Discovery of two novel HLA-B alleles, B*46:13:03 and B*15:189, in two Taiwanese volunteer bone marrow donors by sequence-based typing.

We report here two novel HLA-B alleles, B*46:13:03 and B*15:189, discovered in two Taiwanese volunteer bone marrow donors. The sequence of B*15:189 has a nucleotide sequence possibly derived from a recombination event between HLA-B*39:01:01 and B*15:01:01:01, while the origin of the sequence B*46:13:03 was less obvious to postulate, considering the low frequency of B*46:13 in the general population and the silent mutations involved. Our report here adds further HLA polymorphism to the growing lists of HLA-B*46 and HLA-B*15 and provides an additional HLA information for donor search programme for patients undergoing transplant.

Oriental HLA-A*11:90 detected in a Taiwanese cord blood sample and the haplotype in association with A*11:90 allele.

We report here an HLA-A allele, A*11:90, found in a Taiwanese cord blood sample using DNA sequence-based typing (SBT) protocol after observing an anomalous reaction pattern in a sequence-specific oligonucleotide (SSO) typing exercise. The sequence of A*11:90 is identical to A*11:01:01, the most predominant A*11 variant in Taiwanese, in exon 2 but differs from A*11:01:01 in exon 3 by two nucleotide substitutions at codon 163 (c.487C>G and c.488G>A), resulting R163E. In comparison with the sequence of A*11:02:01, the second most predominant subtype of A*11 in Taiwanese A*11:90 has one nucleotide difference at codon 19 (c.55A>G) in exon 2 resulting K19E and two nucleotides variations at codon 163 (c.487C>G and c.488G>A) in exon 3 resulting R163E. HLA-A*11:90-B*40:02-DRB1*11:01 is the deduced probable HLA haplotype in association with A*11:90. The generation of A*11:90 is thought to involve a DNA recombination event between alleles A*11:01:01 and A*80:01 where A*80:01 donated a fragment of the DNA sequence (from n.t. 487 to n.t. 497) to the recipient sequence of A*11:01:01.

Detection of two HLA-A alleles, A*31:30 and A*26:20, in two Taiwanese volunteer bone marrow donors by sequence-based typing.

We here report detection of a novel sequence of HLA-A*31:30 and a confirmatory sequence of HLA*26:20 from two Taiwanese individuals. The sequence of A*31:30 is identical to that of A*31:01:02 in exons 2 and 3, except one nucleotide (n.t.) substitution c.539T > G resulting in p.Leu180Trp. The sequence of A*26:20 is identical to A*26:01:01 in exons 2 and 3, except a segment of the sequence from n.t. 78 to n.t.102. The mismatched sequence segment is identical to a sequence segment of A*02:03:01, suggesting that the formation of A*26:20 was resulted from a DNA recombination event between A*26:01:01 and A*02:03:01 sequences. A*26:20 differs from A*26:01:01 with c.98A > T resulting in p.Tyr33Phe.

Identification of two novel HLA-B*40 alleles, B*40:137 and B*40:158, in Taiwanese individuals.

Using sequence-based typing method we discovered two new HLA-B*40 variants, B*40:137 and B*40:158, in Taiwanese individuals. The sequence of B*40:137 has three nucleotide (nt) changes from B*40:21 at nt 353 (C→T), nt 355 (C→A) and nt 369 (C→T) resulting two coding changes at residue 94 (T→I) and residue 95 (L→I), whereas the sequence of B*40:158 differs from B*40:01:01 with five nt substitutes at nt 463 (C→A), nt 477 (C→G), nt 499 (T→A), nt 512 (T→G) and nt 527 (T→A) causing five amino acid exchanges at codons 140 (Y→S), 155 (R→S), 168 (S→T), 171 (L→W) and 179 (V→E). Our hypotheses on the generation of the two novel alleles are presented.

Identification of a novel HLA-A allele, A*11:60, in a Taiwanese family.

We report the identification and sequence analysis of a new HLA-A11* variant, A*11:60 allele, found in a Taiwanese leukaemic patient and his siblings. The novel A*11 variant is identical to A*11:03 in exon 2 but differs from A*11:03 in exon 3 by one nucleotide substitution at position 527 (A→T) causing an amino acid change at codon 152 E (Glu)→V (Val) (GAG→GTG). In comparison with HLA-A*11:01:01, allele A*11:60 has two nucleotide differences in exon 3: at nt 524 (A→G) (CAT→CGT) and at nt 527 (C→T) (GCG→GTG) leading to two amino acid variations at residues 151 H (His)→R (Arg) and 152 A (Ala)→V (Val).

Rare HLA alleles and their predicted haplotypes in Tzu Chi Taiwanese marrow donor registry.

The marrow donor registry of Buddhist Tzu Chi Stem Cells Centre (BTCSCC) maintained an HLA database of 291677 bone marrow volunteer donors from 1993 to 2008. From the 291677 donors we identified 28 rare alleles and discovered six novel alleles in Taiwanese. Our criterion of a rare allele was according to the definition, set by the National Marrow Donor Program (NMDP), one having a frequency of 0.002% (1/50,000) or less. The authenticity of the rare alleles was confirmed by sequence specific primer (SSP) typing protocol and/or sequence-based typing (SBT) method. This study reports the number of times the alleles were observed between 1993 and 2008, and the ethnicity of the donors carrying the rare or novel alleles.

Identification of a novel HLA-DQB1 allele, DQB1*0326, in a College of American Pathologists 2009 survey specimen.

We identified a novel DQB1*0326 allele from a proficiency test sample provided by the College of American Pathologists (CAP) medical society. This novel DQB1 allele was unexpectedly discovered by sequence-based typing method in an attempt to resolve a discrepant typing result between the CAP survey report and our laboratory report. This novel DQB1 allele is most similar to DQB1*030302 and DQB1*0311. DQB1*0326 has a nucleotide substitution resulting an amino acid change when compared with DQB1*030302 (M to L) and it differs from DQB1*0311 by one nucleotide variation causing an amino acid replacement (A to D).

A novel HLA-B allele, B*5214, detected in a Taiwanese volunteer bone marrow donor using a sequence-based typing method.

HLA-B*5214, a novel rare allele of HLA-B*52 variant, was found in a Taiwanese volunteer bone marrow donor by sequence-based typing method. The sequence of B*5214 is identical to that of B*520101 in exon 2 but differs from B*520101 in exon 3 at nucleotide positions 419 A-->T and 435 A-->G. Alteration of these two nucleotides resulted an amino acid substitution at amino acid residue 116 Y-->F ( TAC-->TTC) and a silent exchange at residue 121 K-->K (AAA-->AAG).

Detection of a rare Caucasoid HLA-DRB1*0337 in a Taiwanese bone marrow donor using sequence-based typing method.

We here describe the identification of HLA-DRB1*0337, using sequence-based typing (SBT) method, in a Taiwanese bone marrow donor intrigued by a casual curiosity on the donor's racial background. On high-resolution sequence-specific primer (SSP) typing we observed misleading reaction patterns due to similarity of DNA sequences in the exon 2 of DRB1*0301, *0317, *0337 and *1139. When encountering rare alleles in HLA typing, it is important to pay extra attentions to avoid pitfalls and shortcomings of SSP typing kits routinely used and bear in mind that constant up-dating of high-resolution SSP typing kits to be able to distinguish newly reported alleles. SBT may be considered as a back-up HLA typing method to confirm rare alleles.

Unrelated haematopoietic stem cell transplantation in Taiwan and beyond.

Since its inception in October 1993, the world-renowned Buddhist Tzu Chi Marrow Donor Registry has facilitated more than 1800 cases of stem cell donations for patients in 27 countries to date. Under the auspices of the Buddhist Tzu Chi Stem Cells Center (BTCSCC), the Registry (> 310,000 donors) offers, on average, one case of stem cell donation every day to national or international transplantation community. The accomplishment of the Registry stems from the philosophy and spirit of giving without reward that was inspired by its founder Dharma Master Cheng Yen, the Samaritan devotions of selfless voluntary stem cell donors and the efforts from a dedicated network of volunteer workers. Demographically speaking, slightly less than one third of the donations are provided to domestic patients and the rest to mainland China and countries in Asia, North America, Europe, Middle East, Oceania, and South Africa. While most of the patients belong to the Oriental ethnic group, a few of the patients are non-Oriental. In addition to the Registry, a non-profit umbilical cord blood (UCB) bank is operating since 2002 to provide a complimentary role for patients unable to identify appropriate bone marrow stem cell donors in the Registry in time. To date, with an inventory of over 12,000 units of UCB cryopreserved in the Tzu Chi Cord Blood Bank, 47 units have been employed in 37 cases of transplantation for both paediatric and adult patients domestically and internationally. The fact that Buddhist Tzu Chi Marrow Donor Registry and Cord Blood Bank are established and operating without governmental financial support is unique and special. To facilitate haematopoietic stem cells to its domestic patients experiencing financial burdens, the BTCSCC offers financial aids to the underprivileged for their medical relief. This humanitarian approach and compassion is definitely a role model for many countries in the world.

Detection of a novel HLA-B27 allele, B*2740, in Taiwanese volunteer bone marrow donors by sequence-based typing: curiosity rewarded.

We report here a novel HLA-B allele, B*2740, discovered in Taiwanese volunteer marrow donors. The new sequence has nucleotide variation at position 527 (T-->A) as compared to B*2708. The nucleotide change caused an amino acid substitution from valine (V) to glutamic acid (E) at codon 152. Since B*2740 carries sequence confers to HLA-Bw6 public epitope we believe that this novel B*27 allele might have been generated from a gene conversion involving a Bw4-specific allele (probably B*2704) and a Bw6-specific allele.

New allele name of some HLA-DRB1*1401: HLA-DRB1*1454.

The primary function of MHC polymorphism is considered as the foundation of self-defense mechanism of the host in surveillance against countless diverse invading pathogens. However, this biological function can also elicit undesirable immunological responses that jeopardize transplantations when compatibility between donors and recipients is unfavourable.

Identification of a novel HLA-A allele, A*1131, in a Taiwanese.

Here we report the identification and sequence analysis of a new HLA-A11* variant, A*1131 allele, found in a Taiwanese volunteer bone marrow donor. The novel A*11 variant is identical to A*1125 in exon 2 but differs from A*1125 in exon 3 by one nucleotide substitution at position 527 causing an amino acid change at codon 152 E-->V (GAG-->GTG). In comparison with HLA-A*110101, allele A*1131 has three nucleotide differences in exon 3: 527 C-->T, 538 C-->T and 539 A-->T leading to two amino acid variations at residues 152 A-->V and 156 Q-->L.

Dynamic CT perfusion imaging with acetazolamide challenge for evaluation of patients with unilateral cerebrovascular steno-occlusive disease.

BACKGROUND AND PURPOSE: Perfusion CT (PCT) has the ability to measure quantitative values and produce maps of cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT). We assessed cerebral hemodynamics by using these parameters and acetazolamide challenge in patients with cerebrovascular steno-occlusive disease. METHODS: Fifteen patients underwent PCT with acetazolamide challenge. Comparison of mean CBF, CBV, and MTT was determined between hemispheres and before and after acetazolamide challenge. Hemispheric ratio and percent change due to acetazolamide administration were also calculated. Absolute values and percent changes 2 SDs outside the mean from the nonstenotic hemispheres were defined as abnormal. RESULTS: Significant decreases in CBF (-25.1%, P = .003) and significant increases in MTT (47.1%, P < .001) were found in stenotic hemispheres. After acetazolamide challenge, significant changes in CBF (-39.5%, P < .001) and MTT (92.9%, P < .001) were also seen. The acetazolamide test significantly decreased CBF hemispheric ratio (-20.3%, P < .001) and increased MTT hemispheric ratio (30.8%, P = .002), making both maps more asymmetric. Significance in CBF and MTT percent changes (P < .001 and P = .005, respectively) was found between hemispheres. When CBF percent changes were assumed to represent the true determinant of hemodynamic impairment, normal ranges of baseline MTT value and MTT percent changes demonstrated sensitivities of 66.7% and 100% and specificities of 58.3% and 75%, respectively, for detecting patients with hemodynamic impairment. CONCLUSION: Parameters obtained from PCT with acetazolamide are promising for the evaluation of cerebral hemodynamics in patients with cerebrovascular steno-occlusive disease.

The intubating laryngeal mask airway in severe ankylosing spondylitis.

PURPOSE: To evaluate the use of inhalational induction followed by intubation through the intubating laryngeal mask (ILM) for patients with severe ankylosing spondylitis undergoing elective surgery who prefer airway management under anesthesia. METHODS: Nine patients undergoing a total of 11 procedures were enrolled in the study. Fentanyl 2 microg*kg(-1), midazolam 0.035 mg*kg(-1) and sevoflurane in oxygen 100% were used for induction. The ILM was inserted when the end-tidal sevoflurane concentration reached 3%. After an effective airway was established, atracurium 0.5 mg*kg(-1) was given. A polyvinyl chloride tube in the reversed position using a blind technique was used to intubate the trachea. RESULTS: The ILM provided an effective airway on 11/11 occasions at the first attempt. Intubation was successful at the first attempt on 7/11 occasions, at the second attempt on 2/11 and at the third attempt in 1/11. Intubation failed in one patient. The mean (range) minimal oxygen saturation was 99.4% (97-100%). There were no problems with ILM removal. CONCLUSION: Inhalational induction followed by ILM insertion and blind intubation is a reasonable option in patients with severe ankylosing spondylitis undergoing elective surgery who prefer airway management under anesthesia.

Hemodynamic responses of thiopental and propofol in different-aged patients during endotracheal intubation.

BACKGROUND: Thiopental and propofol are 2 popularly used anesthetic induction agents that have different pharmacological and pharmacokinetic actions. It is not clear how the hemodynamic responses differ in different-aged patients using these 2 drugs for anesthetic induction. The aim of this study was to investigate the hemodynamic responses to propofol and thiopental of different-aged patients during endotracheal intubation. METHODS: Sixty patients, 20 to 83 years old, were included in this study. Patients were randomly divided into 6 groups according to age and different inductive anesthetics. Non-invasive blood pressure (NIBP) monitoring was performed every minute before anesthesia, during anesthetic induction with 5 mg.kg-1 thiopental or 2 mg.kg-1 propofol intravenously, and after intubation. RESULTS: Propofol induction produced a smaller mean arterial pressure increase immediately after intubation than did thiopental induction in adult and elderly groups (101.0 +/- 18.5 and 104.0 +/- 21.1 mmHg in the propofol groups vs. 138.3 +/- 17.1 and 138.9 +/- 16.1 mmHg in the thiopental groups at 1 minute after intubation, p < 0.001 and p = 0.001, respectively). Differences in heart rate between propofol and thiopental groups were found in young and adult groups (85.1 +/- 9.2 and 81.7 +/- 10.9 in the propofol groups vs. 94.5 +/- 9.9 and 95.0 +/- 14.0 beats per minute in the thiopental groups at 1 minute after intubation, p = 0.041 and p = 0.029, respectively). CONCLUSION: Propofol induction produced more stable hemodynamics after intubation in adult and elderly patients than did thiopental induction. Therefore, it is safer to use propofol for induction and intubation in adult and elderly patients.

Simultaneous determination of unbound ropivacaine in rat blood and brain using microdialysis.

To investigate the pharmacokinetics of ropivacaine in rat blood and brain, a sensitive HPLC method and microdialysis were developed for the simultaneous determination of unbound ropivacaine in rat blood and brain. Adult, male Sprague-Dawley rats (290-350 g) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Two microdialysis probes were inserted, one into the jugular vein toward right atrium, and one into the brain striatum of rats. Ropivacaine (5 mg/kg, i.v.) was then administered via the femoral vein. Blood and brain dialysates were collected and eluted with a mobile phase containing methanol-acetonitrite-20 mM monosodium phosphoric acid (pH 5.5) (10:40:50, v/v/v) in a liquid chromatographic system. Separation of ropivacaine was achieved by a CN column (Phenomenex Luna, 250x4.6 mm, particle size 5 microm; Torrance, CA, USA) within 10 min. The UV detector wavelength was set at 205 nm and the detection limit of ropivacaine was 20 ng/ml. The intra- and inter-day accuracy and precision of the analyses were less than 10% in the ranges of 0.02-5 microg/ml. The pharmacokinetic data were calculated from the individual animal measurements of dialysate concentration versus time. This method exhibits no endogenous interference and its sensitivity is sufficient for the determination of biological samples. The present results confirm that microdialysis sampling followed by LC separation with UV detection represents a viable approach for the measurement of free ropivacaine in rat brain and plasma.

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells.

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.