Effect of carbohydrates on the ability of bull sperm to bind to bovine oviduct epithelial cells.

In the present study, we investigated the effect of various carbohydrates on the ability of bovine spermatozoa to bind to the bovine oviduct epithelial cells (OECs). We also examined the fertilization competence and motility of spermatozoa that bind to OECs in the presence of carbohydrates. Frozen-thawed spermatozoa were incubated with OECs, with and without various carbohydrates. The sperms were then divided into two fractions: OEC-binding sperms (B-sperm) and non-OEC binding sperms (NB-sperm). The fertilization rate, ability to bind the zona pellucida, and membrane integrity of the spermatozoa as determined using a hypo-osmotic-swelling test (HOST) were lower in NB-sperm than in the unseparated spermatozoa (control). The motility of the B-sperm was maintained for a longer time than that of the control spermatozoa. The addition of N-acetyl-d-glucosamine (GlcNAc, 5 mm) to the sperm-OEC mixture increased the number of B-sperm. D-mannose (5 mm) and D-fucose (5 mm) had no effect on the number of B-sperm. The motility of B-sperm, which bound to OECs in the presence of GlcNAc, however, was not maintained. When either OECs or the spermatozoa were treated with GlcNAc prior to sperm-OEC co-incubation, only sperm-side treatment enhanced sperm-OEC binding, but B-sperm motility was not maintained. The motility of spermatozoa incubated with GlcNAc was lower than that of controls. These results indicate that GlcNAc enhances sperm binding to OECs, probably via sperm surface modification, but does not promote increased sperm survival.

Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos.

The duration of sperm-oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm-oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (>5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1-3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%; P<0.05), and the highest rate of normal fertilization was observed for 5h culture (73.4%; P<0.05). The sex ratio was significantly (P<0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP; P<0.05) and decreased the proportion of HOST positive sperm (65.8-48.6%; P<0.05; experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5-10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1h (treatment 5) or 5h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively; P<0.05), and the 1h co-incubation significantly skewed (P<0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%; P<0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9; 1.0 mg/ml treatment 10) significantly (P<0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus-sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes.

Inclusion of the transcervical approach in video-assisted thoracoscopic extended thymectomy (VATET) for myasthenia gravis: a prospective trial.

BACKGROUND: Because evidence-based data regarding the quality of video-assisted thoracoscopic thymectomy for the treatment of myasthenia gravis are lacking, a prospective trial comparing three different operative approaches was conducted to evaluate their efficacy. METHODS: This prospective study enrolled 20 consecutive patients with nonthymomatous myasthenia gravis. A series of three approaches for bilateral video-assisted thoracoscopic extended thymectomy (VATET) using the anterior chest wall-lifting method (original), the original method with a flexed-neck position (modified), and the original method with a transcervical approach (final) were prospectively performed in each patient for quantitative and pathologic evaluation of the residual thymus after each approach. RESULTS: Complete VATET required 242 +/- 48 min, with the transcervical procedure requiring 23 +/- 12 min. After the modified method, the residual thymus in the cervical region was 1.5 cm in size and weighed 0.8 g (0.8% of the entire thymus), as compared with a size of 2.2 cm and a weight of 1.3 g (3.2%) after the original method. Each value is the result of comparison with the final method. Histopathologic studies showed residual tissue in the germinal center as well as Hassall's corpuscles in more than 70% of cases. CONCLUSION: The findings show that VATET without the transcervical approach could be an immunologically incomplete treatment for myasthenia gravis. Therefore, the transcervical approach should be included in VATET procedures to ensure radicality.

Safer video-assisted thoracoscopic thymectomy after location of thymic veins with multidetector computed tomography.

BACKGROUND: Video-assisted thoracoscopic (VATS) thymectomy has been applied as a surgical option for autoimmune myasthenia gravis. Prior identification and fine division of the thymic veins are critical to the prevention of unexpected severe bleeding that may require conversion to open surgery. Until recently, such bleeding could be avoided only by meticulous dissection of thymic fat tissue away from the left brachiocephalic vein (LBV). With recent advances in computed tomography (CT), multidetector-row computed tomography (MDCT) can readily be obtained and provides three-dimensional (3D) images. This study explored its value for preoperative identification of the thymic veins draining into the LBV, and thus for prevention of injury to these veins during endoscopic thymectomy. METHODS: Five patients with myasthenia gravis, thymoma, or both underwent enhanced MDCT preoperatively. The thymic veins draining into the LBV were visualized using both horizontal and sagittal/coronal CT images. Then 3D images were reconstructed to enable operators to simulate endoscopic views. During each VATS extended thymectomy, the numbers and branching patterns of the thymic veins were compared with the preoperative MDCT images. RESULTS: The thymic veins draining into the LBV were clearly identified with MDCT in all five patients examined. Reconstructed 3D images clearly located their courses in the thymic/fat tissue and their entry routes into the LBV, thus simulating the actual intraoperative endoscopic views. All tributaries divided during surgery were identified preoperatively with MDCT. CONCLUSIONS: Location of thymic veins with MDCT can provide precise preoperative information about thymic venous anatomy. This easy and less invasive examination has the potential to make VATS thymectomy easier and safer.

Cefoselis, a beta-lactam antibiotic, easily penetrates the blood-brain barrier and causes seizure independently by glutamate release.

Cefoselis is a widely used beta-lactam antibiotic, but occasionally induces seizures and convulsion in elder and renal failure patients. However, beta-lactams are known not to pass through the blood-brain barrier (BBB). In this study, we examined the BBB penetration of cefoselis in normal and renal failure rats by means of brain microdialysis. Cefoselis was dose-dependently appeared in brain extracellular fluid in proportion to its blood level. The elimination constant from brain extracellular fluid (apparent) was slightly lower than that from blood. These results indicated that cefoselis might penetrate the BBB or be discharged by a certain transport system. In contrast to the result of cefoselis, cefazolin, a leading drug of cephalosporins, could not be detected in the brain extracellular fluid after an intravenous injection. In renal dysfunction rats, the elimination half-lives of cefoselis from both blood and brain were extensively prolonged. This would be one of responsible factors inducing seizures seen in patients. However, the additional factor, such as decrease in brain function related to aging, would be involved in seizures in patient received cefoselis, because an extremely high dose was required to induce seizures even in renal failure rats. A local administration of cefoselis into the hippocampus through the microdialysis probe caused a striking elevation of extracellular glutamate, with a minimum increase in gamma-aminobutyric acid (GABA). However, a systematic cefoselis administration via the tail vein did not elevate extracellular glutamate and GABA concentrations in the hippocampus of renal failure rats that exhibited marked seizures. These results suggested that not the stimulation of glutamate release, but the blockade of GABA receptors might be responsible for the seizure induced by cefoselis.

Hemi-clamshell approach for advanced primary lung cancer.

BACKGROUND: The hemi-clamshell approach provides a wide anterior view of the mediastinum, apical dome, and cervicothoracic area. However, only a few reports have been made regarding this technique. METHODS: The hemi-clamshell approach was used in 24 patients, of whom 5 had a Pancoast tumor, 15 had mediastinal involvement, and 4 underwent mediastinal lymphadenopathy for left-sided lung cancer. Twenty-one of the patients received preoperative therapy. RESULTS: Twenty-one operations were complete resections. In addition, 12 patients received cardio-vascular reconstruction and 5 a first rib resection. Postoperative major morbidity was 21 % (6/24) and mortality was 4.2 % (1/24). Nine patients died of systemic tumor relapse and 14 patients were alive after a median follow-up of 24 months (range 3 - 68 months) following the initial therapy. The 5-year survival rate of patients with mediastinal involvement was 37 % and that of 13 patients with postoperative stage I or II was 35 %. CONCLUSIONS: The hemi-clamshell approach provides a wide exposure allowing a safe and complete removal of lung cancer that involves the mediastinum and apical thoracic dome, leading to a better long-term survival rate for patients with this disease.

Accelerated binding of autoantibody to red blood cells with increasing anaemia in cattle experimentally infected with Theileria sergenti.

Anaemia is the most important clinical manifestation in cattle infected with Theileria sergenti. In order to determine the mechanism of red blood cells (RBC) destruction in anaemic cattle, we studied the binding of autoantibody (IgG) to RBC during the development of anaemia in T. sergenti infection. The low levels of IgG-bound RBC before the development of anaemia were triggered in proportion with the progression of anaemia and parasitaemia. Our results suggest an accelerated destruction of RBC in anaemic cattle by IgG-dependent phagocytosis.

Scenarios for autoimmunization of T and B cells in myasthenia gravis.

We have studied responses in thymoma patients to interferon-alpha and to the acetylcholine receptor (AChR) in early-onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two-step process: (step 1) professional antigen-presenting cells and thymic epithelial cells prime AChR-specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross-react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen-antibody complexes and the recruitment of professional antigen-presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.

Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats.

The herbicide paraquat is an environmental factor that could be involved in the etiology of Parkinson's disease. We have previously shown that paraquat penetrates through the blood-brain barrier and is taken up by neural cells. In this study, we examined the in vivo toxic mechanism of paraquat to dopamine neurons. GBR-12909, a selective dopamine transporter inhibitor, reduced paraquat uptake into the striatal tissue including dopaminergic terminals. The subchronic treatment with systemic paraquat significantly decreased brain dopamine content in the striatum and slightly in the midbrain and cortex, and was accompanied by the diminished level of its acidic metabolites in rats. When paraquat was administered through a microdialysis probe, a transitory increase in the extracellular levels of glutamate, followed by long-lasting elevations of the extracellular levels of NO(x)(-) (NO(2)(-) plus NO(3)(-)) and dopamine were detected in the striatum of freely moving rats. This dopamine overflow lasted for more than 24 h after the paraquat treatment. Dopamine overflow was inhibited by N(G)-nitro-L-arginine methyl ester, dizocilpine, 6,7-dinitroquinoxaline-2,3-dione and L-deprenyl. The toxic mechanism of paraquat involves glutamate induced activation of non-NMDA receptors, resulting in activation of NMDA receptor-channels. The influx of Ca(2+) into cells stimulates nitric oxide synthase. Released NO would diffuse to dopaminergic terminals and further induce mitochondrial dysfunction by the formation of peroxynitrite, resulting in continuous and long-lasting dopamine overflow. The constant exposure to low levels of paraquat may lead to the vulnerability of dopaminergic terminals in humans, and might potentiate neurodegeneration caused by the exposure of other substances, such as endogenous dopaminergic toxins.

Three-color flow cytometric study on lymphocytes derived from thymic diseases.

BACKGROUND: Anterior mediastinal masses derive from a variety of diseases. Thymomas have been shown to commonly hold CD4(+)CD8(+) double-positive (DP) lymphocytes, and identification of this subset by two-color flow cytometric study was suggested to help diagnosis of thymoma. Several other thymic diseases, however, possibly hold CD4(+)CD8(+) DP lymphocytes. In this study, we utilized the three-color flow cytometric method for further examination of the phenotypes of lymphocytes in the thymic diseases. MATERIALS AND METHODS: One hundred eight specimens (77 primary and 10 metastatic thymomas, 10 thymic carcinomas, 2 thymic carcinoids, 4 malignant lymphomas, 2 seminomas, an inflammatory pseudotumor, and 2 nonneoplastic thymic hyperplasias) were subjected to the study. The expressions of CD3, CD4, and CD8 on tumor-associated lymphocytes were evaluated by three-color flow cytometric study. RESULTS: The proportion of the CD4(+)CD8(+) DP subset was more than 30% in all 78 lymphocyte-rich thymomas, in 2 malignant lymphomas, and in both thymic hyperplasias. CD3 expression of the CD4(+)CD8(+) DP subset ranged from a negative to a high level in thymomas and thymic hyperplasias, while it was restricted to a particular level in CD4(+)CD8(+) DP-type malignant lymphomas. The proportion of CD3(+) cells in the CD4(+)CD8(-) single-positive subset was consistently less than 90% in the lymphocyte-rich thymomas, while it was more than 90% in the thymic hyperplasias. CONCLUSION: Although identification of the CD4(+)CD8(+) DP subset in the tumor-associated lymphocytes does not necessarily indicate thymoma, a further characterization of thymic neoplasms possessing the CD4(+)CD8(+) DP subset was enabled by three-color flow cytometric study, suggesting the utility of this method as an ancillary tool for differential diagnosis of these diseases.

Acquired methemoglobinemia in anemic cattle infected with Theileria sergenti.

To investigate the mechanism of anemia accompanying Japanese bovine theileriosis, we examined whether production of methemoglobin (MetHB), an indicator of erythrocyte oxidation, was associated with anemia in cattle experimentally infected with Theileria sergenti. The percentage of MetHB, which is an oxidized form of hemoglobin, increased according to the onset of anemia. During severe anemia, high levels of acquired methemoglobinemia were observed in all infected cattle. A significant correlation (r=-0.649; P<0.01) between an increase in MetHB concentration and a decrease in packed cell volume (PCV) was observed. It was considered that hemoglobin oxidation may be one of the aggravating factors of anemia in T. sergenti infection.

Norharman, an indoleamine-derived beta-carboline, but not Trp-P-2, a gamma-carboline, induces apoptotic cell death in human neuroblastoma SH-SY5Y cells.

Carbolines, azaheterocyclic amines derived from indoleamines, have various biological activities, such as neurotoxicity of beta-carbolines and potent mutagenicity of gamma-carbolines. In this study, structural significance among these carbolines was investigated in relation to the types of cell death, apoptosis and necrosis, using human neuroblastoma SH-SY5Y cells. DNA damage was quantitatively analyzed by a single-cell gel electrophoresis assay. DNA damage was induced by both beta-carbolines, harman and norharman, and gamma-carbolines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-4-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in a dose dependent manner. Gamma-carbolines were more potent to damage DNA than beta-carbolines. Alkaline lysis of the cells prevented DNA damage induced by beta-carboline, and pre-treatment of the cells with cycloheximide, an inhibitor of protein synthesis, reduced DNA damage caused by norharman. Morphological observation showed condensed and fragmented nuclei typical for apoptosis, in the cells treated with norharman. Thus, DNA damage induced by norharman was proved to be apoptotic. However, harman, which had a methyl substitution at the position 1, might induce necrosis in the cells. On the other hand, gamma-carbolines, Trp-P-1 and Trp-P-2, directly damaged DNA. Thus, the nitrogen atom at the gamma-position and/or an amino group in carboline structure would be required to induce the direct DNA cleavage.

Glutamate is not involved in the MPP+-induced dopamine overflow in the striatum of freely moving C57BL/6 mice.

The role of glutamate in the N-methyl-4-phenyldihydropyridinium (MPP+) toxicity has been argued in the past decade. However, the effects of glutamate efflux and NMDA antagonist on MPP+-induced dopamine overflow have not been documented. To clarify this, we perfused MPP+ through a microdialysis probe in the striatum of freely moving mature C57BL/6 mice. The 60-min perfusion of 10 and 100 microM MPP+ strikingly increased dopamine levels to 28- and 93-fold of the basal values, respectively. In contrast, an administration of MPP+ did not induce marked glutamate release: the MPP+-perfusion slightly increased the glutamate level at 100 microM, but not at 10 microM. The addition of 100 microM (+)-MK-801 or 200 microM (+/-)-AP-7 to the perfusate did not attenuate MPP+-induced dopamine overflow. The extent of dopamine release only depended on the amount of MPP+ accumulation into the cells. These results indicated that, at least in the striatum, neither glutamate release nor the NMDA antagonist, including (+)-MK-801, could regulate MPP+-evoked dopamine overflow.

Somatic hypermutation and selection of B cells in thymic germinal centers responding to acetylcholine receptor in myasthenia gravis.

The muscle weakness in myasthenia gravis (MG) is mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Production of these pathogenic autoantibodies is believed to be associated with germinal centers (GC) and anti-AChR-secreting plasma cells in the hyperplastic thymus of patients with early onset MG (EOMG). Here, we describe the repertoire of rearranged heavy chain V genes and their clonal origins in GC from a typical EOMG patient. Three hundred fifteen rearranged Ig V(H) genes were amplified, cloned, and sequenced from sections of four thymic GC containing AChR-specific B cells. We found that thymic GC contain a remarkably heterogeneous population of B cells. Both naive and circulating memory B cells undergo Ag-driven clonal proliferation, somatic hypermutation, and selection. Numerous B cell clones were present, with no individual clone dominating the response. Comparisons of B cell clonal sequences from different GC and known anti-AChR Abs from other patients showed convergent mutations in the complementarity determining regions. These results are consistent with AChR driving an ongoing GC response in the thymus of EOMG patients. This is the first detailed analysis of B cell clones in human GC responding to a defined protein Ag, and the response we observed may reflect the effects of chronic stimulation by autoantigen.

Characterization of a thrombin-like serine protease, Kangshuanmei, isolated from the venom of a Chinese snake, Agkistrodon halys brevicaudus stejneger.

An enzyme, referred to as Kangshuanmei, was isolated from the venom of the Chinese snake Agkistrodon halys brevicaudus stejneger by gel filtration chromatography followed by affinity chromatography. Kangshuanmei is composed of a single polypeptide chain with a molecular weight of approximately 34,000, estimated by SDS-PAGE. The enzyme hydrolyzed both benzoyl-arginine ethyl ester and H-D-Phe-Pip-Arg-p-nitroanilide, specific substrates for thrombin. The protease activity of Kangshuanmei was inhibited by 4-(2-aminoethyl)-benzensulfonyl fluoride, but was not affected by EDTA. The enzyme acted on human fibrinogen to form a fibrin clot and released three fragments. These fragments were shown to be fibrinopeptide A, fibrinopeptide B, and the Bbeta1-42 peptide of fibrinogen, respectively. These results indicate that Kangshuanmei is a thrombin-like serine protease with coagulant activity. However, the enzyme did not induce activation of blood coagulation factor XIII, unlike thrombin. Moreover, antithrombin-III, the specific thrombin inhibitor in plasma, had no inhibitory effect on the thrombin-like amidolytic activity of Kangshuanmei. The N-terminal amino acid sequence of the enzyme up to 50 residues was determined by a peptide sequencer. The N-terminal sequence of Kangshuanmei was highly homologous to most thrombin-like serine proteases from the venom of the snakes of the crotalidae family.

Carrier-mediated processes in blood--brain barrier penetration and neural uptake of paraquat.

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringer's solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

Geographical north-south decline in DNASE1*2 in Japanese populations.

Allele frequencies for human deoxyribonuclease I (DNase I) phenotypes were determined using blood samples from about 2000 Japanese subjects living in nine prefectures, and compared with one another. DNase I phenotyping was performed principally using isoelectric focusing electrophoresis and activity staining. The DNase I system was shown to have enhanced potential for anthropologic, genetic, and clinical studies of Japanese populations. DNase I phenotypes were analyzed to evaluate the degree of genetic variation at the DNASE1 locus. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNASE1 allele.

Regulation of nuclear import by light-induced activation of caged nuclear localization signal in living cells.

A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine-128 residue in SV40 T-antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78+/-0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm(2)). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally.

Clinical and functional significance of WHO classification on human thymic epithelial neoplasms: a study of 146 consecutive tumors.

We examined the clinical and functional significance of histologic classification of thymic epithelial neoplasms proposed by the World Health Organization (WHO), based on an analysis of 146 consecutive tumors derived from 141 patients and 47 normal thymuses derived from children ranging in age from 1 to 9 years. Invasive tumors were seen in 12.5%, 38.6%, 40.0%, 69.4%, 80.0%, and 100% of type A, AB, B1, B2, B3, and C primary tumors, respectively. All of six recurrent or metastatic lesions were type B2 tumors. Myasthenia gravis was associated in 0%, 6.8%, 40.0%, 55.6%, 10.0%, and 0% in patients with type A, AB, B1, B2, B3, and C tumors, respectively. The average number (x10(6)) of tumor-associated CD4+CD8+ cells present in 1 g of tumor tissue was 1.5, 391.1, 1041.7, 333.9, 24.5, and 0.2 in type A, AB, B1, B2, B3, and C, respectively, and it was 1168.2 in the normal thymuses. Thus, type B1 tumor retained the function to induce CD4+CD8+ double-positive cells at a level comparable to that of the normal thymic cortical epithelial cells, followed by type AB and type B2 tumors. Type A and B3 tumors had this function at a barely detectable level, and type C tumor was nonfunctional. WHO histologic classification was shown to reflect the clinical features and the T-cell-inducing function of thymic epithelial tumors.

Classification of standard alleles of the MN blood group system.

BACKGROUND AND OBJECTIVES: We had classified the M alleles of the MN blood group system into two subtypes, M(G) (standard M) and M(T), based on a G/T substitution in intron 1 of the glycophorin A (GPA) gene. This study provides further study on nucleotide sequences of M(G), M(T) and N alleles to profile the new allele, M(T). MATERIALS AND METHODS: M(G), M(T) and N alleles of the GPA gene were amplified using GPA gene-specific primers to avoid co-amplification of the genes of glycophorins B and E. Then the 5'-flanking region, exons 1-7 and introns 1-4 of the alleles were analyzed by DNA sequencing. RESULTS: There were 17 nucleotide substitutions and deletions between M(G) (standard M) and N alleles. Ten of the M(T) nucleotides were M(G)-type but the other 7 were N-type. M(T) allele also showed one base change and one deletion that were observed in neither the M(G) nor the N allele. Moreover, we found nucleotide substitutions within each allele, allowing further classification of the alleles. CONCLUSION: By the sequence data of M(G), M(T) and N alleles, the three alleles could be further classified into M101 and M102, M201 and M202, and N101 and N102, respectively.