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-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).

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.

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.

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.

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 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.

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).

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).

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.

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.

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.

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.

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.

Leukocyte-endothelial adherence correlates with pancreatic nitric oxide production in early cerulein-induced pancreatitis in rats.

The role of nitric oxide (NO) in microcirculation during the development of acute pancreatitis was not clear. An in vivo microscopic technique was used for evaluating leukocyte-endothelial adherence in the pancreatic microcirculation after induction (cerulein) of acute pancreatitis. Microdialysis was performed to detect pancreatic nitrate concentration (NO level) by high-performance liquid chromatography. Cerulein caused significantly reduced flow velocity in 1 h (31 %) and increased the number of sticking leukocytes in 2 h; both persisted for at least 3 h. Pancreatic NO level was found to be significantly elevated (2.5-fold) in 1 h and also persisted for 3 h. Both microcirculatory changes and NO elevation were significantly alleviated in cerulein-induced animals pretreated with NO synthase inhibitor (NG-nitro-L-arginine), indicating that elevation of NO could precede and account for a major portion of the observed microcirculatory changes. Furthermore, there was a strong positive correlation between numbers of adherent leukocytes and pancreatic NO level, suggesting that during the development of acute pancreatitis, NO could play an adverse role in microcirculation.

Spinal nitric oxide participates in the control of the blood pressure during graded hemorrhage in the conscious rat.

We sought to evaluate the role of spinal nitric oxide (NO) in the control of blood pressure in the conscious animal and determine its possible participation in the progression of hemorrhagic shock. Adult, male Sprague-Dawley rats were chronically prepared with intrathecal, intravenous, and intra-arterial catheters. We first investigated the role of spinal NO on blood pressure control by intrathecal administration of N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) at 0.37 micromol, 0.74 micromol. or 1.48 micromol. A dose-related increase in blood pressure was observed. We next pretreated animals with intrathecal or intravenous L-NAME at 0.37 micromol and induced the animal to shock by graded hemorrhage. Animals that received vehicle control or intravenous L-NAME had a decrease in blood pressure after 12% of the total circulatory blood volume (TBV) had been removed and developed severe hypotension after 24% TBV was bled. On the other hand, intrathecal pretreatment of L-NAME significantly attenuated the decrease in blood pressure. The blood pressure was maintained until 40% TBV had been withdrawn. We concluded that inhibition of NO synthase, in the spinal cord, increased blood pressure in a dose-dependent manner, and hemorrhagic shock induced by graded hemorrhage may involve an upregulation mechanism of spinal NO synthase in producing severe hypotension in conscious rats.

Power spectral analysis of arterial and central venous pressure signals during graded hemorrhage in anesthetized rats.

Based on simultaneous power spectral analysis of systemic arterial pressure (SAP) and central venous pressure (CVP) signals in rats anesthetized with pentobarbital sodium, we assessed the hypotheses that subtle changes in the SAP spectrum exist during hemorrhagic shock, and that the CVP spectrum is a feasible index for central blood volume during acute graded blood loss. During Stage I hemorrhagic shock seen after reduction in 10% of total blood volume (TBV), there was a significant increase in the power of both the very low frequency (VLF, 0-.25 Hz) and low frequency (LF, .25-.8 Hz) components, along with a moderate decrease in the very high frequency (VHF, 5-9 Hz) component, of SAP signals. Substantial reduction in VLF, LF, and VHF components in the SAP spectrum occurred after a blood loss of 25% of TBV (Stage II), which persisted during Stage III hemorrhagic shock when the withdrawn blood reached 50% of TBV and the mean SAP maintained at 40 mm Hg. The depressed SAP-VLF and SAP-LF components sustained the period of spontaneous recovery and subsequent retransfusion of shed blood, although the power of SAP-VHF component gradually elevated during these two periods. The power of the high-frequency (HF, .8-2.4 Hz) component of SAP signals increased discernibly only during Stage III, became significant on spontaneous recovery, and declined during retransfusion. Although CVP and CVP-VHF component progressively declined, the power of the CVP-HF component manifested a gradual increase that was significantly and reversely correlated with the reduction in TBV. We conclude that differential changes in individual components of the SAP spectrum occur during hemorrhagic shock, and that the CVP-HF component may be a reliable indicator for central blood volume during acute graded blood loss.

Prostacyclin analogue (OP-2507) induces delayed ex vivo neutrophil apoptosis and attenuates reperfusion-induced hepatic microcirculatory derangement in rats.

Leukocyte-endothelial adherence and changes of blood flow in microcirculation are associated with the development of ischemia-reperfusion injury in the liver. Polymorphonuclear neutrophil (PMN) apoptosis is essential to maintain homeostasis and plays a major role in limiting the reperfusion-related systemic effects. This study investigates the effects of a prostacyclin analogue (OP-2507) on hepatic ischemia-reperfusion injury. Adult, male Sprague-Dawley rats were used. Five groups were evaluated: (1) sham-operated control, n = 8; (2) ischemia control (1-h ischemia, 5-h reperfusion), n = 8; (3) intravenous infusion with OP-2507 ([15 cis-14-propylcyclohexyl]-16,17,18,19,20-pentanor-9-deoxy-9alpha,6-ni-trilo-PGF, methyl eater) at a dose of 1 microg/kg/min plus ischemia, n = 8; (4) intravenous infusion with OP-2507 at a dose of 0.1 microg/kg/min plus ischemia, n = 8, and (5) sham-operated control and intravenous infusion with OP-2507 at a dose of 1 microg/kg/min, N =8. Laser-Doppler flowmetry and an in vivo microscopy were used to investigate hepatic microcirculation. PMN apoptosis was quantitated by flow-cytometric labeling of DNA strand breaks. Tissue malondialdehyde and adenosine triphosphate were determined at the end of the experiment. Compared with the ischemia control group, OP-2507 significantly improved harmful insults following ischemia-reperfusion. The changes of mean systemic arterial pressure following ischemia-reperfusion have been significantly attenuated by OP- 2507 at both doses. OP-2507 lessened adherent leukocyte count in the post-sinusoid venules, and improved flow velocity in these areas. OP-2507 at both doses reduced malondialdehyde and increased adenosine triphosphate levels and this effect was dose-related. The activity of delayed ex vivo PMN apoptosis was significantly lower in the ischemia group than that of control and treatment groups. OP-2507 induced the activity of PMN apoptosis and its effect is dose-related, also. The PMN apoptosis activity is strongly correlated with parenchymal damages. This study demonstrates that OP-2507 treatment with ischemia may ameliorate the ischemia-reperfusion injury of the liver in the rat model, and increase spontaneous neutrophil apoptosis ex vivo.

Neutrophil elastase inhibitor (ONO-5046) attenuates reperfusion-induced hepatic microcirculatory derangement, energy depletion and lipid peroxidation in rats.

UNLABELLED: Microcirculatory derangement, energy depletion, and lipid peroxidation are associated with the development of ischemia-reperfusion injury in the liver. This study investigated the effects of a neutrophil elastase inhibitor (ONO-5046) on hepatic ischemia-reperfusion injury. Adult, male Sprague-Dawley rats were divided into four treatment groups: 1) sham-operated control (laparotomy only, no ischemia) and saline injection (1 mL/kg), n = 6; 2) ischemia control (1-h ischemia, 2-h reperfusion) and saline injection (1 mL/kg), n = 6; 3) intravenous injection with ONO-5046 at a dose of 1 mg/kg 5 min before ischemia and immediately after reperfusion plus 1-h ischemia and 2-h reperfusion, n = 6; and 4) intravenous injection with ONO-5046 at a dose of 10 mg/kg 5 min before ischemia and immediately after reperfusion plus 1-h ischemia and 2-h reperfusion, n = 6. A laser-Doppler flowmeter and in vivo microscopy were used to investigate hepatic microcirculation. Tissue malondialdehyde (MDA) and adenosine triphosphate (ATP) levels were determined at the end of the experiment. RESULTS: Compared with ischemia alone, ONO-5046 significantly reduced the extent of microcirculatory and hemodynamic derangement after ischemia-reperfusion. ONO-5046 at both doses significantly attenuated decreases in mean arterial pressure. ONO-5046 lessened adherent leukocyte count and improved flow velocity in the sinusoids and postsinusoidal venules. ONO-5046 at the dose of 10m/kg reduced MDA (1.97 +/- 0.54 micromol/g protein vs. 3.58 +/- 1.21 micromol/g protein in the ischemia and reperfusion group) and increased ATP levels (2.62 +/- 0.19 micromol/g wet wt vs. 0.57 +/- 0.37 pmol/g wet wt in the ischemia and reperfusion group), whereas ONO-5046 at a smaller dose (1 mg/kg) had lesser but significant effects on MDA and ATP alterations. This study demonstrates that treatment with ONO-5046, a neutrophil elastase inhibitor, can ameliorate ischemia-reperfusion injury of the rat liver.