Genomic analysis of KEL*03 and KEL*04 alleles among Thai blood donors.

Background: The Kell blood group system is clinically important in transfusion medicine, particularly in patients with antibodies specific to Kell antigens. To date, genetic variations of the Kell metallo-endopeptidase (KEL) gene among Thai populations remain unknown. Objective: This study aimed to determine the frequencies of KEL*03 and KEL*04 alleles among Thai blood donors using an in-house polymerase chain reaction-sequence-specific primer (PCR-SSP) method. Methods: Blood samples obtained from 805 unrelated central Thai blood donors at a blood bank in Pathumthani, Thailand, from March 2023 to June 2023, were typed for Kpa and Kpb antigens using the column agglutination test, and the results for 400 samples were confirmed using DNA sequencing. A PCR-SSP method was developed to detect the KEL*03 and KEL*04 alleles, and genotyping results were validated using known DNA controls. DNA samples obtained from Thai donors in central (n = 2529), northern (n = 300), and southern (n = 427) Thailand were also genotyped using PCR-SSP for comparison. Results: All 805 (100%) donors had the Kp(a-b+) phenotype. The PCR-SSP genotyping results agreed with the column agglutination test and DNA sequencing. All 3256 Thai blood donors had the homozygous KEL*04/KEL*04 genotype. Frequencies of the KEL*03 and KEL*04 alleles among Thai donors differed significantly from those of Japanese, Native American, South African, Brazilian, Swiss, and German populations. Conclusion: This study found a 100% KEL*04 allele frequency in three Thai populations. These data could provide information on KEL*03 and KEL*04 allele frequencies to estimate the risk of alloimmunisation in Thai populations. What this study adds: This study demonstrates that in-house PCR-SSP can be used to determine KEL*03 and KEL*04 alleles to predict Kpa and Kpb antigens. Even though only homozygous KEL*04/KEL*04 genotypes were found among Thai donor populations, the established PCR-SSP method may be useful for estimating the risk of alloimmunisation in other populations.

Production and immunological characterization of the novel single-chain variable fragment (scFv) antibodies against the epitopes on Opisthorchis viverrini cathepsin F (OvCatF).

BACKGROUND: Opisthorchis viverrini infection is a significant health problem in several countries, especially Southeast Asia. The infection causes acute gastro-hepatic symptoms and also long-term infection leading to carcinogenesis of an aggressive bile duct cancer (cholangiocarcinoma; CCA). Hence, the early diagnosis of O. viverrini infection could be the way out of this situation. Still, stool examination by microscopic-based methods, the current diagnostic procedure is restricted by low parasite egg numbers in the specimen and unprofessional laboratorians. The immunological procedure provides a better chance for diagnosis of the infection. Hence, this study aims to produce single-chain variable fragment (scFv) antibodies for use as a diagnostic tool for O. viverrini infection. METHODS: This study uses phage display technologies to develop the scFv antibodies against O. viverrini cathepsin F (OvCatF). The OvCatF-deduced amino acid sequence was analyzed and predicted for B-cell epitopes used for short peptide synthesis. The synthetic peptides were used to screen the phage library simultaneously with OvCatF recombinant protein (rOvCatF). The potentiated phages were collected, rescued, and reassembled in XL1-blue Escherichia coli (E. coli) as a propagative host. The positive clones of phagemids were isolated, and the single-chain variable (scFv) fragments were sequenced, computationally predicted, and molecular docked. The complete scFv fragments were digested from the phagemid, subcloned into the pOPE101 expression vector, and expressed in XL1-blue E. coli. Indirect ELISA and Western analysis were used to verify the detection efficiency. RESULTS: The scFv phages specific to OvCatF were successfully isolated, subcloned, and produced as a recombinant protein. The recombinant scFv antibodies were purified and refolded to make functional scFv. The evaluation of specific recognition of the particular epitopes and detection limit results by both computational and laboratory performances demonstrated that all three recombinant scFv antibodies against OvCatF could bind specifically to rOvCatF, and the lowest detection concentration in this study was only one hundred nanograms. CONCLUSION: Our produced scFv antibodies will be the potential candidates for developing a practical diagnostic procedure for O. viverrini infection in humans in the future.

Characterization of GYP(B-A-B) hybrid glycophorins among Thai blood donors with Mia-positive phenotypes.

BACKGROUND: GYPA and GYPB genes encode the antigens of the MNS blood group system carried on glycophorin A (GPA) and glycophorin B (GPB), or on a hybrid molecule of GPA and GPB. GP hybrid variants are created through unequal crossing over and gene conversion, typically from the parent genes GYPA and GYPB. In the present study, we characterized the GYP(B-A-B) hybrid variants among Thai blood donors with Mia-positive phenotypes using PCR-based coupled to DNA sequencing techniques. MATERIALS AND METHODS: Altogether, 1,020 samples from Thai blood donors were tested with anti-Mia by conventional tube technique (CTT). Polymerase chain reaction with sequence-specific primer (PCR-SSP) was initially used to differentiate normal GYPB, GYP*Vw and groups of GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun and GYP*HF alleles. Subsequently, GYP(B-A-B) hybrid variants were investigated using DNA sequencing. RESULTS: Among 1,020 blood donors, 127 (12.45%) were Mi(a+) phenotypes. The comparison Mia typing results between CTT and PCR-SSP were concordant. All Mi(a+) samples were positive with only group of GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun and GYP*HF alleles by PCR-SSP. Regarding the sequencing results, 115/1,020 (11.27%) donors carried the GYP*Mur, of which 111/1,020 (10.88%) were GYP*Mur/GYPB heterozygotes and the other 4/1,020 (0.39%) donors were GYP*Mur/GYP*Mur homozygotes. The remaining 12 donors included different GYP*Bun-like alleles; 11 of them (1.08%) were GYP*Thai/GYPB heterozygotes, and one (0.10%) was GYP*Thai II/GYPB heterozygotes. With 5.83% (119/2,040) of the total hybrid alleles, GYP*Mur was the predominant allele. The GYP*HF, GYP*Bun, GYP*Hop and GYP*Kip alleles were not observed in this study. DISCUSSION: Regarding the hybrid GP variants, a consensus of observed prevalent GYP*Mur and GYP*Bun-like alleles, respectively, was identified in the Thai population. The introduction of our strategy has allowed us to identify the zygosity for GYP hybrid variants, particularly GYP(B-A-B) hybrid genes, when antisera are unavailable and lacking adequate phenotypic features to determine GP variants.

Genotyping Approach to Predict Coa and Cob Antigens in Thai Blood Donor Populations.

Purpose: Coa and Cob antigens of the Colton (CO) blood group system are implicated in acute and delayed hemolytic transfusion reactions (HTRs). Owing to the inadequate supply of specific antiserum, data on CO phenotypes remain limited. This study aimed to develop genotyping methods to predict Coa and Cob antigens and to estimate transfusion-induced alloimmunization risks in three Thai blood donor populations. Materials and Methods: The study included 2451 blood samples from unrelated healthy Thai blood donors obtained from central, northern, and southern Thailand. DNA sequencing was used to determine the CO*A and CO*B alleles. In-house PCR with sequence-specific primers (PCR-SSP) and high-resolution melting curve (HRM) assays were performed and genotyping results were compared using DNA sequencing. CO*A and CO*B allele frequencies among Thais were determined using PCR-SSP and their frequencies were compared with other populations. The risks of Coa and Cob transfusion-induced alloimmunization among Thai donor populations were calculated. Results: The validated genotyping results by PCR-SSP and HRM assays agreed with DNA sequencing. The CO*A/CO*A was the most common (100.0, 100.0, and 99.3%), followed by CO*A/CO*B (0.0, 0.0, and 0.7%) among central, northern and southern Thais. Homozygous CO*B/CO*B was not found. The CO*A and CO*B allele frequencies among central Thais significantly differed compared among southern Thais (p < 0.01) but not among northern Thais. Those allele frequencies among Thais were similar to those of Taiwanese, Chinese and Malay-Malaysian populations but not to South Asian, Southeast Asian, Korean, Japanese, Filipino, French Basque, and Maltese populations (p < 0.01). A higher risk of anti-Cob production rather than anti-Coa production was particularly noted in the southern Thai population. Conclusion: This study constitutes the first to determine CO*A and CO*B genotypes using PCR-SSP and HRM assays among Thais and this finding would be beneficial in predicting alloimmunization risk and providing safe transfusions among Thais.

Evaluation of Rhophilin Associated Tail Protein (ROPN1L) in the Human Liver Fluke Opisthorchis viverrini for Diagnostic Approach.

Tegumental and excretory-secretory proteins are reported as diagnostic antigens for human opisthorchiasis. Rhophilin associated tail protein1-like (OvROPN1L) protein of Opisthorchis viverrini sperm tail showed potential as a diagnostic antigen. The OvROPN1L recombinant fragments were assayed for diagnostic antigenicity for human opisthorchiasis using indirect ELISA. The strongest antigenic region was a N-terminus peptide of M1 - P56. One synthetic peptide (P1, L3-Q13) of this region showed the highest antigenicity to opisthorchiasis. Sera from other parasitic infections including Strongyloides stercoralis, hookworm, Taenia spp, minute intestinal flukes, Paragonimus spp showed lower reactivity to P1. Peptide P1 is located in the disordered N-terminus of ROPN1L supporting its suitability as linear epitope. In the Platyhelminthes the N-terminal sequence of ROPN1L is diverging with taxonomic distance further suggesting that peptide P1 has potential as diagnostic tool in the genus Opisthorchis/Clonorchis. It should be further evaluated in combination with peptides derived from other O. viverrini antigens to increase its diagnostic power.

Evaluation of Opisthorchis viverrini calreticulin for potential host modulation.

The multifunctional calreticulin (CALR) was identified as a major calcium-binding protein of the endoplasmic reticulum before being recognized as a chaperone in the same place. Only later were activities of calreticulin outside the endoplasmic reticulum described that for example affect cell proliferation and the innate immune system. In the present work we have investigated those extracellular activities of CALR from the cancerogenic human liver fluke Opisthorchis viverrini (OvCALR), as they might be important in host/parasite interaction. We first demonstrate that OvCALR is released from the parasite and stimulates a specific humoral immune response. Recombinant OvCALR is then shown to suppress proliferation of primary endothelial cells, their motility and sprouting activities. The potential of OvCALR to interfere with the complement system is established, firstly by demonstrating its direct binding to C1q and, secondly by suppression of hemolysis of sensitized red blood cells. These findings suggest that OvCALR is an important parasite antigen that could modulate diverse host functions and support parasite survival.

Molecular and Biochemical Characterization of Opisthorchis viverrini Calreticulin.

Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P-, and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion, and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed, and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehlis' gland, prostate gland, and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed.