V(D)J, virtually sparing CH and C lambda. These mutations were more abundant in secondary VHDJH-C gamma than primary VHDJH-C mu transcripts and in V(D)J-C than V lambda J lambda-C lambda transcripts. These mutations were also associated with coding DNA strand polarity and showed an overall rate of 2.42 x 10(-4) base changes/cell division in VHDJH-CH transcripts. Transitions were favored over transversions, and G nucleotides were preferentially targeted, mainly in the context of AG dinucleotides. Thus, in CL-01 cells, Ig somatic hypermutation is readily inducible by stimuli different from those required for class switching and displays discrete base substitution modalities.

CD40 engagement triggers switching to IgA1 and IgA2 in human B cells through induction of endogenous TGF-beta: evidence for TGF-beta but not IL-10-dependent direct S mu-->S alpha and sequential S mu-->S gamma, S gamma-->S alpha DNA recombination.

IgA are major effectors of antimicrobial defense in the respiratory and digestive tracts. We have analyzed the requirements for and the modalities of switching to IgA using our recently identified monoclonal model of human germinal center differentiation, CL-01 B cells. CL-01 cells bear surface IgM (sIgM) and sIgD and switch to all seven downstream isotypes in response to physiologic stimuli. In these cells, CD40 engagement by CD40 ligand induces production of endogenous TGF-beta and IL-10, expression of germline Ialpha1-Calpha1 and Ialpha2-Calpha2 transcripts, mature VHDJH-Calpha1 and VHDJH-Calpha2 transcripts, and IgA secretion. These events are associated with not only direct Smu-->Salpha, but also sequential Smu-->Sgamma, Sgamma-->Salpha DNA recombination, and are ablated by neutralizing anti-TGF-beta but not IL-10 Ab, and indicating that TGF-beta, not IL-10, is a crucial mediator of the transcriptional activation and recombination of human Calpha1 and Calpha2 genes. Our findings in CL-01 cells were reproduced in freshly isolated naive sIgM+ sIgD+ B lymphocytes. Thus, engagement of CD40, in the absence of other (known) stimuli, is sufficient to effectively induce switching to IgA in human B cells. This is effected by direct and sequential DNA recombination events, which are both dependent upon endogenous TGF-beta secreted by the CD40L-induced B cells.

CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B cells.

We used our monoclonal model of germinal center maturation, CL-01 B cells, to investigate the role of CD30 in human B cell differentiation. CL-01 cells are IgM+ IgD+ CD30+ and switch to IgG, IgA, and IgE when exposed to CD40L and IL-4. Switching is hampered by CD30 coengagement, possibly through interference with the CD40-mediated NF-kappaB-dependent transcriptional activation of downstream C(H) genes. The physiological relevance of this phenomenon is emphasized by similar CD30-mediated effects in naive B cells. Expression of CD30 by these cells is induced by CD40L but is inhibited by B cell receptor coengagement and/or exposure to IL-6 and IL-12. Our data suggest that CD30 critically regulates the CD40-mediated differentiation of non-antigen-selected human B cells.

CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM+IgD+ B cell line.

B lymphocytes are induced to undergo Ig class switching and a complex phenotypic differentiation by the milieu of the germinal center. Partly as a result of the lack of a suitable in vitro B cell model, the relationship between these processes in the humans has never been formally established in vitro. We have identified a human monoclonal B cell line, CL-01, that expresses surface IgM and IgD and, upon induction with CD40 ligand, IL-4, and IL-10, switches to all seven downstream isotypes, showing typical DNA switch recombination preceded by germline transcription of targeted CH regions. In CL-01 cells, switch-inducing stimuli trigger concomitant changes in expression of surface IgD, CD23, CD38, and CD77 that parallel those reported in ex vivo isolated tonsillar centroblasts, centrocytes, and memory B cells. Eventually, in the presence of IL-6, CL-01 cells express CD56 and accumulate cytoplasmic IgG and IgA, both traits of plasmacytoid differentiation. Analysis of transcription and recombination of the Ig H locus in sorted CL-01 cells suggest that Ig class switching begins in centroblasts, it extends to all isotypes in centrocytes, and it is extinct in memory B cells. Thus, we have induced coordinated Ig class switching, progression through germinal center phenotypic stages, and differentiation to memory B cells and plasma cells at the level of a single B clonotype. Our data suggest that these processes are likely regulated by a common maturation program, the activation of which may require CD40 ligand, IL-4, IL-10, and IL-6 only.

Temperature-induced expression of human-mouse chimeric Fab.

The temperature-induced expression vector pHZ01 with lambda PRPL promoter for the efficient expression of human-mouse chimeric Fab was constructed. Three kinds of chimeric Fab were expressed in E. coli: anti-prostate specific antigen (PSA) chimeric Fab, anti-lysozyme (HEL) chimeric Fab, and anti-tetanus toxoid (TT) chimeric Fab. All the soluble chimeric Fabs expressed showed specific antigen-binding activities.

[Cloning of variable region genes of anti-tetanus toxoid antibody and their expression as three kinds of engineered antibodies in E. coli].

The heavy and light chain variable region genes of anti-tetanus toxoid (TT) antibody and the heavy chain Fd genes were amplified and cloned through RT-PCR from mouse hybridoma cells. The sequences of VH and VK were determined. Fd gene fragments were expressed in E. coli. The ELISA results indicated that the expressed Fd showed antigen binding activity but was nonspecific. Furthermore, through SOE and PCR techniques, the VH and VK gene fragments together with ScFv linker were assembled into single chain antibody (ScFv) gene fragment. While together with human heavy chain CH 1 gene fragment and Fab linker, they were assembled into chimeric Fab gene fragment. The two assembled gene fragments were separately inserted into phagemid pHEN 1, which was a fd-based vector containing gene 3 encoding the minor coat protein. In presence of helper phage M 13-VCS the anti-TT phage-ScFv or phage-Fab were displayed on the surface of phage particles respectively. Results from phage-ELISA indicated that both phage antibodies were TT-specific.