Ribosomal protein S6 is hyperactivated and differentially phosphorylated in epidermal lesions of patients with psoriasis and atopic dermatitis.

BACKGROUND: The ribosomal protein S6 is part of the translation machinery and is activated by phosphorylation via the mammalian target of rapamycin pathway, which is activated in psoriatic skin. OBJECTIVES: To investigate which S6 sites are phosphorylated in psoriasis and atopic dermatitis (AD), and to study whether S6 phosphorylation is associated with inflammation and/or keratinocyte hyperproliferation. METHODS: Healthy skin and skin lesions of patients with psoriasis and AD were investigated by immunostaining using antibodies that stain proliferating cells, leucocytes and distinct phosphorylated sites of S6. RESULTS: All psoriasis and AD lesions revealed abnormal S6 phosphorylation in the epidermis. The extent of S6 phosphorylation was diverse, generally stronger in psoriasis and correlated, in both diseases, with inflammation. S6 showed differential phosphorylation in distinct epidermal layers, which was most pronounced in hyperproliferative regions. CONCLUSIONS: Differential S6 phosphorylation may have a role in abnormal keratinocyte proliferation/differentiation.

Autoaggressive myocytotoxic T lymphocytes expressing an unusual gamma/delta T cell receptor.

Polymyositis mediated by gamma/delta T cells is a unique disease in which autoaggressive T lymphocytes surround, invade, and destroy muscle fibers. Histochemically, the vast majority of muscle-infiltrating T cells in a patient with polymyositis were reactive with a pan-gamma/delta T cell receptor (TCR)-specific monoclonal antibody (TCR-delta 1+), but unlike > 90% of peripheral blood gamma/delta T cells, these lymphocytes did not react with V delta 1- or V gamma 9-specific antibodies (A13- and Ti gamma A-, respectively). Differential reactivity with two different V delta 2-specific monoclonal antibodies (BB3-/TiV-delta 2+) indicated that the infiltrating T cells express a V delta 2-containing TCR with unusual additional structural features. Using conventional and anchored polymerase chain reaction for the analysis of TCR transcripts, we found a striking predominance of one unusual V delta 2-J delta 3 recombination and one V gamma 3-J gamma 1 recombination. Both the unusual phenotype (TCR-delta 1+/A13-/Ti gamma A-/BB3-/TiV-delta 2+) and the dominance of distinct TCR transcripts are compatible with the assumption that one T cell clone, which expresses a V gamma 3-J gamma 1-C gamma 2/V delta 2-J delta 3-C delta disulfide-linked TCR, dominates among the infiltrating T cells of the polymyositis muscle specimen analyzed.

Crucial role of tumor necrosis factor receptor 1 expression on nonhematopoietic cells for B cell localization within the splenic white pulp.

During immune responses the initial activation of B cells takes place in T cell zones of periarteriolar lymphoid sheaths (PALS) of the splenic white pulp. After initial activation, B cells migrate into the primary follicles and, in association with follicular dendritic cells (FDCs), undergo clonal expansion and differentiation giving rise to germinal centers (GCs). Peanut agglutinin binding (PNA+) cells of the GC differentiate further into memory or plasma cells. Here we report that in tumor necrosis factor receptor 1-deficient mice (TNFR1(-/-)), the location of B cells was altered and that plasma cells were abnormally distributed in the splenic PALS. In contrast to lymphotoxin alpha-deficient mice (LTalpha-/-), bone marrow or fetal liver transplantation did not correct the abnormal organization of the spleen, location of B cells, the lack of an FDC network, nor the antibody response in TNFR1(-/-) mice. These results argue for a crucial role of TNFR1 expression on nonhematopoietic cells for the maintenance of the splenic architecture and proper B cell location. In addition, the lack in development of an FDC network after adoptive transfer suggests that either FDCs are not of bone marrow origin or that they depend on signals from nonhematopoietic cells for maturation.

Phagocytosis of Mycobacterium ulcerans in the course of rifampicin and streptomycin chemotherapy in Buruli ulcer lesions.

BACKGROUND: Infection with Mycobacterium ulcerans involves a devastating skin disease called Buruli ulcer (BU). Currently, dual therapy with rifampicin and streptomycin (R/S) for 8 weeks as well as surgery are the standard treatments. OBJECTIVES: To elucidate the processes taking place in BU lesions in the course of chemotherapy we performed an in-depth histological analysis of lesions after 4 weeks of R/S treatment and compared results with findings in untreated lesions and lesions treated for 8 weeks. METHODS: Tissue specimens were collected from patients who had no treatment and from patients after 4 and 8 weeks of R/S treatment. The main features evaluated were local immune responses, histopathological alterations and bacterial distribution. RESULTS: After 4 weeks of R/S treatment we observed a large proportion of mycobacteria inside macrophages, occasionally forming globus-like aggregations. While distinct bands of inflammatory leucocytes surrounded the necrotic core in an ulcer and early granuloma formation was apparent in the healthy-appearing margins, acute cellular infiltration covering the whole lesion had developed in a nodular lesion. In contrast, ulcerative lesions after 8 weeks of chemotherapy showed intra- and extracellular bacterial debris as well as the presence of extensive chronic infiltrates forming huge granulomas. CONCLUSIONS: R/S treatment of BU results in a rapid onset of local cellular immune responses associated with phagocytosis of the extracellular M. ulcerans. This may be related to declining levels of the macrolide toxin mycolactone in the tissue, thus leading to an enhanced chemotherapy-induced clearance of the infection.

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains.

AIMS: Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium. METHODS AND RESULTS: The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide--anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate-protein conjugates containing the synthetic tetrasaccharide, an anthrose-rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested. CONCLUSIONS: Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.

ST2859 serogroup A meningococcal meningitis outbreak in Nouna Health District, Burkina Faso: a prospective study.

We analysed cerebrospinal fluid samples from suspected meningitis cases in Nouna Health District, Burkina Faso, during the meningitis seasons of 2004-2006. Serogroup A ST2859 meningococci belonging to the ST5 clonal complex of subgroup III meningococci were the predominant causative agent. ST2859 bacteria were associated with focal outbreaks in the north of the district. While >10% of the population of an outbreak village carried ST2859, the population in the south of the district was predominantly colonised by serogroup Y ST4375 meningococci, which were associated with only sporadic cases of meningitis. Colonisation with the less virulent Y meningococci may interfere with the spread of the ST2859 to the south of the district, but there are concerns that this serogroup A clone may cause a third wave of subgroup III meningococcal disease in the African Meningitis Belt.

Possible healthcare-associated transmission as a cause of secondary infection and population structure of Staphylococcus aureus isolates from two wound treatment centres in Ghana.

We have previously shown that secondary infections of Buruli ulcer wounds were frequently caused by Staphylococcus aureus. To gain understanding into possible routes of secondary infection, we characterized S. aureus isolates from patient lesions and surrounding environments across two Ghanaian health centres. One hundred and one S. aureus isolates were isolated from wounds (n = 93, 92.1%) and the hospital environment (n = 8, 7.9%) and characterized by the spa gene, mecA and the Panton-Valentine leucocidin toxin followed by spa sequencing and whole genome sequencing of a subset of 49 isolates. Spa typing and sequencing of the spa gene from 91 isolates identified 29 different spa types with t355 (ST152), t186 (ST88), and t346 dominating. Although many distinct strains were isolated from both health centres, genotype clustering was identified within centres. In addition, we identified a cluster consisting of isolates from a healthcare worker, patients dressed that same day and forceps used for dressing, pointing to possible healthcare-associated transmission. These clusters were confirmed by phylogenomic analysis. Twenty-four (22.8%) isolates were identified as methicillin-resistant S. aureus and lukFS genes encoding Panton-Valentine leucocidin were identified in 67 (63.8%) of the isolates. Phenotype screening showed widespread resistance to tetracycline, erythromycin, rifampicin, amikacin and streptomycin. Genomics confirmed the widespread presence of antibiotic resistance genes to ß-lactams, chloramphenicol, trimethoprim, quinolone, streptomycin and tetracycline. Our findings indicate that the healthcare environment probably contributes to the superinfection of Buruli ulcer wounds and calls for improved training in wound management and infection control techniques.

Titration calorimetry study of an anti-idiotypic antibody cascade in a human melanoma-associated antigen system.

The thermodynamic parameters of interactions between six variants of the anti-idiotypic monoclonal antibody (mAb) CGP 60686 produced by the hybridoma MK2-23 with an idiotypic mAb and five different anti-anti-idiotypic mAb were studied with high sensitivity titration calorimetry. CGP 60686 recognizes an epitope in the antigen-combining region of the human high-molecular-weight-melanoma-associated antigen (HMW-MAA)-specific mouse mAb CGP 76873 produced by the hybridoma 763.74. The five HMW-MAA-specific anti-anti-idiotypic mAbs GH 464, GH 518, GH 149, GH 386 and GH 586 were generated from mice immunization with mAb CGP 60686. All interactions between the anti-idiotypic mAb and the idiotypic mAb or the anti-anti-idiotypic mAb showed large exothermic binding enthalpies between -15 and -23 kcal/mol and binding affinities larger than 6 x 10(9) M-1. Four of the five anti-anti-idiotypic mAbs tested exhibited significantly higher binding enthalpies for the interaction with the anti-idiotypic than the idiotypic mAbs. Replacement of either the heavy or the light chain variable region of the anti-idiotypic mAbs with an unrelated variable region abolished the idiotype to anti-idiotype interaction. Thus, both the heavy and the light chain variable region of the anti-idiotypic mAbs are required for binding to the idiotype. The values of the binding enthalpy showed only small variations when binding of the idiotypic mAb CGP 76873 to four variants of the anti-idiotypic mAb CGP 60686 with different immunoglobulin constant regions, but identical variable regions were compared. Furthermore, Fab fragments of the idiotypic mAbs showed almost the same binding enthalpy per binding site as the whole IgG molecules. Immunoglobulin constant regions thus had little influence on the idiotype to anti-idiotype interactions. Taken together, the observed thermodynamic parameters suggest that the idiotype to anti-idiotype interactions studied here are enthalpy-driven processes with only minor entropic contributions. High sensitivity titration calorimetry was used to monitor protein-protein interactions within an anti-idiotypic antibody cascade. It was found that the direct measurement of the interaction enthalpy allowed a quantitative characterization of the binding processes studied.

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.

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.

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.

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.

Oligoclonality and skewed T cell receptor V beta gene segment expression in in vivo activated human intestinal intraepithelial T lymphocytes.

Intraepithelial intestinal T lymphocytes (IEL) bearing alpha beta or gamma delta T cell receptors (TCR) are positioned to serve as a first line of defense against enteric pathogens. To investigate whether intestinal IEL are subject to antigenic selective forces distinct from that influencing (xp T cells in the peripheral blood (PBL), we performed a comparative analysis of V beta gene segment usage in IEL and PBL of immunologically normal donors by quantitative PCR. Primers for 22 different human TCR V beta gene segments of V beta gene segments families were used to analyze the repertoire of TCR beta chain transcripts in colonic IEL (c-IEL), in corresponding colonic lamina propria lymphocytes (c-LPL), and in peripheral blood lymphocytes. In each of the three individuals examined, a limited number (1-4 out of 22) of TCR V beta families predominated and accounted for more than 50% of the total beta chain transcripts from c-IEL, whereas in PBL and c-LPL a more even distribution of V beta gene families was observed. The dominating V beta gene families were V beta 2, V beta 3, V beta 6, V beta 8 and V beta 14. In one individual, V beta 3 comprised more than 40% of the entire repertoire of c-IEL beta chain transcripts. Sequence analysis of the predominant V beta 3 family in this individual revealed identical sequences in 13 of 17 clones analyzed. Human alpha beta TCR+ c-IEL could not be driven to proliferate or exhibit cytotoxic function in vitro however, PCR analysis for detection of lymphokine mRNA revealed constitutive production of several lymphokines known to exert trophic effects on intestinal epithelial cells and pro-inflammatory activities. Taken together, the striking degree of oligoclonality may indicate that the intraepithelial intestinal immune system is targeted to a limited set of hitherto unknown self- or foreign antigens present in the intestine and orchestrates intramucosal inflammatory and regenerative processes.

Analysis of T cell receptor V beta regions expressed by rheumatoid synovial T lymphocytes.

The T cell receptor (TCR) V beta gene segment repertoire of T lymphocytes derived from peripheral blood of two healthy individuals and synovial tissue, synovial fluid and peripheral blood of three rheumatoid arthritis (RA) patients was analyzed. A sensitive assay based on the amplification of cDNA by the polymerase chain reaction (PCR) was used to analyze the levels of expression of 20 TCR V beta gene segment families. The relative expression of V beta gene segments in lymphocytes derived from peripheral blood, synovial tissue and synovial fluid was conserved over 155 days in one patient. V beta 9 transcripts were undetectable in the cells of this individual. In the two other patients the frequency of V beta 2 transcripts in synovial T cells of affected joints was significantly higher than in their peripheral blood lymphocytes. Dominance of distinct rearrangements among the V beta 2 transcripts from the synovial cells of these patients support the idea that the synovial T cell response is driven by antigen.

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.

Risk factors for meningococcal meningitis in northern Ghana.

Meningococcal meningitis is a major cause of morbidity and mortality in the meningitis belt of sub-Saharan Africa where it occurs in epidemics every 8-12 years. Risk factors for the disease in this setting remain largely unknown. We carried out a case-control study to investigate possible risk factors among survivors of a meningitis epidemic occurring in 1997 in northern Ghana. A structured questionnaire on socio-economic factors, housing and household overcrowding, smoking and exposure to smoke and close contact with a case was administered to 505 of the survivors and 505 of age-, sex- and location-matched controls. Cooking in kitchens with firewood stoves (OR 9.00, CI 1.25-395) and sharing a bedroom with a case (OR 2.18 CI 1.43-3.4) were found to be risk factors for disease. Socio-economic factors, overcrowding, smoking and passive exposure to tobacco smoke were not found to be risk factors. Exposure to smoke from cooking fires or close contact with a case puts people at risk of contracting meningococcal meningitis. In the hot dry months, exposure to smoke from cooking fires should be minimized by encouraging alternatives to cooking over wood fires, or cooking outside. If wood-burning stoves cannot be avoided, kitchens should be made larger with improved ventilation. Meningitis cases should be nursed in well-ventilated rooms and the number of people sharing a room with a case kept at a minimum.

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.

Application of genetically-engineered anti-CEA antibodies for potential immunotherapy of colorectal cancer.

Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors, and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for "humanization" of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or used for therapeutic strategies which similarly take advantage of the ability of the antibodies to target cytotoxic agents selectively to tumor cells. The application of these novel procedures for manipulating MAb structure presents entirely new opportunities for diagnosis and treatment of human colorectal cancer.

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.