In this study, we demonstrate that constitutive activation of Raf-1
oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic
kinase that ultimately drives the transition from an epithelial to a highly
invasive mesenchymal phenotype in estrogen receptor α-positive
(ERα+) breast cancer cells. The transition from an
epithelial- to a mesenchymal-like phenotype was characterized by reduced
expression of ERα, HER-2/Neu overexpression and loss of CD24 surface
receptor (CD24 –/low). Importantly, expression of key
epithelial-to-mesenchymal transition (EMT) markers and upregulation of the
stemness gene SOX2 was linked to acquisition of stem cell-like
properties such as the ability to form mammospheres in vitro
and tumor self-renewal in vivo. Moreover, aberrant Aurora-A
kinase activity induced phosphorylation and nuclear translocation of SMAD5,
indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in
the development of EMT, stemness and ultimately tumor progression. Importantly,
pharmacological and molecular inhibition of Aurora-A kinase activity restored a
CD24+ epithelial phenotype that was coupled to ERα
expression, downregulation of HER-2/Neu, inhibition of EMT and impaired
self-renewal ability, resulting in the suppression of distant metastases. Taken
together, our findings show for the first time the causal role of Aurora-A
kinase in the activation of EMT pathway responsible for the development of
distant metastases in ERα+ breast cancer cells. Moreover, this
study has important translational implications because it highlights the mitotic
kinase Aurora-A as a novel promising therapeutic target to selectively eliminate
highly invasive cancer cells and improve the disease-free and overall survival
of ERα+ breast cancer patients resistant to conventional
breast cancer; stemness; metastases
Interstitial cystitis is a chronic pelvic pain syndrome of which the origin and mechanisms involved remain unclear. In this study Ca2+ transients in the bladder wall of domestic cats diagnosed with naturally occurring feline interstitial cystitis were examined.
Materials and Methods
Cross-sections of full-thickness bladder strips from normal cats and cats with feline interstitial cystitis were examined by optically mapping Ca2+ transients and recording tension. Responses of Ca2+ activity and detrusor contractions to pharmacological interventions were compared. In addition, pharmacological responses were compared in mucosa denuded preparations.
Optical mapping showed that feline interstitial cystitis bladders had significantly more spontaneous Ca2+ transients in the mucosal layer than control bladders. Optical mapping also demonstrated that feline interstitial cystitis bladders were hypersensitive to a low dose (50 nM) of the muscarinic receptor agonist arecaidine when the mucosal layer was intact. This hypersensitivity was markedly decreased in mucosa denuded bladder strips.
In feline interstitial cystitis cat bladders there is increased Ca2+ activity and sensitivity of muscarinic receptors in the mucosal layer, which can enhance smooth muscle spontaneous contractions.
urinary bladder; cystitis, interstitial; cats; cat diseases; receptors, muscarinic
The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams–Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date.
A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated.
We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1.
These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.
TFII-I; BRCA1; interaction; activation; DNA damage repair
Increased gap junction expression in lamina propria myofibroblasts and urothelial cells may be involved in detrusor overactivity, leading to incontinence. Immunohistochemistry was used to compare connexin (Cx) 26, 43, and 45 expression in the bladders of neonatal, adult, and spinal cord-transected rats, while optical imaging was used to map the spread of spontaneous activity and the effects of gap junction blockade. Female adult Sprague-Dawley rats were deeply anesthetized, a laminectomy was performed, and the spinal cord was transected (T8/T9). After 14 days, their bladders and those of age-matched adults (4 mo old) and neonates (7-21 day old) were excised and studied immunohistochemically using frozen sections or optically using whole bladders stained with voltage- and Ca2+-sensitive dyes. The expression of Cx26 was localized to the urothelium, Cx43 to the lamina propria myofibro-blasts, and Cx45 to the detrusor smooth muscle. While the expression of Cx45 was comparable in all bladders, the expression of Cx43 and Cx26 was increased in neonate and transected animals. In the bladders of adults, spontaneous activity was initiated at multiple sites, resulting in a lack of coordination. Alternatively, in neonate and transected animals spontaneous activity was initiated at a focal site near the dome and spread in a coordinated fashion throughout the bladder. Gap junction blockade (18β-glycyrrhetinic acid, 1 μM) abolished this coordinated activity but had no effect on the uncoordinated activity in adult bladders. These data suggest that coordinated spontaneous activity requires gap junction upregulation in urothelial cells and lamina propria myofibroblasts.
lamina propria myofibroblasts; optical mapping; urothelium; voltage-and Ca2+-sensitive dyes
This study examined the origin of spontaneous activity in neonatal and adult rat bladders and the effect of stretch and muscarinic agonists and antagonists on spontaneous activity. Rats were anesthetized and their bladders were excised, cannulated, and loaded with voltage- and Ca2+-sensitive dyes. Intracellular Ca2+ and membrane potential transients were mapped using photodiode arrays in whole bladders, bladder sheets, or cross-section preparations at 37°C. Intravesical pressure was recorded from whole bladders. In neonatal bladders and sheets, spontaneous Ca2+ and electrical signals arose at a site near the dome and spread in a coordinated manner throughout the bladder with different dome-to-neck conduction velocities (Ca2+: 3.7 ± 0.4 mm/s; membrane potential: 46.2 ± 3.1 mm/s). In whole bladders, optical signals were associated with spontaneous contractions (10–20 cmH2O). By contrast, in adult bladders spontaneous Ca2+ and electrical activity was uncoordinated, originating at multiple sites and was associated with smaller (2–5 cmH2O) contractions. Spontaneous contractions and optical signals were insensitive to tetrodotoxin (2 μM) but were blocked by nifedipine (10 μM). Stretch or low carbachol concentrations (50 nM) applied to neonatal whole bladders enhanced the amplitude (to 20–35 cmH2O) of spontaneous activity, which was blocked by atropine. Bladder cross sections revealed that Ca2+ and membrane potential transients produced by stretch or carbachol began near the urothelial-suburothelial interface and then spread to the detrusor. In conclusion, spontaneous activity in neonatal bladders, unlike activity in adult bladders, is highly organized, originating in the urothelium-suburothelium near the dome. Activity is enhanced by stretch or carbachol and this enhancement is blocked by atropine. It is hypothesized that acetylcholine is released from the urothelium during bladder filling to enhance spontaneous activity.
optical mapping; pacemaker activity; urothelium; voltage- and Ca2+-sensitive dyes
Aims: To characterise a novel strain of adenovirus (Ad) type Ad8 (genome type Ad8I) involved in an epidemic keratoconjunctivitis (EKC) outbreak in Hiroshima city using serological testing and sequence analysis of the fibre and hexon gene.
Methods: A neutralisation test (NT) was performed in microtitre plates containing a confluent monolayer of A549 cells using 100 tissue culture infectious doses of virus and type specific antisera. The haemagglutination inhibition test was also carried out in microtitre plates with rat erythrocytes using four haemagglutination units of virus and twofold dilutions of serum. The fibre gene was sequenced by generating overlapping polymerase chain reaction products or by direct sequencing of genomic DNA. Primer selection was based on alignment of the fibre genes of human adenovirus serotypes Ad8, Ad19, Ad37, Ad9, and Ad15 available from Gene Bank.
Results: The virus strain was specifically neutralised by anti-Ad8 antibodies, although there was a major crossreaction with anti-Ad9 antibodies. Haemagglutination was equally inhibited by anti-Ad8 and anti-Ad9 antibodies. The predicted amino acid sequences of the hypervariable regions (HVRs) of the Ad8I hexon gene showed higher homology with Ad9 (83.3%) than with Ad8 (62.0%). However, the Ad8I fibre knob was more homologous to Ad8 (94.4%) than to Ad9 (91.6%).
Conclusions: Ad8I is a unique strain of adenovirus because of its lower genomic homology with Ad8, major crossreactivity with Ad9 in NT, and mixed genetic organisation of HVRs of the hexon gene. These factors may have enabled the virus to circumvent acquired immunity, resulting in the outbreak.
adenovirus; epidemic keratoconjunctivitis; hexon gene; fibre gene; sequence analysis
Aims: To investigate the genetic differences among the strains of adenovirus type 8 (Ad8) circulating in Hiroshima city, Japan, and to study their circulation pattern.
Methods: One hundred and twenty nine strains of adenovirus type 8 (Ad8) were isolated in Hiroshima City over a 15 year period (1983–97) from patients with keratoconjunctivitis, and analysed with six restriction enzymes—BamHI, HindIII, PstI, SacI, SalI, and SmaI—to investigate possible relations among the isolates and their genetic variability. Seven hypervariable regions of the hexon gene that carry the type specific epitope were also sequenced to investigate the variation among the genome types.
Results: Restriction endonuclease analyses yielded three known genome types (Ad8A, 13 samples; Ad8B, seven samples; and Ad8E, 35 samples) and a novel genome type (Ad8I, 74 samples). Ad8A, Ad8B, and Ad8E were closely related, with 96% homology, whereas Ad8I had only 71% homology. Ad8A, Ad8B, and Ad8E shared 91.8% and 96.4% homology with regard to their amino acid and nucleotide sequences, respectively, with the isolate 1127 (accession no X74663). However, when compared with Ad8A, Ad8B, Ad8E, and isolate 1127, Ad8I shared only 62.7% and 69.9% homology with regard to amino acid and nucleotide sequences, respectively. Ad8A, Ad8B, and Ad8E had a unique 31 amino acid deletion in the hypervariable region 1 of the hexon gene, whereas Ad8I had a 33 residue deletion. The Ad8E strain that circulated from 1984 to 1995 was stable among the study population. Ad8I was isolated from an outbreak of epidemic keratoconjunctivitis in 1995 and was also isolated from sporadic cases until 1997.
Conclusions: These results confirmed that genetic variability occurs in Ad8 in the microenvironment and revealed the emergence of a new genome type (Ad8I).
adenovirus; type 8; genetic characterisation; Hiroshima
Methods: Serum samples from 1048 Japanese patients with various autoimmune diseases were screened for anti-Th/To antibodies using RNA and protein immunoprecipitation assays. The reactivity to RNase P subunits was examined by immunoprecipitating 35S labelled recombinant Rpp38, Rpp30, and hPop1 produced by in vitro transcription and translation. HLA-DRB1, DQB1, and DPB1 alleles were identified using a polymerase chain reaction followed by a restriction fragment length polymorphism assay.
Results: Serum anti-Th/To antibodies were detected in 14 of 303 patients with SSc and seven of 745 patients without SSc (4.6% v 0.9%; p=0.0003). Similar percentages of patients with and without SSc showed immunoreactivity to Rpp38 and Rpp30, but more patients with SSc than patients without SSc showed a reactivity to hPop1 (93% v 14%; p=0.002). In patients with anti-Th/To antibodies DRB1*1502 or *0802 was detected more often, and the DRB1*0405-DQB1*0401 haplotype less often in patients with SSc than in patients without SSc (79% v 14%, p=0.02, and 7% v 71%, p=0.01, respectively).
Conclusions: Anti-Th/To antibodies were detected in a small proportion of autoimmune patients lacking clinical features related to SSc. A close relationship between disease expression and anti-hPop1 reactivity as well as HLA class II alleles in anti-Th/To positive patients suggests that the process of anti-Th/To antibody production may be different between patients with and those without SSc.
chemoradiotherapy; docetaxel; cisplatin; 5-fluorouracil; squamous cell carcinoma of the head and neck (SCCHN)
Elucidation of genetic alterations is an approach to understanding the underlying molecular mechanisms of progression of human prostate cancers. We have searched for genes differentially expressed in advanced prostate cancers using cDNA-representational difference analysis, and thereby isolated the Lsm1 as one of down-regulated gene. An Lsm1 expression vector was transfected into PC3 cells, normally featuring down-regulated Lsm1, and four transfectants were established. No differences in morphology or cell proliferation were evident in comparison with parent PC3 or PC3/mock-transfectants. In contrast, significant suppression of invasive potential or metastatic ability of Lsm1 transfectants was observed in the Matrigel chemoinvasion assay and in nude mice, respectively. With human prostate cancers, almost all of informative prostatectomised cases without neoadjuvant therapy showed allelic retention in the Lsm1 region, whereas refractory cancers frequently showed allelic loss in this region. No critical gene mutations were found in open reading frame of Lsm1 in prostate cancers examined by PCR–SSCP analysis, including localised and refractory cancers. These results suggest that Lsm1 is deeply involved in prostate cancer progression through its down-regulation, independent of any gene mutation.
British Journal of Cancer (2002) 86, 940–946. DOI: 10.1038/sj/bjc/6600163 www.bjcancer.com
© 2002 Cancer Research UK
Lsm1; prostate cancer; tumour progression
AIMS—To determine the incidence, natural course, and severity of dry eye occurring or worsening after haematopoietic stem cell transplantation (SCT).
METHODS—At a tertiary care hospital, 53 patients undergoing allogeneic or autologous SCT followed by at least 180 days of follow up were studied prospectively. Examination included grading of symptoms of dry eye, evaluation of ocular surface, tear break up time, and Schirmer tests with and without nasal stimulation. Meibomian gland secretion was also examined using a slit lamp while applying steady digital pressure.
RESULTS—Of the 53 patients, 44 received allografts. Half of these patients (22) developed dry eye or their pre-existing dry eye worsened after SCT, while none of nine autograft recipients did. Onset of dry eye was 171 (SD 59) days after SCT. Two types of dry eye occurred. One (n=10) was severe with ocular surface findings resembling Sjögren's syndrome and reduction of reflex tearing soon after onset. A mild type (n=12) had unimpaired reflex tearing. Meibomian gland dysfunction (MGD) was more frequent and severe in patients with dry eye and chronic graft versus host disease (GVHD), and overall severity of dry eye was greater in patients with MGD and chronic GVHD.
CONCLUSIONS—Dry eye after SCT occurred only in allograft recipients, and was not evident in autograft recipients. The severe form of dry eye had a tendency to develop rapidly. Further study on the prediction and treatment of severe dry eye after SCT is necessary.
Vascular endothelial growth factor-C (VEGF-C) functions specifically to induce lymphangiogenesis. We examined the relationship between expression of VEGF-C and clinicopathological features in patients with colorectal cancer. The expression of VEGF-C in the 99 primary tumours and 18 metastatic lymph nodes from colorectal cancer patients was examined immunohistochemically. To verify VEGF-C mRNA expression, reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out. The expression of VEGF-C correlated with lymphatic involvement, lymph nodes metastasis, and depth of invasion. On the other hand, correlations were nil with regard to gender of the patients, histologic type, venous involvement, and liver metastasis. The expression of VEGF-C in metastatic lymph nodes was fairly consistent with this expression in the primary tumour. Survival time was shorter for VEGF-C positive groups than for VEGF-C negative ones, but with no statistically significant difference. RT-PCR findings revealed that the expression of VEGF-C mRNA correlated mostly with that of VEGF-C protein expression. VEGF-C may play an important role in lymphatic spread of colorectal cancer. © 2000 Cancer Research Campaign
VEGF-C; lymphatic involvement; lymph nodes metastasis; colorectal carcinoma
We investigated levels of p53 protein expression in Japanese patients with oesophageal squamous cell carcinoma. A significantly larger proportion of heavy alcohol drinkers and cigarette smokers was evident in the p53-positive group. The combination of drinking and smoking was associated with a high frequency of p53 protein accumulation. © 2000 Cancer Research Campaign
alcohol; cigarettes; p53; oesophageal squamous cell carcinoma
To understand the mechanisms and identify novel approaches to overcoming retinoic acid (RA) resistance in acute promyelocytic leukaemia (APL), we established the first human RA-resistant APL model in severe combined immunodeficiency (SCID) mice. UF-1 cells, an RA-resistant APL cell line established in our laboratory, were transplanted into human granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing SCID (hGMTg SCID) mice and inoculated cells formed subcutaneous tumours in all hGMTg SCID mice, but not in the non-transgenic control SCID mice. Single-cell suspensions (UF-1/GMTg SCID cells) were similar in morphological, immunological, cytogenetic and molecular genetic features to parental UF-1 cells. All-trans RA did not change the morphological features of cells or their expression of CD11b. RA did not alter the growth curve of cells as determined by MTT assay, suggesting that UF-1/GMTg SCID cells are resistant to RA. These results demonstrate that this is the first RA-resistant APL animal model that may be useful for investigating the biology of this myeloid leukaemia in vivo, as well as for evaluating novel therapeutic approaches including patients with RA-resistant APL.
T cell proliferative responses to platelet membrane GPIIb-IIIa were examined in 14 patients with chronic immune thrombocytopenic purpura (ITP), 7 systemic lupus erythematosus (SLE) patients with or without thrombocytopenia, and 10 healthy donors. Although peripheral blood T cells from all subjects failed to respond to the protein complex in its native state, reduced GPIIb-IIIa stimulated T cells from three ITP patients and one SLE patient with thrombocytopenia, and tryptic peptides of GPIIb-IIIa stimulated T cells from nearly all subjects. The specificity of the responses for GPIIb-IIIa was confirmed by activation of GPIIb-IIIa-primed T cells by a recombinant GPIIbalpha fragment in secondary cultures. Characterization of T cell response induced by modified GPIIb-IIIa showed that the response was restricted by HLA-DR, the responding T cells had a CD4(+) phenotype, and the proliferation was accelerated only in ITP patients, suggesting in vivo activation of these T cells. In vitro IgG anti-GPIIb-IIIa synthesis in PBMC cultures was induced by modified GPIIb-IIIa specifically in ITP patients with platelet-associated anti-GPIIb-IIIa antibody. Anti-GPIIb-IIIa antibody produced in supernatants was absorbed by incubation with normal platelets. In summary, CD4(+) and HLA-DR-restricted T cells to GPIIb-IIIa are involved in production of anti-platelet autoantibody in ITP patients and are related to the pathogenic process in chronic ITP.
AIMS--Immunostaining of chromogranin identifies gastrointestinal mucosal endocrine cells. The detailed distribution and significance of chromogranin positive cells in colorectal carcinomas and in transitional mucosa remain unclear. The aim of this study was to clarify these aspects. METHODS--The distribution of chromogranin positive cells was studied by immunohistochemical methods in normal epithelium remote from carcinoma, in transitional mucosa, and in carcinomas of the colorectum. In selected cases northern or western blot analyses were performed. RESULTS--Chromogranin positive cells were seen in the lower third of the normal crypts and less frequently in transitional mucosa. Thirty five per cent (n = 38) of colorectal carcinomas showed immunohistochemically positive carcinoma cells in the tumour tissue. Northern and western blot analyses showed similar results. There was no difference in clinicopathological factors, including prognosis, between chromogranin positive cases of colorectal carcinoma (n = 38) and chromogranin negative cases (n = 70). CONCLUSIONS--Neuroendocrine cell differentiation is controlled in transitional mucosa and the presence of chromogranin positive cells in carcinoma tissue does not influence the patient's prognosis.
AIMS: To examine the detection limit of the ligase chain reaction kit for Chlamydia trachomatis, to study the inhibitory effect of phosphate on the ligase chain reaction, and to clarify the mechanism of inhibition. METHODS: Three reference serovars of C trachomatis--D/UW-3/Cx, F/UW-6/Cx, and L2/434/Bu--were used to test the sensitivity of the chlamydia ligase chain reaction. Comparison was made of the inhibition by phosphate before and after DNA amplification. Phosphate in up to 2.4 mM concentration was added to specimens of C trachomatis serovar D (1 to 50 inclusion forming units (IFU)/reaction) before DNA amplification to examine the concentration dependency of phosphate inhibition of the ligase chain reaction. RESULTS: The detection limits were 0.6 IFU/reaction for serovar D/UW-3/Cx and F/UW-6/Cx, and 0.4 IFU/reaction for L2/434/Bu. Phosphate inhibited the ligase chain reaction only when it was added before the amplification stage. The specimens containing chlamydia at 1 to 50 IFU/reaction were negative when the concentration of phosphate added at the prethermocycle stage was more than 1.2 mM. CONCLUSIONS: Ligase chain reaction analysis is a reliable method of diagnosing C trachomatis infection because of its high sensitivity. It would be clearly superior to the currently used methods if the problem of inhibitors could be eliminated. The mechanism of inhibition of the ligase chain reaction by phosphate was thought to be blockade of the amplification of the target DNA. The efficacy of the ligase chain reaction could be inhibited by phosphate in the urine, so duplicate dilution analysis of some negative specimens should be useful.
AIMS--To determine whether neural invasion in advanced gastric cancer is of clinicopathological significance. METHODS--The study population comprised 121 cases of primary advanced gastric carcinoma. Two paraffin wax embedded blocks taken from the central tissue slice in each primary tumour were used. For definitive recognition of neural invasion, immunostaining for S-100 protein was applied to one slide; the other slide was stained with haematoxylin and eosin. RESULTS--Neural invasion was recognised in 34 of 121 (28%) primary gastric carcinomas. There were significant differences in tumour size, depth of tumour invasion, stage, and curability between patients with and without neural invasion. The five year survival rates of patients with and without neural invasion were 10 and 50%, respectively. Multivariate analysis, however, demonstrated that neural invasion was not an independent prognostic factor. CONCLUSIONS--Neural invasion could be an additional useful factor for providing information about the malignant potential of gastric carcinoma. This may be analogous to vessel permeation which is thought to be important, but is not an independent prognostic factor.
Platelet aggregation contributes to arresting bleeding at wound sites, but may cause occlusion of atherosclerotic vessels, thus curtailing blood flow to vital organs. According to current dogma, the integrin alphaIIbbeta3 plays an exclusive role in linking platelets to one another through interactions with fibrinogen or vWf. We demonstrate here that, depending on shearing flow conditions, this process may require vWf binding to glycoprotein Ibalpha, even when alphaIIbbeta3 is competent to bind adhesive ligands. Platelet activation induced solely by high shear stress is initiated by glycoprotein Ibalpha interaction with vWf, but results in aggregation only if the latter can bind concurrently to alphaIIbbeta3. In contrast, platelets exposed to high shear rate after activation by exogenous agonists such as ADP and epinephrine can aggregate when fibrinogen is the alphaIIbbeta3 adhesive ligand, yet only if vWf binding to glycoprotein Ibalpha can also occur. Thus, the latter interaction appears to provide a bond with biomechanical properties necessary to overcome the effects of high shear rate and initiate interplatelet cohesion. These findings highlight the distinct function of two adhesive receptors mediating platelet aggregation under varying fluid dynamic conditions, and modify the current interpretation of a crucial event in hemostasis and thrombosis.
CS-834, (+)-[pivaloyloxymethyl (4R,5S,6S)-6-[(R)-1-hydroxyethyl]-4-methyl-7-oxo-3-[[(R)-5-oxopyrroli din-3-yl]thio]-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate], is an ester-type oral carbapenem prodrug, and an active metabolite is R-95867, which has antibacterial activity. CS-834 was administered orally to healthy male volunteers at single doses of 50, 100, 200, and 400 mg and at a multiple dose of 150 mg three times a day for 7 days to investigate its safety and pharmacokinetic profiles. Other studies were conducted to examine the effect of food intake on the bioavailability of CS-834 and also the effect of the coadministration of probenecid on the pharmacokinetics of CS-834. In the fasting state, the concentration of R-95867 in plasma reached maximum levels from 1.1 to 1.7 h after the oral administration of CS-834, followed by a monoexponential decrease. The maximum concentrations of R-95867 in serum (C[max]s) after the administration of CS-834 at doses of 50, 100, 200, and 400 mg were 0.51, 0.97, 1.59, and 2.51 microg/ml, respectively. The half-lives (t1/2s) were almost constant, approximately 0.7 h. The areas under the concentration-time curves (AUCs) were proportional to the doses, ranging from 50 to 400 mg x h/ml. The cumulative recoveries in urine were approximately 30 to 35% until 24 h after drug administration. The C(max), AUC, t1/2, and recovery in urine were not affected by food intake. Probenecid coadministration prolonged the t1/2, and it increased the C(max) and AUC for R-95867 by approximately 1.5- and 2.1-fold, respectively. The multiple-dose study showed no change in the pharmacokinetics from those for the single doses and no drug accumulation in the body. A mild transient soft stool was observed in one volunteer in the study with a single dose of 400 mg. In the multiple-dose study, mild transient soft stools were observed in six volunteers, one volunteer had mild transient diarrhea, and one volunteer had elevated serum glutamic oxalacetic transaminase and serum glutamic pyruvic transaminase levels (1.4- and 2.8-fold compared with the upper limits of normal, respectively). There were no other abnormal findings for objective symptoms or laboratory findings, including blood pressure, heart rate, electrocardiogram, body temperature, hematology, blood chemistry, and urinalysis.
AIMS--To determine the maturity of reticulocytes in patients with anaemia as a result of various haematological disorders including those with qualitative abnormalities such as ineffective erythropoiesis or dyserythropoiesis. METHODS--The number of mature reticulocytes was measured with flow cytometry in venous blood samples from 122 patients with haematological disorders and 100 healthy controls. Reticulocytes were classified into three categories by the fluorescence intensity of auramin O staining: low fluorescence ratio (LFR), medium fluorescence ratio (MFR), and high fluorescence ratio (HFR). Immature reticulocytes were determined as the aggregate of MFR and HFR (%). RESULTS--The mean (2SD) number of immature reticulocytes in 100 normal subjects was 9.0 (7.0)%. Significantly high mean values of immature reticulocytes with a normal or reduced reticulocyte count were shown in 90 patients with dyserythropoietic or ineffective erythropoietic conditions, such as acute myeloid leukaemia (AML) (n = 37), myelodysplastic syndrome (MDS) (n = 35), aplastic anaemia (AA) (n = 8), or megaloblastic anaemia (MA), (n = 6). Reticulocyte ratios returned to normal after successful treatment of patients with AML (n = 10) and MA (n = 3). However, high percentages of immature reticulocytes with increased reticulocyte counts were consistently observed in patients with enhanced erythropoiesis such as those with acquired autoimmune haemolytic anaemias (AIHA) (n = 4) or acute blood loss (ABL) (n = 4). Reticulocyte maturity was within the normal range in patients with reduced erythropoiesis such as occurs in chronic renal failure (CRF) (n = 11), or in iron deficiency anaemia (IDA) (n = 13). CONCLUSIONS--The evaluation of reticulocyte maturity with total reticulocyte count seems to be clinically useful for estimating the qualitative impairment of erythropoiesis, and so could help differentiate haematological disorders.