Seroprotection rate in adults after 1-dose, 2-dose, and 3-dose series of a reduced-diphtheria-tetanus toxoid vaccine (Td) during a diphtheria outbreak in Thailand.

To evaluate seroprotection of different dosing strategies of reduced-diphtheria-tetanus-toxoid vaccine (Td) for adults during a diphtheria outbreak in Thailand, we enrolled 160 healthcare workers and 161 adults aged 20-60 years old and measured diphtheria antitoxin (DAT) level before administration of a Td vaccine. We scheduled a second Td at 4-8 weeks and a third Td at 6-12 months interval. DAT was measured 4 weeks after each dose. DAT levels of ≥0.1 and ≥1 IU/mL were considered as seroprotective and long-term seroprotective. Persons achieving long-term seroprotection were not given a further dose. The baseline seroprotection rate was 32.6%, which increased to 87.1% (95% confidence interval, 83.4-90.8%) after one dose. The seroprotection rate increased slightly with additional doses. The immune response was lowest among persons 30-49 years of age. We suggest 1-dose Td for adults during a diphtheria outbreak, and a 2-dose series being considered for those born before 1980.

SLC39A4 mutation in zinc deficiency patients.

OBJECTIVE: To analyze the clinical presentation and SLC39A4 mutations in zinc deficiency patients. MATERIAL AND METHOD: The authors conducted a cross-sectional study on all cases of zinc deficiency treated at Queen Sirikit National Institute of Child Health between January 2004 and December 2012. Demographic data, clinical manifestations, laboratory results, treatment and outcome were analyzed. Genetic, SLC39A4 for acrodermatitis enteropathic (AE), mutation analysis was performed in all cases. RESULTS: There were 15 cases, 10 males and 5 females. The age of onset was between 2 and 10 months (median 3 months). Duration of the disease ranged between 3 days and 17 months (median 2 months). Acral and periorificial dermatitis, diarrhea and alopecia were present in 15 cases (100%), 12 cases (80%) and 8 cases (53%) respectively. The characteristic triad of acral and periorificial dermatitis, diarrhea and alopecia was observed in only 6 patients (40%). Serum zinc level ranged between 10 and 111 mcg/dl (mean 49.69 ± 33.87 mcg/100 ml). Low serum zinc level was observed in 10 cases (67%). All of the patients were treated with zinc sulfate 5 mg/kg/day. All cutaneous lesions and diarrhea had resolved within 7 days of starting therapy. A genetic study of SLC39A4 gene in our 15 patients revealed that 3 patients had homozygous c.1878_1879ins21 (p.G627_T633dup) in exonl2. These three patients have to receive lifelong zinc supplementation to prevent recurrence of the disease. The other twelve patients, who did not carry the gene mutation, did not have symptoms after discontinuance of oral zinc therapy. This is the first report of genetically confirmed acrodermatitis enteropathic in Thailand. CONCLUSION: Acrodermatitis enteropathica is a rare disease, which needs lifelong zinc supplementation. A genetic study of SLC39A4 gene will confirm the diagnosis. Most of patients presenting with characteristic triad of acral and periorificial dermatitis, diarrhea and alopecia in Thailand were acquired zinc deficiency. Early recognition and treatment of the disease will decrease morbidity and mortality.

Molecular analysis of factor XII gene in Thai patients with factor XII deficiency.

Factor XII is the initiating protein of the intrinsic coagulation pathway. It activates factor X after being activated by the so called 'contact system'. Both congenital factor XII deficiency, usually without bleeding symptoms, and several factor XII polymorphisms with possible thrombotic tendency have been described. In the presented work, two Thai patients with congenital factor XII deficiency have been studied by utilizing a PCR single-stranded conformation polymorphism (PCR-SSCP) method followed by direct sequencing. A new mutation of factor XII gene in the exon 7, c.583delC or p.H195fsX250, has been discovered and variable factor activities resulting from different mutations with or without polymorphisms are demonstrated. The other case had a homozygous missense mutation, c.G218C. Heterozygotes of this mutation, found in 1.9% (2/107) of healthy Thai volunteers, showed low factor XII activities suggesting that it is a deleterious mutation.

Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation.

Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC50) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a better treatment for viper bites.

Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain.

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell-matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5'-RACE, the 2040bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3mg/L of culture medium. The molecular weights were 11kDa in reduced and 22kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC(50) of 976nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.