Biomedical subjects

G Pluschke

Publications and source records attributed to G Pluschke.

At least 19 recordsLinked to original sources

PubMed2005

Estimation of the sequestered parasite load in severe malaria patients using both host and parasite markers.

The virulence of the malaria parasite Plasmodium falciparum is due, in part, to its ability to cytoadhere in deep vascular beds. Our inability to quantify the load of sequestered parasites hampers our understanding of the pathophysiological mechanisms involved in disease progression and complicates diagnosis. In this study we evaluate potential biochemical markers of sequestered load by comparing them with estimates of the sequestered load from a statistical model fitted to longitudinal patterns of peripheral parasite densities in a series of 22 patients with severe Plasmodium falciparum malaria. The markers comprised the host factors: haematocrit, circulating host DNA, sTNF-R75 and parasite derived products HRP2, pLDH, pigments and circulating parasite DNA. We investigated the suitability of these markers in determining sequestered loads in patients on quinine treatment. Observed peripheral parasitaemia, plasma levels of sTNF-R75 and circulating parasite DNA were most strongly correlated with estimates of sequestered loads on admission. However the dynamics of both sTNF-R75 and circulating parasite DNA during follow-up were very different from those of the estimated sequestered mass. These analyses suggest that none of the markers gave reliable estimates of the current sequestered load, though they may reflect the history of infection. Longitudinal analyses are needed that allow for the clearance rates of the marker molecules and for variations between hosts in the history of parasitaemia.

Animals↗

PubMed2003

Comparison of analytical methods for the evaluation of antibody responses against epitopes of polymorphic protein antigens.

Surface exposed protein antigens of the malaria parasite Plasmodium falciparum frequently harbor multiple dimorphic amino acid positions. These are associated with parasite immune evasion and represent a major obstacle for subunit vaccine design. Here, we have analyzed the flexibility of the humoral immune response against a semiconserved sequence (YX(44)LFX(47)KEKMX(52)L) of the key malaria blood stage vaccine candidate merozoite surface protein-1 (MSP-1). Monoclonal antibodies (mAbs) raised against one of the six described natural sequence variants of MSP-1(43-53) were analyzed for cross-reactivity with the other allelic forms, which differ in one to three positions from the immunizing sequence. Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy demonstrated marked differences in mAb binding avidity to the variant sequences and isothermal titration calorimetry (ITC) provided evidence for a very low affinity of some of the interactions. In immunofluorescence analysis (IFA) and Western blotting analysis, the mAbs nevertheless stained all analyzed parasite clones expressing MSP-1(43-53) variant sequences. When used for the evaluation of humoral immune responses in clinical malaria vaccine trials, these two commonly used methods may thus not be suitable to distinguish biologically functional high affinity antibody responses from irrelevant low-affinity cross-reactivities.

Amino Acid Sequence↗

PubMed2003

Development and application of real-time PCR assay for quantification of Mycobacterium ulcerans DNA.

Buruli ulcer, an infection caused by Mycobacterium ulcerans, is, after tuberculosis and leprosy, the third most common mycobacterial disease. The mode of transmission of M. ulcerans is not exactly known, but since Buruli ulcer often occurs in focalized swampy areas, it is assumed that there is a reservoir of the pathogen in stagnant water. Buruli ulcer usually starts as a painless nodule and can lead to massive destruction of skin, subcutaneous tissue, and eventually muscle and bone. Currently the only recommended treatment is wide surgical excision. In this report we describe the development of a real-time PCR method for the quantification of M. ulcerans DNA (IS2404 TaqMan). The highly specific assay is based on the detection of the M. ulcerans specific insertion sequence IS2404. The IS2404 TaqMan assay turned out to be about 10 times more sensitive than the available conventional PCR-based diagnostic test. It is demonstrated that the IS2404 TaqMan assay is suitable for the quantitative assessment of the dissemination of the mycobacteria in Buruli ulcer lesions. Prototype results obtained with excised tissue from a patient with a late preulcerative Buruli ulcer lesion reconfirmed earlier histopathological findings indicating that tissue damage occurs far beyond the regions in which large numbers of mycobacteria are detectable. The IS2404 TaqMan assay should be a useful tool for both diagnosis and research into the pathology and mode of transmission of this still inadequately investigated mycobacterial disease.

Child↗

PubMed2001

Functional and structural similarity of V gamma 9V delta 2 T cells in humans and Aotus monkeys, a primate infection model for Plasmodium falciparum malaria.

Gammadelta T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human gammadelta T cells carrying the Vgamma9Vdelta2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vgamma9Vdelta2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vgamma9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human gammadelta T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vgamma9Vdelta2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.

Amino Acid Sequence↗

PubMed2001

Exploiting conformationally constrained peptidomimetics and an efficient human-compatible delivery system in synthetic vaccine design.

Peptide and protein mimetics are potentially of great value in synthetic vaccine design. The mimetics should function by stimulating the immune system to produce antibodies that recognize the intact parasite. Also the mimetics should be presented to the immune system in a way that leads to efficient antibody production. Here we investigate the application of cyclic peptidomimetics presented on immunopotentiating reconstituted influenza virosomes (IRIVs), a form of antigen delivery that is licensed already for human clinical use, in synthetic vaccine design. We focus on the central (NPNA)(n) repeat region of the circumsporozoite (CS) protein of the malaria parasite Plasmodium falciparum as a model system. Cyclic peptidomimetics of the NPNA repeats were incorporated into both an IRIV and (for comparison) a multiple-antigen peptide (MAP). Both IRIV and MAP delivery forms induced mimetic-specific humoral immune responses in mice, but only with the mimetic-IRIV preparations did a significant fraction of the elicited antibodies cross-react with sporozoites. The results demonstrate that IRIVs are a delivery system suitable for the efficient induction of antibody responses against conformational epitopes by use of cyclic template-bound peptidomimetics. Combined with combinatorial chemistry, this approach may have great potential for the rapid optimization of molecularly defined synthetic vaccine candidates against a wide variety of infectious agents.

Animals↗

PubMed2001

Herpesvirus saimiri transformed T cells and peripheral blood mononuclear cells restimulate identical antigen-specific human T cell clones.

Panels of human antigen-specific T cell clones (TCC) have been established by limiting dilution using Herpesvirus saimiri (HVS) subtype C transformed T cells as antigen presenting cells (APC). They showed antigen-specific proliferation when peripheral blood mononuclear cells (PBMC), HVS-transformed T cells and Epstein Barr Virus transformed lymphoblastoid B cell lines (EBV-LCL) were used as APC. All T cell clones were CD4+ and HLA class II restricted. For a detailed analysis, two panels of T cell clones specific for an epitope located in the N-terminus of the Merozoite Surface Protein 1 (MSP-1) of Plasmodium falciparum were established from the same founder T cell line using either PBMC or HVS-transformed T cells as APC. TCR analysis of the two panels of TCC demonstrated that the same founder cells could be propagated in both culture systems. Furthermore, no difference in the cytokine expression pattern or antigen processing and co-stimulatory requirements was observed between TCC established on PBMC or HVS-transformed T cells. Based on the finding that HVS-transformed T cells can replace PBMC as APC for isolation and propagation of antigen-specific TCC, a protocol was developed and successfully executed, which allows to establish and maintain vaccine-specific T cell clones from 20 ml of blood. This method might be particularly significant in clinical trials of immune intervention strategies.

Amino Acid Sequence↗

PubMed2001

Survival and sequelae of meningococcal meningitis in Ghana.

BACKGROUND: Meningococcal meningitis epidemics are frequent in the Sahel zone of Africa but there is little information on the frequency of long-term sequelae. We analysed excess mortality in the two years following the 1997 epidemic in northern Ghana and carried out a case-control study to assess sequelae in the survivors. METHODS: Two-year survival of 696 meningitis cases recorded at the War Memorial Hospital, Navrongo, was analysed using data from a demographic surveillance system. A structured questionnaire on disability and on psychiatric, neuropsychological and behavioural problems was administered to 505 of the survivors and 505 age- sex- and location-matched controls as well as to their respective relatives. Cases and controls underwent full neurological and neuropsychological examination and were evaluated for hearing impairment by audiometry. RESULTS: Survival rates after the first month following the attack were similar in cases and controls. Hearing impairment was the major sequela, and was reported in 6 per cent of cases and 2 per cent of controls (odds ratio [OR] = 3.10; 95% CI : 1.48-7.09). Audiometry detected severe and profound hearing loss in the worse affected ear (> or =70 db) in 8/496 (1.6%) survivors but in only one control. Survivors of meningitis were more likely to suffer from feelings of tiredness (OR = 1.47; 95% CI : 1.03-2.11) and were more often reported by relatives to have insomnia (OR = 2.31; 95% CI : 1.17-4.82) and daily alcohol consumption. INTERPRETATION: Meningococcal meningitis annually causes approximately 10 000 cases of deafness in sub-Saharan Africa; there is a need for early detection of affected survivors and promotion of simple hearing devices. There is a sizeable burden of depressive disorders secondary to meningitis which should be identified and looked after appropriately.

Activities of Daily Living↗

PubMed2001

Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro.

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.

Amino Acid Sequence↗

PubMed2000

Identification and recombinant expression of glyceraldehyde-3-phosphate dehydrogenase of Plasmodium falciparum.

The gene coding for the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was isolated from Plasmodium falciparum. The gene contains 1 intron and the A+T content is characteristic for the codon usage of P. falciparum. The predicted open reading frame codes for 337 amino acids (36651Da) and is 63.5% identical to the human erythrocytic GAPDH. GAPDH sequences from several field isolates of P. falciparum displayed 100% conservation. Phylogenetic analysis supports the hypothesis that dinoflagellates and Plasmodium are closely related. The protein encoded by the pfGAPDH was expressed recombinantly in Escherichia coli and exhibited enzymatic activity with NAD(+) but not with NADP(+) as cofactor. Antiserum raised against the recombinantly expressed enzyme detected specifically all developmental stages of cultured P. falciparum blood-stage parasites.

Amino Acid Sequence↗

PubMed2000

Sequence diversity of the merozoite surface protein 1 of Plasmodium falciparum in clinical isolates from the Kilombero District, Tanzania.

Merozoite surface protein 1 of Plasmodium falciparum (PfMSP-1) is regarded as a key candidate antigen for malaria vaccine development. It exhibits significant antigenic polymorphism and has been divided into 17 building blocks based on the analysis of sequence diversity. Differences in the antigenic composition of PfMSP-1 in local P. falciparum populations may result in differences in the efficacy of vaccines, which contain sequences of particular allelic variant(s) of PfMSP-1. To contribute to the required knowledge of genetic diversity of malaria parasites in geographically diverse regions, we have used the polymerase chain reaction (PCR) to analyze the sequence diversity of blocks 1-4 of PfMSP-1 in disease isolates from the Kilombero District in Tanzania. In the semi-conserved block 1, in which dimorphic amino acid variances have been described at three positions, we found three of the five previously described combinations of these three pairs of amino acids. In addition one combination was found, which has not been reported before in parasite isolates from different locations worldwide. Of the two sequence variants, which were dominating, one (S44-Q47-V52) corresponded to the 83.1 sequence incorporated into the SPf66 malaria peptide vaccine, while the other one (G44-H47-I52) differed from the previous in all three dimorphic amino acids. The partial protection observed in a phase III SPf66 trial conducted in the Kilombero District in children aged 1-5, thus does not seem to be associated with a clear dominance of favourable variants of block 1 of PfMSP-1 in this area. All three different principle types of block 2, the major polymorphic region of PfMSP-1, were found in the Tanzanian isolates. Most of the sequences contained K1-type tripeptide repeats, but clones with MAD20-type repeats or no repetitive sequence (RO33-type block 2) were also present. K1- and MAD20-type tripeptide repeat motifs were never mixed within one parasite clone. In one sequence a hexapeptide repeat was found at the end of block 2, which has not been reported before. Dimorphism in 13 of the 17 previously described variable positions of the semi-conserved block 3 and three of four recombination types of block 4 (K/K, M/K and M/M) were found among the Tanzanian isolates. Apart from previously described dimorphic amino acid positions, polymorphism was rare in the non-repeated building blocks. Selection and spreading of parasite variants, which contain amino acid exchanges at other than the dimorphic positions thus, is not a common event. Parasite isolates frequently harboured more than one PfMSP-1 allele. Three of the four heterogeneous isolates analysed contained two different general types of sequences. One isolate contained at least four distinct clones, demonstrating the high endemicity of malaria in the Kilombero District, which is a well-established site for malaria vaccine field trials.

Amino Acid Sequence↗

PubMed2000

Sequence and diversity of MHC DQA and DQB genes of the owl monkey Aotus nancymaae.

The New World primate Aotus nancymaae has been recommended by the World Health Organization (WHO) as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and Plasmodium vivax. We present here the nucleotide sequences of the complete cDNA of MHC-DQA1 and of the polymorphic exon 2 segments of MHC-DQB1/DQB2. In a group of three nonrelated animals captured in the wild, five alleles of MHC-DQA1 could be identified. They all belong to one lineage, namely Aona-DQA1*27. This lineage has not been described in any other New World monkey species studied. In a group of 19 unrelated animals, 14 Aona-DQB1 alleles could be identified which are grouped into the two lineages Aona-DQB1*22 and Aona-DQB1*23. These lineages have been described previously in the common marmoset and cotton-top tamarin. In addition, two Aona-DQB2 sequences could be identified which are highly similar to HLA-DQB2 sequences. Essential amino acid residues contributing to MHC DQ peptide binding pockets number 1 and 4 are conserved or semi-conserved between HLA-DQ and Aona-DQ molecules, indicating a capacity to bind similar peptide repertoires. These results fully support the use of Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.

Alleles↗

PubMed2000

Immunoglobulin kappa light-chain V, J, and C gene sequences of the owl monkey Aotus nancymaae.

Sequences of Aotus nancymaae immunoglobulin kappa light-chain rearrangements were analyzed after reverse transcription polymerase chain reaction. Among 22 in-frame rearrangements analyzed, 12 IGKV genes belonging to the families 1, 2, or 3 were identified. Aotus counterparts for all five human IGKJ genes were found. The identity of the deduced human and Aotus amino acid sequences was between 83% and 92% for junctional regions and 74% for the constant region. Sequence comparisons between rearrangements indicated that somatic mutations, the addition of nongermline-encoded nucleotides, and exonuclease trimming contribute to the generation of diversity of Aotus immunoglobulin kappa chains. The high identity of Aotus and human IGK genes is comparable to that of T-cell receptor genes and further supports the proposal to use the Aotus Plasmodium falciparum infection model for the evaluation of malaria vaccine candidates.

Amino Acid Sequence↗

PubMed2000

Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites.

The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the beta-pleated sheets and by frequent exchange of fixed sequence motifs in the alpha-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA-DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.

Alleles↗

PubMed2000

Microheterogeneity of serogroup A (subgroup III) Neisseria meningitidis during an outbreak in northern Ghana.

During a meningitis outbreak in the eastern subdistrict of the Kassena-Nankana District of the Upper East Region of Ghana, we analysed cerebrospinal fluid from suspected meningitis cases for the most common causative organisms. In 50 of 92 samples analysed, serogroup A Neisseria meningitidis were detected. The ages of serogroup A N. meningitidis patients ranged from 4 months to 64 years. The case fatality ratio was 20%. Coma or stupor on presentation worsened the prognosis. All serogroup A N. meningitidis isolates recovered revealed the A: 4: P1.9, 20 phenotype characteristic for the subgroup III clonal grouping. No evidence for resistance to penicillin G, chloramphenicol, cefotaxime, ciprofloxacin, rifampicin or tetracycline was found. All strains were resistant to sulphadiazine. Restriction analysis patterns of opa, iga and ingA genes were characteristic for the majority of N. meningitidis serogroup A subgroup III bacteria isolated in Africa after the 1987 epidemic in Mecca. Differences in pulsed-field gel electrophoresis patterns of NheI and SpeI digested DNA revealed microheterogeneity among the Ghanaian isolates.

Adolescent↗

PubMed1999

Sequence and diversity of T-cell receptor beta-chain V and J genes of the owl monkey Aotus nancymaae.

The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and has therefore been recommended by the World Health Organization as a model for the evaluation of malaria vaccine candidates. Recently, we have shown that Aotus TCRVA genes and TCRJA segments exhibit a high degree of similarity to human counterparts. In the present report we used reverse transcription polymerase chain reaction to analyze the sequences of A. nancymaae TCR beta-chain gene rearrangements. Alignment with human sequences and phylogenetic comparison identified 18 distinct Aotus TCRBV genes homologous to the human TCRBV gene families 2, 4, 5, 6, 7, 9, 12, 15, 24, and 28. Multiple Aotus genes were found in the TCRBV4, 5, 6, and 7 families. Some of these TCRBV genes aligned best to the same human gene and thus do not seem to have separate human homologues. Amino acid sequences of the Aotus TCRBV genes were 77 to 90% identical to their closest human counterparts. Ten distinct Aotus TCRBJ segments homologous to the human segments J1-1, J1-2, J1-4, J1-5, J1-6, J2-1, J2-2, J2-3, J2-4, J2-5 were found. In some cases the amino acid sequences of Aotus and human TCRBJ segments were completely identical. A comparison of the proportion of synonymous and non-synonymous substitutions in Aotus vs human beta-chain-encoding genes revealed a dominance of synonymous substitutions in TCRBJ segments and of nonsynonymous substitutions in TCRBV segments. Dominance of nonsynonymous substitutions was more pronounced in TCRBV CDR1 and CDR2 regions than in the framework regions. No evidence for the emergence of new TCRBJ segments or TCRBV families was found. These results confirm that the TCR repertoire in primates is remarkably stable and support the concept of using Aotus monkeys as an infection model for the evaluation of future subunit vaccine candidates.

Amino Acid Sequence↗

PubMed1999

Use of reconstituted influenza virus virosomes as an immunopotentiating delivery system for a peptide-based vaccine.

Immunopotentiating reconstituted influenza virosomes (IRIV) were used as a delivery system for the synthetic peptide-based malaria vaccine SPf66. The reduced SPf66 peptide molecules containing terminal cysteine residues were covalently attached to phosphatidylethanolamine with the heterobifunctional crosslinker gamma-maleimidobutyric acid N-hydroxysuccinimide ester. The SPf66-phosphatidylethanolamine was incorporated into IRIV and BALB/c mice were immunized twice by intramuscular injection with peptide-loaded virosomes. Titres of elicited anti-SPf66 IgG were determined by ELISA. These titres were significantly higher and the required doses of antigen were lower, when mice had been preimmunized with a commercial whole virus influenza vaccine. After preimmunization with the influenza vaccine, SPf66-IRIV elicited far more consistently anti-SPf66 antibody responses than SPf(66)n adsorbed to alum. MoAb produced by four B cell hybridoma clones derived from a SPf66-IRIV-immunized mouse cross-reacted with Plasmodium falciparum blood stage parasites in immunofluorescence assays. All four MoAbs were specific for the merozoite surface protein-1 (MSP-1)-derived 83.1 portion of SPf66. Sequencing of their functionally rearranged kappa light chain variable region genes demonstrated that the four hybridomas were generated from clonally related splenic B cells. Biomolecular interaction analyses (BIA) together with these sequencing data provided evidence for the selection of somatically mutated affinity-matured B cells upon repeated immunization with SPf66-IRIV. The results indicate that IRIV are a suitable delivery system for synthetic peptide vaccines and thus have a great potential for the design of molecularly defined combined vaccines targeted against multiple antigens and development stages of one parasite, as well as against multiple pathogens.

Amino Acid Sequence↗

PubMed1999

Sequence analysis of the MSP 1 gene of Plasmodium falciparum isolates from Hainan, China.

AIM: To obtain the complete sequence and analyze the diversity of the MSP 1 molecule from the Chinese isolates of Plasmodium falciparum. METHODS: Genomic DNA was prepared directly from blood samples spotted on filter papers from 2 malaria patients from Baoting County, Hainan Province. PCR amplification of the target gene was carried out using 5 pairs of oligonucleotides specific for the MSP 1 gene. Direct sequencing of the target gene fragments was performed using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) in a automatic ABI PRISM 310 Genetic Analyzer. RESULTS: For the first time, two complete sequences of the MSP1 gene from two Chinese isolates of Plasmodium falciparum were obtained. A comparison with the previously reported sequences identified them as members of the MAD20 allelic family. The deduced amino acid sequences of the MSP1 from this two Chinese isolates were identical with each other except for Blocks 2, 4 and 8. CONCLUSION: The sequences of the MSP 1 from two Chinese isolates of P. falciparum belong to the MAD20 allelic family. Minor variations through the whole sequences exist compared with the MAD20 sequence. The results provide the first evidence of the diversity of the MSP 1 molecule from Chinese isolates of Plasmodium falciparum.

Amino Acid Sequence↗

PubMed1998

Generation of chimeric monoclonal antibodies from mice that carry human immunoglobulin Cgamma1 heavy of Ckappa light chain gene segments.

Gene targeting in mouse embryonic stem (ES) cells was used to replace (i) the mouse immunoglobulin heavy chain (IgH) Cgamma2a gene segment (mCgamma2a) with the human Cgamma1 gene segment (hCgamma1), and (ii) the mouse immunoglobulin light chain (IgL) Ckappa gene segment (mC kappa) with its human counterpart (hC kappa). ES cells carrying these gene conversions were used to generate chimeric mice that transmitted the human alleles through the germ line. Mice homozygous for both gene alterations were generated by breeding. Serum from homozygous mutant mice contained comparable amounts of antibodies with chimeric kappa or mouse lambda light chains but only small fractions of basal serum IgG or antibodies elicited against immunizing agents contained chimeric heavy chains. A relative increase in immunogen-specific hCgamma1 antibodies was seen following immunization in combination with the saponin adjuvant QS-21. The effect of this was to shift the IgG1-dominated response to an IgG subclass profile that included significant amounts of IgG2a, IgG2b and IgG3 and chimeric IgG. The amounts of antibody secreted by hybridomas derived from mutant and wild-type mice were similar. Sequencing confirmed correct splicing of hCgamma1 and hCkappa gene segments to mouse J gene segments in hybridoma Ig gene transcripts. In conclusion, IgHhCgamma1/IgLhCkappa double mutant mice provide a useful animal model for deriving humanized antibodies with potential applications in immunotherapy and diagnostics in vivo as well as for investigating hCgamma1 associated functions.

Animals↗