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.

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.

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.

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.

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.

Regulation of uridine diphosphate glucuronosyltransferase expression by phenolic antioxidants.

Dietary antioxidants protect laboratory animals against the induction of tumors by a variety of chemical carcinogens. Among possible mechanisms, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes. Therefore, we investigated the effects of two commonly used food preservatives, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), on the expression of UDP-glucuronosyltransferase isoforms in rat liver. Male Wistar rats were fed a control diet or diets containing BHA (0.75%) or BHT (0.5%) for 2 weeks. BHT and BHA increased UDP-glucuronosyltransferase activities in liver microsomes for p-nitrophenol (236 and 218%, respectively), 3-hydroxybenzo(a)pyrene (246 and 175%, respectively), and androsterone (269 and 152%, respectively). Immunoblots showed changes in the amounts of UDP-glucuronosyltransferase isoforms corresponding to the changes in enzyme activities. Northern blot analysis showed that the concentration of UDP-glucuronosyltransferase mRNA paralleled the concentration of enzyme proteins and their respective levels of enzyme activity. BHT, for example, caused about a 250% increase in mRNA using a probe that recognizes the common 3'-domain of bilirubin/phenol UDP-glucuronosyltransferase mRNAs. In addition to inducing hepatic enzyme activities, BHT and BHA increased the activity of UDP-glucuronosyltransferase in the small intestine and kidney.

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.

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 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.

Dietary lipids induce phase 2 enzymes in rat small intestine.

We examined the role of dietary lipids in determining the activities of glutathione S-transferase (GST) and UDPglucuronosyl-transferase (UGT) in rat small intestine. Male Wistar rats were fed a fat-free (FF) diet or isocaloric control diet containing 5% corn oil (CO) or 5% fish oil (FO) for 3 weeks. The activities of these enzymes were about 2-fold higher in rats fed the FO diet vs. the FF diet. Intermediate levels of enzyme activity were found in rats fed the CO diet. Diet-induced differences in enzyme levels were shown by immunoblotting. The highest levels of glutathione S-transferase and UDPglucuronosyltransferase were detected in rats fed the FO diet. The lowest levels of these enzymes were found in rats fed the FF diet. Intermediate levels of enzyme were detected in rats fed the CO diet. Thus, diet-induced differences in enzyme activities were paralleled by changes in enzyme levels. Fatty acid analysis of mucosal lipids showed that the FF and FO diets were associated with decreased levels of linoleic and arachidonic acids as compared with the CO diet.

Dietary lipids coinduce xenobiotic metabolizing enzymes in rat liver.

We examined the role of dietary lipids in regulating the activities and amounts of epoxide hydrolase, UDP-glucuronosyltransferase and glutathione S-transferase in rat liver. Male Wistar rats were fed a fat-free (FF) diet or isocaloric control diet containing 5% corn oil (CO) or 5% fish oil (FO) for 3 weeks. The activities of these enzymes were approx. 2-fold higher in rats fed the FO diet vs. the FF diet. Intermediate levels of enzyme activity were found in rats fed the CO diet. Diet-induced differences in enzyme levels were shown by immunoblotting. The highest levels of epoxide hydrolase, UDP-glucuronosyltransferase and glutathione S-transferase were detected in rats fed the FO diet. The lowest levels of these enzymes were found in rats fed the FF diet. Intermediate levels of enzyme were found in rats fed the CO diet. Thus, diet-induced differences in enzyme activities were paralleled by changes in enzyme levels. Fatty acid analysis of microsomal lipids showed that the FF diet was associated with decreased levels of n-6 fatty acids vs. the CO diet. The FO diet resulted in increased levels of n-3 fatty acids vs. the other diets.

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.

Effect of dietary lipids on levels of UDP-glucuronosyltransferase in liver.

Others have shown recently that dietary fish oil protects against acetaminophen-induced liver injury in vivo. Fish oil was protective because it increased the clearance of acetaminophen via glucuronidation. This work left unresolved the basis for increased rates of glucuronidation in animals fed fish oil. We therefore have determined how the amount and type of lipid in the diet affect the activity of liver microsomal UDP-glucuronosyltransferase activity. Male Wistar rats were fed a fat-free diet or isocaloric diets containing 5% corn oil, olive oil or fish oil for 4 weeks. The activity of UDP-glucuronosyltransferase was highest in rats fed fish oil and lowest in rats fed the fat-free diet. Treatment with corn oil and olive oil resulted in intermediate levels of activity. Diet-induced differences in amounts of UDP-glucuronosyltransferase were shown by immunoblotting and kinetic measurements. Treatment with fish oil resulted in a 3-fold increase in the amount of UDP-glucuronosyltransferase versus the fat-free diet. Corn oil and olive oil diets caused 2-fold increases in the amount of UDP-glucuronosyltransferase versus the fat-free diet. Fatty acid analysis of microsomal lipids showed that the fat-free diet was associated with decreased levels of arachidonic acid versus the corn oil or olive oil diets. The fish oil diet resulted in increased levels of omega-3 fatty acids versus the other diets.

Gender-related differences in the amount and functional state of rat liver UDP-glucuronosyltransferase.

The basis for gender-dependent differences in rates of glucuronidation of xenobiotics is uncertain. To clarify this issue, the glucuronidation of p-nitrophenol was compared in liver microsomes from adult male and female rats. The activity of native UDP-glucuronosyltransferase was 47% higher in microsomes from male than from female rats. Immunoblotting of microsomal protein with anti-UDP-glucuronosyltransferase antiserum revealed 66% more immunoreactive protein in male microsomes. A kinetic method for measuring glucuronidating enzyme content confirmed the result of the immunoblot. Responses of UDP-glucuronosyltransferase to activation by palmitoyllysophosphatidylcholine or high pressure indicated that the activity of the enzyme was more latent in male than in female microsomes. Differences in enzyme latency could be due to differences in membrane structure. A comparison of microsomal fatty acid composition revealed significantly higher levels of oleic and linoleic acids and lower levels of stearic and docosahexaenoic acids in male than in female microsomes. The phospholipid composition, ratio of cholesterol:phospholipid, and membrane fluidity were similar in male and female microsomes. These results indicate that gender-dependent differences in UDP-glucuronosyltransferase activity are due to differences in both the amount and functional state of the enzyme.

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.

Effect of brain angiotensin II AT1, AT2, and cholinergic receptor antagonism on drinking in water-deprived rats.

The physiological role of brain Ang II and acetylcholine in mediating water deprivation-induced drinking was assessed in male Sprague-Dawley rats. Specific receptor antagonists were intracerebroventricularly (i.c.v.) administered in 48-h water-deprived rats. When water was given 20 min after i.c.v. injection, PD 123319 almost totally blocked the drinking response. However, losartan and CGP 42112A produced an approx. 20% inhibition of water intake. Central blockade of AT1 receptor with KR 31080 and cholinergic receptor with atropine attenuated water intake more than 50% which was significantly greater than inhibition produced by losartan and CGP 42112A. Atropine given alone or mixed with losartan and CGP-42112A produced a similar magnitude of inhibition of water intake. When water was given 90 min after i.c.v. injection, losartan or CGP-42112A produced a significantly greater inhibition of water intake than when water was given 20 min after injection. The present results suggest that both the central angiotensinergic and cholinergic system play an important role in the physiological drinking response after water deprivation. Both brain AT1 and AT2 receptors are involved in dehydration-induced drinking, but relative contribution of the receptors remains to be clarified.