Identification of an HLA-B*27 variant, B*27:120, by sequence-based typing in a Taiwanese bone marrow stem cell donor.

One nucleotide substitution at residue 577 of HLA-B*27:04:01 results in a novel allele, HLA-B*27:120.

Detection of a novel HLA-DRB1*12 variant, HLA-DRB1*12:68, in a Taiwanese individual.

One nucleotide substitution at residue 628 of HLA-DRB1*12:01:01:01 results in a novel allele, HLA-DRB1*12:68.

HLA-C*07:566, a novel HLA-C*07 variant, detected in a Taiwanese hematopoietic stem cell donor.

One nucleotide replacement in codon 334 of HLA-C*07:02:01:01 results in a novel allele, HLA-C*07:566.

A dispermic chimerism detected in a Taiwanese potential unrelated hematopoietic stem cell donor.

Chimerism is defined as the presence of 2 or more than 1 genetically distinct cell populations in an organism. Dispermic chimeras are derived from the fertilization of 1 or 2 matured nuclei by 2 sperms. We here report detection of a healthy and phenotypically normal female with normal ABO red blood cell typing in whom dispermic chimerism was suspected after 3 alleles were identified at multiple human leukocyte antigen (HLA) loci using molecular HLA analysis. Molecular HLA typing showed the donor to have 3 HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles in blood, saliva and nail samples. In addition, 3 of her 9 short tandem repeat loci also showed to have 3 distinct alleles in blood, nail and saliva specimens. In all investigations, the third alleles were attributed to a dual paternal contribution. This case represents a dispermic chimerism, with 2 paternal and 1 maternal haplotypes variably distributed throughout body tissues in a healthy and phenotypically normal female without abnormalities in erythrocyte ABO blood group. The origin of this chimerism is probably due to the fertilization of a single egg and its polar body, or a parthenogenetic egg, by 2 sperms.

Detection of HLA-C*04:212, a novel HLA-C*04 variant, in a Taiwanese hematopoietic stem cell donor.

Recombination and point mutation may result the formation of HLA-C*04:212.

Discovery of HLA-A*11:01:69, a novel HLA-A*11:01 variant, in a Taiwanese hematopoietic stem cell donor.

One nucleotide replacement at residue 210 of HLA-A*11:01:01:01 results in a novel allele, HLA-A*11:01:69.

Detection of a novel variant of HLA-A*02, HLA-A*02:570, in a Taiwanese unrelated hematopoietic stem cell donor.

Two nucleotide changes at residue 142 (G → A) and residue 144 (A → C) of HLA-A*02:01:01:01 result in a novel allele, HLA-A*02:570.

Detection of two HLA-B*27 alleles, B*27:25 and B*27:86, in two Taiwanese blood donors by sequence-based typing.

We report here two HLA-B*27 alleles, B*27:86 and B*27:25, found in two Taiwanese blood donors. The new sequence of B*27:86 is identical to B*27:04:01 in exons 2 and 3, except at nucleotide 602 (A → G) in exon 3. The nucleotide change caused an amino acid substitution from E to G at amino acid residue 177. The sequence of B*27:25 is identical to B*27:04:01 in exons 2, 3, 4, 5, 6 and 7 except at nucleotides 538, 539, 559 and 560 in exon 4. The nucleotide changes caused amino acid substitutions from L to W and from E to L at residues 156 and 163, respectively. The generation of B*27:86 was probably resulted by a point mutation while the generation of B*27:25 may have been derived from a sequence recombination event between B*46:01:01 and B*27:04:01. The probable HLA-A, -B and -DRB1 haplotypes in association with B*27:86 and B*27:25 may be deduced as A*11:53-B*27:86-DRB1*12 and A*11:01-B*27:25-DRB1*04:05, respectively.

Generation of anti-tumour immune response using dendritic cells pulsed with carbonic anhydrase IX-Acinetobacter baumannii outer membrane protein A fusion proteins against renal cell carcinoma.

Carbonic anhydrase IX (CA9), a specific molecular marker for renal cell carcinoma (RCC), serves as a potential target for RCC-specific immunotherapy using dendritic cells (DCs). However, pulsing of DCs with CA9 alone is not sufficient for generation of a therapeutic anti-tumour immune response against RCC. In this study, in order to generate a potent anti-tumour immune response against RCC, we produced recombinant CA9-Acinetobacter baumannii outer membrane protein A (AbOmpA) fusion proteins, designated CA9-AbOmpA, and investigated the ability of DCs pulsed with CA9-AbOmpA fusion proteins in a murine renal cell carcinoma (RENCA) model. A recombinant CA9-AbOmpA fusion protein was composed of a unique proteoglycan-related region of CA9 (1-120 amino acids) fused at the C-terminus with transmembrane domain of AbOmpA (1-200 amino acids). This fusion protein was capable of inducing DC maturation and interleukin (IL)-12 production in DCs. Interaction of DCs pulsed with CA9-AbOmpA fusion proteins with naive T cells stimulated secretion of IL-2, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in T cells. Lymphocytes harvested from mice immunized with DCs pulsed with CA9-AbOmpA fusion proteins secreted IFN-γ and showed a specific cytotoxic activity against CA9-expressing RENCA (RENCA-CA9) cells. Administration of CA9-AbOmpA-pulsed DC vaccine suppressed growth of RENCA-CA9 cells in mice with an established tumour burden. These results suggest that DCs pulsed with CA9-AbOmpA fusion proteins generate a specific anti-tumour immune response against RCC, which can be utilized in immunotherapy of RCC.

Peripherally acting CB1-receptor antagonist: the relative importance of central and peripheral CB1 receptors in adiposity control.

OBJECTIVE: To investigate whether drugs targeting peripheral cannabinoid-1 (CB1) receptor ameliorate adiposity comparable to central CB1-receptor antagonist or not. MEASUREMENTS: Receptor binding assay and functional assay in vitro. Pharmacokinetic parameters in mice, brain uptake clearance of compounds in rats and antagonism on the CB1-agonist-induced hypothermia in mice. Diet consumption, body weight changes, hepatic gene expression of sterol-regulatory element-binding protein-1 (SREBP-1) and plasma/tissue concentrations of compounds in HF diet-induced obese (HF-DIO) mice after acute and chronic treatment. RESULTS: Compound-1, an SR141716A derivative, is a peripheral CB1-receptor-selective antagonist that is 10 times less potent than SR141716A in in vitro evaluations. Although the plasma concentrations of Compound-1 are five times higher than those of SR141716A, its potency is still 10 times lower than that of SR141716A in reducing the consumption of normal or HF diet by mice. Through evaluations of brain uptake and the effect on CB1-agonist-induced hypothermia, it was verified that the blood-brain barrier (BBB) penetration of Compound-1 is much lower than that of SR141716A. In HF-DIO mice, chronic treatment by Compound-1 showed dose-dependent antiobesity activities, while its brain distribution was very low as compared with that of SR141716A. Compound-1's effective doses for antiobesity activity were just over 30 mg kg(-1). However, Compound-1 completely suppressed the elevated hepatic SREBP-1 expression even at 10 mg kg(-1). CONCLUSION: These results suggest that (1) central CB1 receptors mediate anorectic response of CB1-receptor antagonists and (2) peripheral modulations, including SREBP-1 expression, are not major mechanisms in the antiobesity effects of CB1-receptor antagonists.

Altered expression of potassium channel subunit mRNA and alpha-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy.

Previous studies have raised the possibility that a decrease in voltage-gated K+ currents may contribute to hyperexcitability of injured dorsal root ganglion (DRG) neurons and the emergence of neuropathic pain. We examined the effects of axotomy on mRNA levels for various Kv1 family subunits and voltage-gated K+ currents in L4-L5 DRG neurons from sham-operated and sciatic nerve-transected rats. RNase protection assay revealed that Kv1.1 and Kv 1.2 mRNAs are highly abundant while Kv1.3, Kv1.4, Kv1.5 and Kv1.6 mRNAs were detected at lower levels in L4-L5 DRGs from sham and intact rats. Axotomy significantly decreased Kv1.1, Kv1.2, Kv1.3 and Kv1.4 mRNA levels by approximately 35%, approximately 60%, approximately 40% and approximately 80%, respectively, but did not significantly change Kv1.5 or Kv1.6 mRNA levels. Patch clamp recordings revealed two types of K+ currents in small-sized L4-L5 DRG neurons: sustained delayed rectifier currents elicited from a -40 mV holding potential and slowly inactivating A-type currents that was additionally activated from a -120 mV holding potential. Axotomy decreased both types of K+ currents by 50-60% in injured DRG neurons. In addition, axotomy increased the alpha-dendrotoxin sensitivity of the delayed rectifier, but not slow A-type K+ currents in injured DRG neurons. These results suggest that Kv1.1 and Kv1.2 subunits are major components of voltage-gated K+ channels in L4-L5 DRG neurons and that the decreased expression of Kv1-family subunits significantly contributes to the reduction and altered kinetics of Kv current in axotomized neurons.

The association of HLA -A, -B, and -DRB1 genotypes with Graves' disease in Taiwanese people.

Graves' disease has been associated with different human leukocyte antigen (HLA) genes in different races. To evaluate the association of HLA type in Taiwanese with Graves' disease, the HLA-A, -B, and -DRB1 alleles in a total of 236 Taiwanese adults with Graves' disease and 533 racially matched normal control subjects were examined using the PCR-SSOP (sequence specific oligonucleotide probe) technique. The prevalence of HLA-A*0207, -B*2704, -B*4601, and -DRB1*0901 among patients with Graves' disease was found to be increased, with odds ratios (OR) of 2.21, 3.82, 1.76 and 1.62, respectively. However, after correction for multiple comparisons, the relative risk of HLA-A*0207 susceptibility to Graves' disease remained statistically significant and the haplotype HLA-A*3303 -B*5801 -DRB1*0301 had a significantly protective effect. None of the other 2- or 3-locus haplotypes showed any significantly increased risk. Although HLA-DRB1*1405 showed an increased relative risk in patients with GO (Graves' opthalmopathy) (OR 4.61) when compared with patients without GO, the relative risk after adjusting for the number of comparisons was not significant. Taiwanese patients with Graves' disease have HLA-associated susceptibility genes which are similar to those found in Chinese patients in Hong Kong and Singapore. However, the finding in this study of a higher frequency of HLA-A*0207 in Taiwanese with Graves' disease has not been documented in any other ethnic group.

Kvbeta subunits increase expression of Kv4.3 channels by interacting with their C termini.

Auxiliary Kvbeta subunits form complexes with Kv1 family voltage-gated K(+) channels by binding to a part of the N terminus of channel polypeptide. This association influences expression and gating of these channels. Here we show that Kv4.3 proteins are associated with Kvbeta2 subunits in the brain. Expression of Kvbeta1 or Kvbeta2 subunits does not affect Kv4.3 channel gating but increases current density and protein expression. The increase in Kv4.3 protein is larger at longer times after transfection, suggesting that Kvbeta-associated channel proteins are more stable than those without the auxiliary subunits. This association between Kv4.3 and Kvbeta subunits requires the C terminus but not the N terminus of the channel polypeptide. Thus, Kvbeta subunits utilize diverse molecular interactions to stimulate the expression of Kv channels from different families.

Assessment of toxic potential of industrial chemicals using a cultured human bioartificial skin model: production of interleukin 1alpha and hydroxyeicosatetraenoic acids.

Cytotoxicity assays using artificial skin are proposed as alternative methods for in vitro tests to minimize animals used in ocular and dermal irritation testing. The responses of the artificial skins were studied to a well-characterized chemical irritant, such as toluene, glutaraldehyde and sodium lauryl sulfate (SLS), and a nonirritant, such as polyethylene glycol. The evaluation of irritating and nonirritating test chemicals was also compared with responses seen in human dermal fibroblasts and human epidermal keratinocytes grown in monolayer culture. The responses monitored included the MTT mitochondrial functionality assay. In order to better understand the local mechanisms involved in skin damage and repair, the productions of several mitogenic proinflammatory mediators such as interleukin-1alpha (IL-1alpha), 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE were investigated. Dose-dependent increases in the levels of IL-1alpha and HETEs were observed in the underlying medium of the skin systems exposed to two skin irritants, glutaraldehyde and SLS. The results of the present study show that both human artificial skins can be used as efficient testing models for the evaluation of skin toxicity in vitro and for screening the contact skin irritancy in vitro.

Tissue engineered artificial skin composed of dermis and epidermis.

We made an artificial skin comprised of a stratified layer of keratinocytes and a dermal matrix with a type I collagen containing fibroblasts. In this work, we showed keratinocyte behavior under primary culture, gel contractions varying with concentration of collagen solution, and cell growth plots in the collagen gel. The optimum behavior of dermal equivalent could be obtained using 3.0 mg/ml collagen solution and attached gel culture. The attached gel culture had a jumping effect of growth factor on cell growth at the lag phase. To develop the artificial skin, 1x10(5) cells/cm2 of keratinocytes were cultured on the dermal equivalent at air-liquid interface. Finally, to overcome the problem that artificial skin of collagen gel was torn easily during suturing of grafting, we prepared histocompatible collagen mesh and attached the mesh to the bottom of the gel. Cultured artificial skins were successfully grafted onto rats.

Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer.

A large body of evidence suggests that cyclooxygenase-2 (COX-2) is important in gastrointestinal cancer. The purpose of this study was to determine whether COX-2 was expressed in adenocarcinoma of the human pancreas. Quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry were used to assess the expression of COX-2 in pancreatic tissue. Levels of COX-2 mRNA were increased by >60-fold in pancreatic cancer compared to adjacent nontumorous tissue. COX-2 protein was present in 9 of 10 cases of adenocarcinoma of the pancreas but was undetectable in nontumorous pancreatic tissue. Immunohistochemical analysis showed that COX-2 was expressed in malignant epithelial cells. In cultured human pancreatic cancer cells, levels of COX-2 mRNA and protein were induced by treatment with tumor-promoting phorbol esters. Taken together, these results suggest that COX-2 may be a target for the prevention or treatment of pancreatic cancer.

Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck.

The purpose of this study was to determine whether cyclooxygenase-2 (COX-2) was overexpressed in squamous cell carcinoma of the head and neck (HNSCC). Quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry were used to assess the expression of COX-2 in head and neck tissue. Mean levels of COX-2 mRNA were increased by nearly 150-fold in HNSCC (n = 24) compared with normal oral mucosa from healthy volunteers (n = 17). Additionally, there was about a 50-fold increase in amounts of COX-2 mRNA in normal-appearing epithelium adjacent to HNSCC (n = 10) compared with normal oral mucosa from healthy volunteers. Immunoblotting demonstrated that COX-2 protein was present in six of six cases of HNSCC but was undetectable in normal oral mucosa from healthy subjects. Immunohistochemical analysis showed that COX-2 was expressed in both HNSCC and adjacent normal-appearing epithelium. Taken together, these results suggest that COX-2 may be a target for the prevention or treatment of HNSCC.

Phenotypic abnormalities in macrophages from leptin-deficient, obese mice.

Obesity is a complex syndrome that involves defective signaling by a number of different factors that regulate appetite and energy homeostasis. Treatment with exogenous leptin reverses hyperphagia and obesity in ob/ob mice, which have a mutation that causes leptin deficiency, proving the importance of this factor and its receptors in the obesity syndrome. Cells with leptin receptors have been identified outside of the appetite regulatory centers in the brain. Thus leptin has peripheral targets. Because macrophages express signaling-competent leptin receptors, these cells may be altered during chronic leptin deficiency. Consistent with this concept, the present study identifies several phenotypic abnormalities in macrophages from ob/ob mice, including decreased steady-state levels of uncoupling protein-2 mRNA, increased mitochondrial production of superoxide and hydrogen peroxide, constitutive activation of CCAAT enhancer binding protein (C/EBP)-beta, an oxidant-sensitive transcription factor, increased expression of interleukin-6 and cyclooxygenase (COX)-2, two C/EBP-beta target genes, and increased COX-2-dependent production of PGE2. Given the importance of macrophages in the general regulation of inflammation and immunity, these alterations in macrophage function may contribute to obesity-related pathophysiology.

Inhibition of cyclooxygenase-2 expression. An approach to preventing head and neck cancer.

Cyclooxygenase (COX) catalyzes the formation of prostaglandins (PG) from arachidonic acid. A large body of evidence has accumulated to suggest that COX-2, the inducible form of COX, is important in carcinogenesis. In this study, we determined whether (1) COX-2 was overexpressed in squamous cell carcinoma of the head and neck (HNSCC) and whether (2) retinoids, a class of chemopreventive agents, blocked epidermal growth factor (EGF)-mediated activation of COX-2 expression. Levels of COX-2 mRNA were determined in 15 cases of HNSCC and 10 cases of normal oral mucosa. Nearly a 100-fold increase in amounts of COX-2 mRNA was detected in HNSCC. By immunoblot analysis, COX-2 protein was detected in 6 of 6 cases of HNSCC but was undetectable in normal mucosa. Because retinoids protect against oral cavity cancer, we investigated whether retinoids could suppress EGF-mediated induction of COX-2 in cultured oral squamous carcinoma cells. Treatment with EGF led to increased levels of COX-2 mRNA, COX-2 protein, and synthesis of PG. These effects were suppressed by a variety of retinoids. Based on the results of this study, it will be important to establish whether newly developed selective COX-2 inhibitors are useful in preventing or treating HNSCC. Moreover, the anticancer properties of retinoids may be due, in part, to inhibition of COX-2 expression. Combining a retinoid with a selective COX-2 inhibitor may be more effective than either agent alone in preventing cancer of the upper aerodigestive tract.