SurePath® LBC improves the diagnostic accuracy of intrahepatic and hilar cholangiocarcinoma.

INTRODUCTION: The current study aimed to compare cytology using SurePath® (SP)-LBC and biliary tissue histology (BTH) for the diagnosis of biliary disease. METHODS: Between January 2014 and December 2016, 57 patients underwent endoscopic retrograde cholangiopancreatography for the diagnosis of biliary disease. Biliary cytological samples were processed using SP-LBC and subsequently BTH was performed. A final diagnosis was confirmed by surgery (23 malignant cases) and clinical follow-up (34 benign and malignant cases): 18 extrahepatic cholangiocarcinoma; 17 intrahepatic/hilar cholangiocarcinoma (intra/H-CC); eight other malignant disease; and 14 benign biliary disease. The diagnoses made using SP-LBC and BTH were classified into four categories: (1) benign; (2) indeterminate; (3) suspicious for malignancy/malignant; and (4) inadequate. In addition, diagnostic accuracy was compared between SP-LBC and BTH. RESULTS: Although 23% (13/57) of BTH samples were classified as inadequate, all SP-LBC cases were classified as adequate. Among 43 malignant cases, 11 normal, four indeterminate and 28 suspicious for malignancy/malignant were found using SP-LBC (26%, 9% and 65%, respectively), in contrast to 10 inadequate, nine normal, 10 indeterminate and 14 suspicious for malignancy/malignant observed using BTH (23%, 21%, 23%, and 33%, respectively). The identification of malignant cells was strikingly different between SP-LBC and BTH. Furthermore, limited to intra/H-CC, accuracy was significantly higher using SP-LBC than using BTH (P < .001). CONCLUSIONS: SP-LBC of the biliary tract is a useful and reliable method for diagnosing biliary malignant disease and has an advantage over BTH for detecting malignant cells and accurately diagnosing intra/H-CC.

Genome-wide microRNA expression profiling in renal cell carcinoma: significant down-regulation of miR-141 and miR-200c.

We investigated expression profiles of microRNA (miRNA) in renal cell carcinoma [clear cell carcinomas (CCC) and chromophobe renal cell carcinomas (ChCC)] and in normal kidneys by using a miRNA microarray platform which covers a total of 470 human miRNAs (Sanger miRBase release 9.1). Unsupervised hierarchical cluster analysis revealed that CCC and ChCC were separable and that no subgroups were identified in CCCs. We found that 43 miRNAs were differentially expressed between CCC and normal kidney, of which 37 were significantly down-regulated in CCC and the other 6 were up-regulated. We also found that 57 miRNAs were differentially expressed between ChCC and normal kidney, of which 51 were significantly down-regulated in ChCC and the other 6 were up-regulated. Together, these observations indicate that expression of miRNAs tends to be down-regulated in both CCC and ChCC compared with normal kidney. We observed that miR-141 and miR-200c were the most significantly down-regulated miRNAs in CCCs. Indeed, in all cases of CCC analysed, both miR-141 and miR-200c were down-regulated in comparison with normal kidney. Microarray data and quantitative RT-PCR showed that these two miRNAs were expressed concordantly. TargetScan algorithm revealed that ZFHX1B mRNA is a hypothetical target of both miR-141 and -200c. We established by quantitative RT-PCR that, in CCCs in which miR-141 and miR-200c were down-regulated, ZFHX1B, a transcriptional repressor for CDH1/E-cadherin, tended to be up-regulated. Furthermore, we found that overexpression of miR-141 and miR-200c caused down-regulation of ZFHX1B and up-regulation of E-cadherin in two renal carcinoma cell lines, ACHN and 786-O. On the basis of these findings, we suggest that down-regulation of miR-141 and miR-200c in CCCs might be involved in suppression of CDH1/E-cadherin transcription via up-regulation of ZFHX1B.

Genome-wide analysis of DNA copy number alterations and gene expression in gastric cancer.

Genomic copy number aberrations (CNAs) are believed to play a major role in the development and progression of human cancers. Although many CNAs have been reported in gastric cancer, their genome-wide transcriptional consequences are poorly understood. In this study, to reveal the impact of CNAs on genome-wide expression in gastric cancer, we analysed 30 cases of gastric cancers for their CNAs by array comparative genomic hybridization (array CGH) and 24 of these 30 cases for their expression profiles by oligonucleotide-expression microarray. We found that with the application of laser microdissection, most CNAs were detected at higher frequency than in previous studies. Notably, gain at 20q13 was detected in almost all cases (97%), suggesting that this may play an important role in the pathogenesis of gastric cancer. By comparing the array CGH data with expression profiles of the same samples, we showed that both genomic amplification and deletion strongly influence the expression of genes in altered genomic regions. Furthermore, we identified 125 candidate genes, consisting of 114 up-regulated genes located in recurrent regions (>10%) of amplification and 11 down-regulated genes located in recurrent regions of deletion. Up-regulation of several candidate genes, such as CDC6, SEC61G, ANP32E, BYSL and FDFT1, was confirmed by immunohistochemistry. Interestingly, some candidate genes were localized at genomic loci adjacent to well-known genes such as EGFR, ERBB2 and SMAD4, and concordantly deregulated by genomic alterations. Based on these results, we propose that our list of candidate genes may contain novel genes involved in the pathogenesis of advanced gastric cancer.

High-resolution analysis of DNA copy number alterations and gene expression in renal clear cell carcinoma.

We analysed chromosomal copy number aberrations (CNAs) in renal cell carcinomas by array-based comparative genomic hybridization, using a genome-wide scanning array with 2304 BAC and PAC clones covering the whole human genome at a resolution of roughly 1.3 Mb. A total of 30 samples of renal cell carcinoma were analysed, including 26 cases of clear cell carcinoma (CCC) and four cases of chromophobe renal cell carcinoma (ChCC). In CCCs, gains of chromosomes 5q33.1-qter (58%), 7q11.22-q35 (35%) and 16p12.3-p13.12 (19%), and losses of chromosomes 3p25.1-p25.3 (77%), 3p21.31-p22.3 (81%), 3p14.1-p14.2 (77%), 8p23.3 (31%), 9q21.13-qter (19%) and 14q32.32-qter (38%) were detected. On the other hand, the patterns of CNAs differed markedly between CCCs and ChCCs. Next, we examined the correlation of CNAs with expression profiles in the same tumour samples in 22/26 cases of CCC, using oligonucleotide microarray. We extracted genes that were differentially expressed between cases with and without CNAs, and found that significantly more up-regulated genes were localized on chromosomes 5 and 7, where recurrent genomic gains have been detected. Conversely, significantly more down-regulated genes were localized on chromosomes 14 and 3, where recurrent genomic losses have been detected. These results revealed that CNAs were correlated with deregulation of gene expression in CCCs. Furthermore, we compared the patterns of genomic imbalance with histopathological features, and found that loss of 14q appeared to be a specific and additional genetic abnormality in high-grade CCC. When we compared the expression profiles of low-grade CCCs with those of high-grade CCCs, differentially down-regulated genes tended to be localized on chromosomes 14 and 9. Thus, it is suggested that copy number loss at 14q in high-grade CCC may be involved in the down-regulation of genes located in this region.

Functional assessment of microencapsulated porcine islets with agarose polystyrene sulfonic acid in vitro and in xenotransplantation.

We evaluated the functional efficacy of microencapsulated porcine islet xenografts transplanted into nonobese diabetic (NOD) mice. Islets were isolated from the pancreata of CSK miniature swine by manual collagenase digestion and Ficoll purification. Purified porcine islets were immediately encapsulated into microbeads of agarose polystyrene sulfonic acid (Ag-PSSa). They remained morphologically intact by dithizone staining after 7 days in culture. Insulin secretion from encapsulated islets was determined in response to glucose challenge during perifusion. When encapsulated islets were exposed to 200 mg/dL glucose, within 5 minutes, insulin release became 5-fold greater than that at 80 mg/dL. However, a second phase insulin secretion appeared in response to 250 mg/dL glucose challenge. In xenotransplantation, microencapsulated porcine islets (1000 to 1800 MC islets) were transplanted into the peritoneal cavity of diabetic NOD mice (n = 4) without immunosuppression. The survival times after the onset of diabetes were observed after both MC islets transplanted NOD mice and nontransplanted NOD mice (n = 4). MC islets transplant recipients had significantly (P < .05) longer survival (47.5 +/- 18.6; mean +/- SD) than nontransplanted NOD mice (21.0 +/- 9.31), although random blood glucose levels were not normalized.

Acute interstitial pneumonitis during chemotherapy for haematological malignancy.

Fourteen adult patients with haematological malignancies (eight non-Hodgkin's lymphoma, one multiple myeloma, one chronic lymphocytic leukaemia, two acute lymphoblastic leukaemia and two acute myeloid leukaemia) developed acute interstitial pneumonitis (IP) during the course of chemotherapy. All patients manifested high fever over 38 degrees C, bilateral diffuse pulmonary interstitial infiltrates in the chest radiograph and severe hypoxia without hypercapnia in the arterial blood gas analysis. Pathogenic microorganisms were not detected in repeated examinations in any patient. Chemotherapy given included various anti-neoplastic drugs. Five patients had received granulocyte colony-stimulating factor (G-CSF) for chemotherapy-induced leucopenia. The onset was associated with an increase of leucocytes in 10 patients. All patients were treated with high dose steroid hormone and broad spectrum antibiotics with or without anti-fungal agents, and three required mechanical ventilation. Eleven patients quickly recovered from these situations, whereas three died. Autopsies were done in two patients and disclosed pneumocystis carinii (PC) pneumonitis in one and non-specific pulmonary congestive oedema and fibrosis in the other. In conclusion, IP of unknown cause could develop in patients with various haematological malignancies especially at the recovery phase of chemotherapy-induced leucopenia irrespective of the previous G-CSF administration. High dose steroid hormone should be used as therapy for such patients as soon as possible after exclusion of an infective aetiology.

Diurnal variation and age-related changes of bone turnover markers in female Göttingen minipigs.

We investigated diurnal variation and age-related changes in bone turnover markers in female Gottingen minipigs. Ten females, 6-9 months of age, were used for confirmation of diurnal variation. Blood was collected at 3 h intervals for 24 h, and bone-specific alkaline phosphatase and intact osteocalcin (OC) levels were determined by enzyme immunoassay and radioimmunoassay, respectively. Urine was collected at 3 h intervals for 24 h using a tray attached to the bottom of the cage. The levels of N-terminal telopeptide of type I collagen (NTX) were determined by enzyme immunoassay. Pyridinoline and deoxypyridinoline were measured by high performance liquid chromatography. OC and NTX exhibited diurnal variation (Kruskal-Wallis test, P < 0.05), with the highest and lowest levels at 18:00 h (76.7 +/- 26.2 ng/ml) and 06:00 h (44.3 +/- 10.3 ng/ml), and at 03:00-05:59 h (550.4 +/- 82.4 nmol/micromol Cr) and 12:00-14:59 h (297.8 +/- 152.5 nmol/micromol Cr), respectively. In the study of age-related changes, blood and urine samples from 66 females (age range, 3-76 months) were examined to determine the bone turnover markers. All markers showed high correlations with age (0.569 < R(2) < 0.818). High levels of bone turnover markers were observed in young animals, decreasing with age (Kruskal-Wallis test, P < 0.01). The diurnal variation and age-related changes revealed in the present study will be useful in studies of bone diseases using female Gottingen minipigs.

Wiener filter-magnetoencephalography of visual cortical activity.

This paper reports a revised Wiener filter to resolve the inverse problem for magnetoencephalograms (MEGs) according to the structural and functional constraints based on magnetic resonance imaging (MRI) and functional magnetic resonance imaging (fMRI). Wiener filter-MEG imaging for half field stimulation with the chromatic stimulus resolved fast, slow and late responses in V1, V4 and the inferotemporal cortex, respectively. The time courses of these responses were roughly comparable with those reported by unit recording studies of the corresponding monkey visual cortical areas. Wiener filter-MEG imaging had comparable spatial resolution and better signal to noise ratio than fMRI. The background noise was robust in fMRI responses, but became virtually eliminated in Wiener filter responses. Wiener filter-MEG imaging with upper and lower quadrant field stimulation demonstrated V1 responses differentially distributed respectively in the lower and upper banks of the calcarine sulcus. These results demonstrate that responses in two cortical areas facing close to each other can be resolved by Wiener filter-MEG. The present method provides a way to image brain activities with millisecond- and millimeter-order spatiotemporal resolution.

Exposure time-dependent bactericidal activities of amoxicillin against Actinobacillus pleuropneumoniae; an in vitro and In vivo pharmacodynamic model.

The pharmacodynamic effects of amoxicillin against Actinobacillus pleuropneumoniae at exposure concentration above and below minimum inhibitory concentration (MIC) were evaluated in both in vitro and in vivo. In vitro, the growth and morphological change of A. pleuropneumoniae in culture medium was observed. In vivo, the efficacy of amoxicillin on experimentally induced A. pleuropneumoniae infection in disease-free pigs was evaluated. Fifteen pigs were divided into three groups (n = 5 per group). After the onset of clinical respiratory disease symptoms, 6 h post-infection, amoxicillin sustained-release injectable formulation was injected intramuscularly at 7.5 mg/kg/day (group I) and 15 mg/kg/day (group II). Then the serum concentration of amoxicillin was measured. An untreated infected group served as controls. In each amoxicillin administration group, if symptoms were not absent after 48 h, the pig was injected with the amoxicillin sustained-release injectable formulation again using the same dosage. In vitro, the growth of A. pleuropneumoniae inhibited by amoxicillin exposure at the concentration above the MIC (1.28 x MIC), and the inhibition time was in directly proportion to the time of amoxicillin exposure. Moreover, all the cells were lysed. Whereas the bacterial growth inhibition at the amoxicillin exposure concentration below the MIC (0.25 x MIC) was not done, and the shape of cells were normal or long filamentous. In vivo, the group I clinical and pathological score was higher than the group II, and the group I weight gain was significantly less than the group II. Performance with respect to weight gain corresponded with clinical signs. The infected control group was severely affected with an 80% (4/5) mortality rate 24-96 h post-challenge. The duration of time above MIC (T > MIC) of serum amoxicillin concentration in the group I was less than group II. The present studies suggest that amoxicillin has exposure time-dependent bactericidal activity against A. pleuropneumoniae.

Effects of subminimal inhibitory concentrations of amoxicillin against Actinobacillus pleuropneumoniae.

The bactericidal effects of amoxicillin at below minimum inhibitory concentration (MIC) against Actinobacillus pleuropneumoniae NB001 were studied in vitro and in vivo. In vivo, the efficacy of amoxicillin on experimentally induced A. pleuropneumoniae infection in disease-free pigs was evaluated. Nine pigs were divided into three groups and all three groups were housed in the same room. Group I pigs were given long-acting amoxicillin injection 22 h prior to A. pleuropneumoniae challenge. Group II pigs were also A. pleuropneumoniae challenged but not given long-acting amoxicillin. Group III pigs were not treated. In vitro, A. pleuropneumoniae growth was suppressed in porcine blood with amoxicillin at below MIC. In vivo, clinical signs of disease were absent or mild in group I during 50 h post-challenge, and serum amoxicillin concentration was already less than MIC from 15 h post-challenge. Infected group II controls were severely affected by the infection, and mortality reached 100% within 50 h post-challenge. All non-treated pigs in group III became infected with NB001 from infected control pigs, and they displayed severe clinical signs of disease within 24 h post-challenge of groups I and II, and died within 50 h post-challenge of groups I and II.

Classification of abomasal displacement in cows according to histopathology of the liver and clinical chemistry.

Histopathological features of livers and blood chemical values in cows with abomasal displacement were investigated. Liver biopsy samples were collected during redressment operations in 92 cows with abomasal displacement, and the samples were stained with haematoxylin and eosin or periodic acid Schiff (PAS). Blood was collected for chemical tests. Livers were histopathologically divided into the following four types: normal histology cases (21%), fatty degeneration cases (36%), cloudy swelling cases (19%) and fatty degeneration cases with cloudy swelling (24%). The number of PAS-positive samples was significantly higher in the normal histology group and significantly lower in the severe fatty degeneration group and severe cloudy swelling group. Cows with fatty degeneration had significantly higher levels of serum 3-hydroxybutyric acid, non-esterified fatty acid and aspartate aminotransferase than did those with cloudy swelling or normal histology. The results indicate that the morbid conditions of cows with abomasal displacement can be classified into four types.

Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses.

Acclimatization and response of minipigs toward humans.

As a first step toward making an efficient acclimatization methodology for minipigs, the reaction of Göttingen minipigs, 3-24 months of age, toward humans was investigated. All minipigs were kept in an individual cage, and the reaction toward humans (acclimatization index) was evaluated by simple observations. The acclimatization index was evaluated as the total number of points scored (0-30 points) based on the following criteria: (1) the position of the minipig when the cage door was opened; (2) the reaction of the minipig when the observer approached; and (31 the reaction of the minipig when the observer touched it. Subsequently, each animal was ranked by total points scored: 30 points = AA, 20 points < or = A < 30 points, 10 points < or = B < 20 points, 0 points < or = C < 10 points. Based on this evaluation, the reactions of minipigs under three conditions were investigated. The following findings were confirmed: first, minipig reaction to humans was influenced by monthly age; second, taming was possible under ordinary conditions of care, but we had to wait until 10 months of age on average for this to occur; third, if simple contact was made during care time, minipigs became tame within less than 4 weeks after the commencement of contact. We therefore consider it possible to artificially control the reaction of minipigs toward humans, and to make minipigs more available for experiments by adding control of the hereditary factors that influence this reaction.

Experience of vein grafting in Göttingen minipigs.

We experimented with vein grafting surgery on Göttingen minipigs. Using the internal jugular vein for the tissue graft, we performed side-to-side anastomosis to the carotid artery, to which it runs parallel. One key point in this surgery was to prevent vasospasm of the carotid artery so as to keep the lumen sufficiently patent during anastomosis. The histopathological findings in the grafts which remained patent resembled those of vein grafts in humans. We therefore considered that this technique in minipigs can be applied for the study of coronary artery bypass surgery in humans.

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.

[Aggressive transformation and extramedullary tumor formation in IgA-lambda multiple myeloma].

A 52-year-old woman complained of lower back pain and gluteal pain in April 1997, and was found to have anemia, hypercalcemia and renal disorder. In September of the same year, she was diagnosed as having IgA-lambda myeloma (stage IIIA). VMMD-IFN therapy was started in November, 1997, and this resulted in improvement of the M-protein level, and relief of the pain in the lower back and gluteal region. A second course of VMMD-IFN therapy was also effective. In April 1998, however, the back pain worsened, and in July the patient suffered a fall and fractured her left femur. Upon readmission to our hospital, the level of M-protein was lower, and high fever, hypercalcemia, renal disorder, elevation of the LDH level, anemia and thrombocytopenia were observed. Bone marrow examination revealed 30% atypical large-sized CD19-, CD38+, CD56+ myeloma cells and chromosomal abnormalities. Although the symptoms were improved temporarily after a third course of VMMD therapy, disease aggravation occurred again, and extramedullary masses appeared on the head, face and pelvis. VAD therapy was performed without effect, and the patient died about 2 months after recurrence. This was a comparatively rare case of fulminant multiple myeloma occurring in the terminal stage.

Enhancement of nitric oxide generation by low frequency electromagnetic field.

Oxidative stress is implicated in the intracellular signal transduction pathways for nitric oxide synthase (NOS) induction. The electromagnetic field (EMF) is believed to increase the free radical lifespan [S. Roy, Y. Noda, V. Eckert, M.G. Traber, A. Mori, R. Liburdy, L. Packer, The phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst in rat peritoneal neutrophils is increased by a 0.1 mT (60 Hz) magnetic field, FEBS Lett. 376 (1995) 164-6; F.S. Prato, M. Kavaliers, J.J. Carson, Behavioural evidence that magnetic field effects in the land snail, Cepaea nemoralis, might not depend on magnetite or induced electric currents, Bioelectromagnetics 17 (1996) 123-30; A.L. Hulbert, J. Metcalfe, R. Hesketh, Biological response to electromagnetic fields, FASEB 12 (1998) 395-420]. We tested the effects of EMF on endotoxin induced nitric oxide (NO) generation in vivo. Male BALB/C mice were injected with lipopolysaccharide (LPS) intraperitoneously (i.p.), followed by the exposure to EMF (0.1 mT, 60 Hz). Five hours and 30 min after the LPS administration, mice were administered with a NO spin trap, ferrous N-methyl-D-glucaminedithiocarbamate (MGD-Fe). Thirty minutes later, mice were sacrificed, and their livers were removed. The results were compared to three control groups: group A (LPS (-) EMF(-)); group B (LPS(-) EMF(+)); group C (LPS(+) EMF(-)). The ESR spectra of obtained livers were examined at room temperature. Three-line spectra of NO adducts were observed in the livers of all groups. In groups A and B very weak signals were observed, but in groups C and D strong spectra were observed. The signal intensity of the NO adducts in Group D was also significantly stronger than that in Group C. EMF itself did not induce NO generation, however, it enhanced LPS induced NO generation in vivo.

Murine cytomegalovirus infection causes apoptosis of uninfected retinal cells.

PURPOSE: To determine the role of apoptosis in prevention and/or exacerbation of retinal disease in a mouse model of cytomegalovirus retinitis. METHODS: Immunocompetent or T-cell- depleted BALB/c mice were injected with murine cytomegalovirus (MCMV) by supraciliary injection. On sequential days after infection, mice were killed, and eyes were harvested for cryosectioning or for DNA extraction. Ocular sections were stained with monoclonal antibodies specific for MCMV or for T cells or used in the TdT-dUTP terminal nick-end labeling (TUNEL) assay to detect apoptotic cells. RESULTS: In immunocompetent BALB/c mice, TUNEL assays revealed that a large area of the retina was apoptotic in relation to the relatively small number of MCMV-infected cells that were observed in the subjacent choroid and/or retinal pigment epithelium. In infected eyes from T-cell- depleted mice, there were more TUNEL-positive cells, and the areas of apoptosis were more extensive than in immunocompetent mice. These observations correlated with the increased extent of MCMV infection that is observed in the eyes of T-cell- depleted mice. However, irrespective of immune status, TUNEL-positive apoptotic cells were present mainly in areas of the retina overlying areas of MCMV-infected choroid and/or retinal pigment epithelium. More intense DNA laddering, indicative of increased apoptosis, was observed in the posterior segments of the eyes of T-cell- depleted mice after supraciliary inoculation with murine cytomegalovirus compared with less intense DNA laddering in the posterior segments of eyes of immunocompetent MCMV-infected mice. CONCLUSIONS: The ability of the mouse's immune system to control MCMV infections in some tissues depends on induction of apoptosis in virus-infected cells. However, in the retina, cells undergoing apoptosis were not virus-infected, a finding that suggests that apoptosis of uninfected retinal cells may play a role in the pathogenesis of MCMV retinitis.

HTLV-1 unrelated adult T-cell leukemia/lymphoma with unique phenotype and karyotype.

We describe a unique case of adult T-cell leukemia/lymphoma (ATL). The patient had typical clinicohematological features as ATL, but showed a lack of antibody to human T-cell leukemia virus type-1 (HTLV-1) and was negative for HTLV-1 proviral DNA in the peripheral mononuclear cells by means of polymerase chain reaction. The phenotype of tumor cells revealed CD7+, CD5+, CD2+, CD3+, WT31-, TcR delta 1-, CD4-, CD8-, CD25-, and the karyotype showed a 5q-, t(12;18). HTLV-1 unrelated ATL is very rare, and the karyotype as in our case has not been reported previously.

Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy.

Attempts were made to apply atomic force microscopy (AFM) imaging to the detection and mapping of the sites of base substitutions in DNA molecules. In essence, DNA fragments to be examined for possible base substitutions were mixed with an equal amount of a corresponding DNA standard and subjected to heat denaturation and subsequent annealing. The reassociated DNA was incubated with MutS protein, a protein that recognizes and binds to mismatched base pairs in duplex DNA. Bound MutS protein molecules were then detected by AFM and their positions along the DNA molecules were determined by calculating the distance from one of the DNA termini, which had been tagged with a biotin-avidin complex. Base substitutions present in DNA molecules >1 kb were effectively detected by this procedure, and the positions determined were in good agreement with the actual mutation sites. This method is quite simple, has virtually no limitations on the size of DNA fragments to be examined and requires only a very small amount of DNA sample.