Neurogenic cardiomyopathy in rabbits with experimentally induced rabies.

Cardiomyopathies have been rarely described in rabbits. Here we report myocardial necrosis of the ventricular wall in rabbits with experimentally induced rabies. Myocardial lesions were found only in rabbits with brain lesions, and the severity of the cardiac lesions was proportional to that of the brain lesions. Neither the frequency nor the cumulative dose of anesthesia was related to the incidence or the severity of the myocardial lesions. The myocardial lesions were characterized by degeneration and/or necrosis of myocardial cells and were accompanied by contraction band necrosis, interstitial fibrosis, and infiltration of inflammatory cells. The brain lesions due to rabies virus infection were most prominent in the cerebral cortex, thalamus, hypothalamus, brainstem, and medulla. Rabies virus antigen was not found in the hearts of any rabbits. Based on these findings, the myocardial lesions were classified as neurogenic cardiomyopathy.

Genetic polymorphism in IFNL4 and response to pegylated interferon-α and ribavirin in Japanese chronic hepatitis C patients.

A genetic polymorphism of the newly discovered interferon-λ 4 (IFNL4) gene was associated with hepatitis C virus (HCV) clearance in individuals of African ancestry. To assess whether a dinucleotide variant of IFNL4 (ss469415590) also affected treatment outcome of antiviral therapy in Japan, we genotyped 213 patients with chronic genotype 1 HCV infection and 176 healthy subjects. The ΔG allele was associated with treatment failure [odds ratio (OR) 4.73, P = 0.019], as was the IFL3 rs8099917 single nucleotide polymorphism (SNP) (OR 5.06, P = 0.068). The correlation between ss469415590 and rs8099917 was high (r(2) = 0.92, D' = 0.98). Multivariate analysis revealed that the rs8099917 SNP was independently associated with treatment failure (OR 5.28, P = 0.009). Therefore, ss469415590 may be another predictive marker of antiviral therapy outcome in the Japanese population.

Chronic cadmium treatment induces tubular nephropathy and osteomalacic osteopenia in ovariectomized cynomolgus monkeys.

In an attempt to establish a primate model of chronic cadmium toxicosis, we ovariectomized cynomolgus monkeys and treated them with CdCl2 by repeated intravenous injections for 13 to 15 months. The animals showed normocytic-normochromic anemia. The cadmium treatment resulted in increases of urinary enzyme activity indicative of renal tubular degeneration. Histopathology of the kidney revealed renal proximal tubular atrophy accompanied by interstitial fibrosis. Decreased bone mineral density was evident in the trabecular and cortical zones of the lumbar vertebra and femur, with osteoid accumulation around the trabeculae and Haversian canals. Iron deposition at the mineralization front and osteoclasts hyperplasia were indicative of impairment of bone mineralization and an increase of resorption. Blood inorganic phosphorus and 1α,25(OH)2 vitamin D3 levels decreased and urinary deoxypyridinoline level increased in cadmium-treated animals. The renal and bone lesions closely resemble those of itai-itai disease patients, the most severe case of cadmium toxicosis in terms of clinical chemistry and histopathology. Thus, ovariectomized monkeys chronically exposed to cadmium can serve as a primate itai-itai disease model, which is beneficial for developing novel therapeutic methods, investigating the mechanisms of the renal and bone lesions, and establishing more clearly defined criteria for diagnosing the disease.

Flumequine enhances the in vivo mutagenicity of MeIQx in the mouse liver.

The combined effects of various carcinogens found in food products are a concern for human health. In the present study, the effects of flumequine (FL) on the in vivo mutagenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the liver were investigated. Additionally, we attempted to clarify the underlying mechanisms through comprehensive gene analysis using a cDNA microarray. Male gpt delta mice were fed a diet of 0.03 % MeIQx, 0.4 % FL, or 0.03 % MeIQx + 0.4 % FL for 13 weeks. The effects of cotreatment with phenobarbital (PB) were also examined. Treatment with MeIQx alone increased gpt and Spi(-) mutant frequencies, and cotreatment with FL, but not with PB, further exacerbated these effects, despite the lack of in vivo genotoxicity in mice treated with FL alone. FL caused an increase in Cyp1a2 mRNA levels and a decrease in Ugt1b1 mRNA levels, suggesting that the enhancing effects of FL may be due in part to modification of MeIQx metabolism by FL. Moreover, FL induced an increase in hepatocyte proliferation accompanied by hepatocellular injury. Increases in the mRNA levels of genes encoding cytokines derived from Kupffer cells, such as Il1b and Tnf, and cell cycle-related genes, such as Ccnd1 and Ccne1, suggested that FL treatment increases compensatory cell proliferation. Thus, the present study clearly demonstrated the combined effects of 2 different types of carcinogens known as contaminants in foods.

Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers.

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

ADC Level is Related to DWI Reversal in Patients Undergoing Mechanical Thrombectomy: A Retrospective Cohort Study.

BACKGROUND AND PURPOSE: In patients with ischemic stroke, DWI lesions can occasionally be reversed by reperfusion therapy. This study aimed to ascertain the relationship between ADC levels and DWI reversal in patients with acute ischemic stroke who underwent recanalization treatment. MATERIALS AND METHODS: We conducted a retrospective cohort study in patients with acute ischemic stroke who underwent endovascular mechanical thrombectomy with successful recanalization between April 2017 and March 2021. DWI reversal was assessed through follow-up MR imaging approximately 24 hours after treatment. RESULTS: In total, 118 patients were included. DWI reversal was confirmed in 42 patients. The ADC level in patients with reversal was significantly higher than that in patients without reversal. Eighty-three percent of patients with DWI reversal areas had mean ADC levels of ≥520 × 10-6 mm2/s, and 71% of patients without DWI reversal areas had mean ADC levels of <520 × 10-6 mm2/s. The mean ADC threshold was 520 × 10-6 mm2/s with a sensitivity and specificity of 71% and 83%, respectively. In multivariate analysis, the mean ADC level (OR, 1.023; 95% CI, 1.013-1.033; P < .0001) was independently associated with DWI reversal. Patients with DWI reversal areas had earlier neurologic improvement (NIHSS at 7 days) than patients without reversal areas (P < .0001). CONCLUSIONS: In acute ischemic stroke, the ADC value is independently associated with DWI reversal. Lesions with a mean ADC of ≥520 × 10-6 mm2/s are salvageable by mechanical thrombectomy, and DWI reversal areas regain neurologic function. The ADC value is easily assessed and is a useful tool to predict viable lesions.

Impact of hospital environmental cleaning with a potassium peroxymonosulphate-based environmental disinfectant and antimicrobial stewardship on the reduction of hospital-onset Clostridioides difficile infections.

BACKGROUND: A 1% potassium peroxymonosulphate-based environmental disinfectant (PPED) produces sodium hypochlorite when combined with sodium chloride, which functions as a disinfectant. However, little is known about the impact of hospital cleaning with PPED on hospital-onset Clostridioides difficile infection (HO-CDI). AIM: To reduce HO-CDI, we promoted antimicrobial stewardship and hospital ward cleaning with PPED: this study was conducted to evaluate their impact. METHODS: We began a promotion of post-prescription review with feedback for broad-spectrum antimicrobials and hospital ward cleaning with PPED. We reviewed the ratio of HO-CDI, PPED consumption, and days of therapy (DOT) of broad-spectrum antimicrobials between July 2014 and March 2018, dividing this time into the pre-promotion (July 2014 to June 2015) and post-promotion periods (July 2015 to March 2018). FINDINGS: Using interrupted time series analysis, an immediate significant change in HO-CDI was observed after intervention (P=0.03), although a downward trend was not observed over this period (P=0.19). Trends in PPED consumption significantly changed over this period (P=0.02). DOT of carbapenems decreased immediately after the intervention began (P<0.01). A Poisson regression analysis showed that PPED consumption and DOT of carbapenems were independent factors affecting HO-CDI (P=0.039 and 0.016, respectively). CONCLUSION: We revealed that DOT of carbapenems and use of PPED were associated with the HO-CDI ratio and that both interventions reduced the rate of HO-CDI. This is the first report on the impact of hospital ward cleaning with PPED on the reduction of HO-CDI.

Endothelial and inflammatory markers in relation to progression of ischaemic cerebral small-vessel disease and cognitive impairment: a 6-year longitudinal study in patients with type 2 diabetes mellitus.

BACKGROUND: Progression of silent brain infarctions (SBIs) and white-matter lesions (WMLs) seen on brain MRI is associated with an increased risk of cognitive impairment, but their relation to endothelial and inflammatory markers is unknown in type 2 diabetes mellitus. METHODS: In 190 type 2 diabetic outpatients (mean age 62.7 years), the authors related baseline levels of soluble intercellular adhesion molecule-1 (sICAM-1) and high-sensitivity C-reactive protein (hs-CRP) to subsequent brain MRI findings and cognitive function. The authors assessed incident SBIs and changes in periventricular and subcortical WMLs (PVWMLs and SCWMLs) on MRI performed at baseline and 3 and 6 years. Neuropsychological tests were administered to 83 patients older than 65 years at 6 years. This present study represents an extension of the authors' previously published study. RESULTS: SBIs were observed in 46 patients (24.2%), PVWMLs in 93 (48.9%) and SCWMLs in 87 (45.8%) on baseline MRI. After adjustment for age, gender, hypertension, duration of diabetes, baseline MRI findings and medication use, the relative odds associated with a 1SD increase in sICAM-1 levels at baseline were 1.67 (95% CI 1.02 to 3.05) for SBI progression and 2.17 (95% CI 1.29 to 3.62) for PVWML progression at 6 years. In contrast, baseline hs-CRP levels were significantly associated with SBI progression only at 3 years. Significant trends were observed between quartiles of sICAM-1 at baseline and scores in Digit Symbol substitution (p for trend=0.01). CONCLUSIONS: The findings suggest that higher sICAM-1 levels are associated with SBI and PVWML progression, and may predict impairment in psychomotor function in type 2 diabetes.

A novel heterophilic antibody interaction involves IgG4.

IgG4 has been implicated in a diverse set of complex pathologies - e.g. autoimmune pancreatitis (AIP), idiopathic membranous nephropathy - and carries unique features including lack of activation of the classical complement pathway and a dynamic Fab-arm exchange. We recently showed that the rheumatoid factor (RF)-like activity of IgG4 is achieved through a hitherto unknown, Fc-Fc (and not Fab-Fc as is the case in classical RF; CRF) interaction; hence the name, novel RF (NRF). Here, we further explore the resemblance/difference between CRF and NRF. As heterophilic interactions of human IgM RF (CRF) are well known, we checked whether this is the case for IgG4. Human IgG4 showed variable reactivity to animal IgGs: reacting intensely with rabbit and mouse IgGs, but weakly with others. The binding to rabbit IgG was not through the Fab (as in CRF) but via the Fc piece, as was recently shown for human IgG (NRF). This binding correlates with the IgG4 concentration per se and could therefore be of diagnostic usage and incidentally explain some observed interferences in biological assays. In conclusion, here is defined a novel heterophilic antibody interaction and is established the universality of the unique Fc-Fc binding, both involving IgG4.

Pathology of lethal peripartum broad ligament haematoma in 31 Thoroughbred mares.

REASONS FOR PERFORMING STUDY: Broad ligament haemorrhage in peripartum mares is a life-threatening disease and there are few reports on the aetiology and pathogenesis of broad ligament haematoma. OBJECTIVES: To obtain information regarding the sites for the early diagnosis and pathogenesis of broad ligament haematoma of mares. METHODS: Thirty-one mares that died of broad ligament haematoma peripartum were examined pathologically for bleeding sites. The arterial distribution of 5 young mares with several parities served as negative controls. RESULTS: Age and/or multiparity were the predisposing factors for the disease. Arterial injuries were most commonly observed in the uterine artery (24 of 31 mares). Among these, the proximal uterine artery that lies within 15 cm of the bifurcation of the iliac artery was the most frequent site for rupture (18 mares). The lesions occurred preferentially at the bifurcations, lateral part of curvatures and abrupt flexures of the artery. The morphology of the injuries was classified into 3 types: ruptures with and without longitudinal fissures, and transections. Histologically, the arterial wall adjacent to the rupture showed atrophy of smooth muscle cells with fibrosis of the tunica media and disruption and/or calcification of the internal elastic lamina. CONCLUSIONS: Arterial injuries that led to broad ligament haematoma in peripartum mares occurred most frequently in the proximal uterine artery, and atrophy of smooth muscle cells with fibrosis of the arterial wall was as one of the predisposing factors in aged and multiparous mares. POTENTIAL RELEVANCE: Monitoring small aneurysms, mural tearing, medial fibrosis at the proximal uterine artery by transrectal echography could provide useful information for the early diagnosis and possible prevention of broad ligament haematoma of peripartum mares.

Safety assessment of ellagic acid, a food additive, in a subchronic toxicity study using F344 rats.

Ellagic acid is a phenolic acid compound, used as a food additive for its antioxidative properties. Because of its chemical characteristics, use is also to be expected in cosmetics. The present 90-day subchronic toxicity study was performed in F344 rats at dose levels of 0, 1.25, 2.5 and 5% in powdered basal diet, with actual doses of 9.4, 19.1, 39.1 g/kg b.w., respectively, in males, and 10.1, 20.1, 42.3 g/kg b.w. in females. No mortality or treatment-related clinical signs were observed throughout the experimental period. Body weight gain was significantly reduced from weeks 3 (5% group), 6 (2.5% group) and 7 (1.25% group) to the end of the experiment (except week 8 in the lowest group) in the treated females, the final body weights being decreased in the 5% (92.5%), 2.5% (94.2%) and 1.25% (94.8%) treated groups as compared to the control. Changes in MCV and serum AST, ALP, Ca, Cl and P were sporadically observed, but these were not considered to be treatment-related alterations. There were no obvious histopathological changes in any of the groups. The no-observed-effect level (NOEL) was estimated to be 5% (3011 mg/kg b.w./day) for males and the no-observed-adverse-effect level (NOAEL) and NOEL in females were estimated to be 5% (3254 mg/kg b.w./day) and <1.25% (778 mg/kg b.w./day), respectively.

Safety assessment of dietary administered paprika color in combined chronic toxicity and carcinogenicity studies using F344 rats.

Combined chronic toxicity and carcinogenicity studies of paprika color, used as a food additive in various countries, were performed in male and female F344 rats. Dietary concentrations of 0%, 0.62%, 1.25%, 2.5% and 5% were applied in a 52-week toxicity study and 0%, 2.5% and 5% in a 104-week carcinogenicity study. Treatment with paprika color caused a significant increase in incidence of hepatocellular vacuolation in 5% males, but no toxicological effects were found with reference to survival rates, body weights, hematological or serum biochemical parameters and organ weights at any dose level in either sex in the chronic toxicity study. Also, paprika color did not induce specific tumors nor did it exert significant influence on the development of spontaneous tumors in any of the organs examined in the carcinogenicity study. In conclusion, based on slight histopathological changes observed in 5% male livers, the no-observed-effect level (NOEL) was estimated to be 2.5% in the diet (1,253 mg/kg bw/day) and the no-observed-adverse-effect level (NOAEL) was determined to be 5% in the diet (2,388 mg/kg bw/day) for male rats, and for females, the NOEL was concluded to be 5% in the diet (2,826 mg/kg bw/day). Additionally, paprika color was not carcinogenic to male and female F344 rats under the present experimental conditions.

Evaluation of bovine viral diarrhoea virus control strategies in dairy herds in Hokkaido, Japan, using stochastic modelling.

Bovine viral diarrhoea virus (BVDV) infection in cattle can result in growth retardation, reduced milk production, reproductive disorders and death. Persistently infected animals are the primary source of infection. In Hokkaido, Japan, all cattle entering shared pastures in summer are vaccinated before movement for disease control. Additionally, these cattle may be tested for BVDV and culled if positive. However, the effectiveness of this control strategy aiming to reduce the number of BVDV-infected animals has not been assessed. The aim of this study was to evaluate the effectiveness of various test-and-cull and/or vaccination strategies on BVDV control in dairy farms in two districts of Hokkaido, Nemuro and Hiyama. A stochastic model was developed to compare the different control strategies over a 10-year period. The model was individual-based and simulated disease dynamics both within and between herds. Parameters included in the model were obtained from the literature, the Hokkaido government and the Japanese Ministry of Agriculture, Forestry and Fisheries. Nine different scenarios were compared as follows: no control, test-and-cull strategies based on antigen testing of either calves or only cattle entering common pastures, vaccination of all adult cattle or only cattle entering shared pastures and combinations thereof. The results indicate that current strategies for BVDV control in Hokkaido slightly reduced the number of BVDV-infected animals; however, alternative strategies such as testing all calves and culling any positives or vaccinating all susceptible adult animals dramatically reduced those. To our knowledge, this is the first report regarding the comparison of the effectiveness between the current strategies in Hokkaido and the alternative strategies for BVDV control measures.

Lack of chemopreventive effects of alpha-eleostearic acid on 7,12-dimethylbenz[a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis in female Sprague-Dawley rats.

alpha-Eleostearic acid is one of the conjugated linolenic acids from tung oil, which is obtained from the seeds of Aleurites fordii. The effects of dietary alpha-eleostearic acid (18:3, n-5) on the post-initiation period of 7,12-dimethylbenz[a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis were examined using female Sprague-Dawley (SD) rats. For initiation, rats were given subcutaneous injections of 40mg/kg body weight (5 times) and 20mg/kg body weight (3 times) of DMH during the age of 6-8 weeks and a single intragastric administration of 50mg/kg body weight of DMBA at 9 weeks. Then, the animals were treated with 0%, 0.01%, 0.1% or 1.0% alpha-eleostearic acid for 34 weeks. Control rats received the basal diet alone or 1.0% alpha-eleostearic acid without prior initiation treatment. All surviving animals were killed at week 37 of the experiment. There were no statistically significant alterations in any of the parameters for either mammary or colon tumors. These results thus indicate that alpha-eleostearic acid does not exert clear modification effects on DMBA and DMH-induced mammary and colon carcinogenesis, at least under the present experimental conditions.

A subchronic toxicity study of dunaliella carotene in F344 rats.

Dunaliella carotene, extracted from dunaliella alga (Dunaliella bardawil or Dunaliella salina), for use as a food-coloring agent, has beta-carotene as its mainly constituent. As there have been no reports of toxicological evaluation, a 90-day subchronic toxicity study was here performed in F344 rats at dose levels of 0 (control), 0.63%, 1.25%, 2.5% and 5% in powdered basal diet. The average daily intakes of dunaliella carotene were 352, 696, 1420 and 2750 mg/kg/day, respectively, for males, and 370, 748, 1444 and 2879 mg/kg/day for females. No mortality or treatment-related clinical signs were observed throughout the experimental period in any of the groups. Body weight gain was slightly but significantly (p < 0.05) reduced from week 5 to the end of the experiment in 2.5% and 5% males. Increased PLT were observed in 1.25% and 5% males, and 2.5% and 5% females. Significant elevations or tendencies for increase in serum T. Cho and Ca were observed in all treated males and females, with clear dose-dependence in males. Organ weight measurement and histopathological observation revealed no toxicological changes. Based on growth suppression, no-observed-adverse-effect-levels (NOAELs) were estimated to be 1.25% (696 mg/kg/day) for males and 5% (2879 mg/kg/day) for females. As increases in serum Ca were observed in the lowest group in both sexes, a no-observed-effect level (NOEL) could not be determined in this study.

Diabetes mellitus prevents ischemic preconditioning in patients with a first acute anterior wall myocardial infarction.

OBJECTIVES: This study was undertaken to assess whether prodromal angina could have beneficial effects in diabetic patients with acute myocardial infarction (AMI). BACKGROUND: Prodromal angina occurring shortly before the onset of AMI is associated with favorable outcomes by the mechanism of ischemic preconditioning. However, little is known about the impact of diabetes on ischemic preconditioning. METHODS: We studied 611 patients with a first anterior wall AMI who underwent emergency catheterization within 12 h after the onset of chest pain: 490 patients without diabetes and 121 patients with non-insulin treated diabetes. Prodromal angina was defined as angina episode(s) occurring within 24 h before the onset of AMI. Serial contrast left ventriculograms were obtained in 424 patients at the time of acute and predischarge catheterization. RESULTS: In non-diabetic patients, prodromal angina was associated with lower peak creatine kinase (CK) value (3,068 +/- 2,647 IU/l vs. 3,601 +/- 2,462 IU/l, p = 0.037), larger increase in left ventricular ejection fraction (LVEF) (10.1 +/- 13.0% vs. 5.8 +/- 13.4%, p = 0.004) and lower in-hospital mortality (3.4% vs. 9.3%, p = 0.015). On the contrary, in diabetic patients, there was no significant difference in peak CK value (3,382 +/- 2,520 IU/l vs. 3,233 +/- 2,412 IU/l, p = NS), the change in LVEF (6.7 +/- 13.8% vs. 7.1 +/- 12.4%, p = NS) and in-hospital mortality (8.8% vs. 11.0%, p = NS) between patients with and patients without prodromal angina. CONCLUSIONS: Prodromal angina limited infarct size, enhanced recovery of LV function and improved survival in non-diabetic patients with AMI. However, such beneficial effects of prodromal angina were not observed in diabetic patients, suggesting that diabetes might prevent ischemic preconditioning.

Evolution of the archaeal rhodopsins: evolution rate changes by gene duplication and functional differentiation.

The amino acid sequences of 25 archaeal retinal proteins from 13 different strains of extreme halophiles were analyzed to establish their molecular phylogenetic relationship. On the basis of amino acid sequence similarity, these proteins apparently formed a distinct family designated as the archaeal rhodopsin family (ARF), which was not related to other known proteins, including G protein-coupled receptors. The archaeal rhodopsin family was further divided into four clusters with different functions; H+ pump (bacteriorhodopsin), Cl- pump (halorhodopsin), and two kinds of sensor (sensory rhodopsin and phoborhodopsin). These four rhodopsin clusters seemed to have occurred by gene duplication(s) before the generic speciation of halophilic archaea, based on phylogenetic analysis. Therefore, the degrees of differences in amino acid sequences within each cluster simply reflected the divergent evolution of halophilic archaea. By comparing the branch lengths after speciation points of the reconstituted tree, we calculated the relative evolution rates of the four archaeal rhodopsins bacteriorhodopsin:halorhodopsin:sensory rhodopsin: phoborhodopsin to be 5:4:3:10. From these values, the degrees of functional and structural restriction of each protein can be inferred. The branching topology of four clusters grouped bacteriorhodopsin and halorhodopsin versus sensory rhodopsin and phoborhodopsin by likelihood mapping. Using bacteriorhodopsin (and halorhodopsin) as an outgroup, the gene duplication point of sensory rhodopsin/phoborhodopsin was determined. By calculating the branch lengths between the gene duplication point and each halophilic archaea speciation point, we could speculate upon the relative evolution rate of pre-sensory rhodopsin and pre-phoborhodopsin. The evolution rate of pre-sensory rhodopsin was fivefold faster than that of pre-phoborhodopsin, which suggests that the original function of the ancestral sensor was similar to that of phoborhodopsin, and that sensory rhodopsin evolved from pre-sensory rhodopsin by the accumulation of mutations. The changes in evolution rate by gene duplication and functional differentiation were demonstrated in the archaeal rhodopsin family using the gene duplication date and halobacterial speciation date as common time stamps.

Detection of bcr/abl mRNA in stained bone marrow smears.

We describe the polymerase chain reaction (PCR) method using stained bone marrow smears as sources of RNA. The amount of extractable RNA decreased during the process of making and staining bone marrow smears. The sensitivity of the reverse transcriptase-based polymerase chain reaction (RT-PCR) method for detecting target mRNA-positive cells in 5 x 10(5) suspended cells and stained bone marrow smears were 1:10(5) and 1:5000, when we used K562 cells. The bone marrow smears of 21 patients with chronic myelogenous leukemia (CML) were examined using this method. We extracted RNA from stained specimens stored at room temperature for 5-14 years. Twelve of 21 (57%) smears showed positive results for bcr/abl. The carrier RNA improved the recovery when added at the step of RNA extraction. These data indicate that mRNA is present in stained bone marrow smears for at least 14 years and that the sensitivity of RT-PCR is adequate for molecular analysis.

P190-type bcr/abl expressed in myeloid colonies in a patient with Ph1-positive acute lymphoblastic leukemia.

In Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL), some cytogenetic studies have suggested clonal derivation from a multipotential stem cell. The role of the product of the chimeric gene, P190, is not, however, well understood. We examined the expression of P190-type bcr/abl in single hematopoietic colonies obtained at various clinical stages of a patient with Ph1-positive ALL, using the polymerase chain reaction (PCR). Seven out of 58 colonies examined expressed P190-type bcr/abl. Five out of seven colonies were granulocyte/macrophage (GM) colonies and two were erythroid colonies. The cell lineages of these colonies were confirmed by testing for the expressions of the myeloperoxidase (MPO) gene in the GM colonies and the beta-globin gene in the erythroid colonies. These results suggest transformation of multipotential stem cell in this patient and confirm that expression of the P190-type bcr/abl fusion gene permits stem cell differentiation leading to Ph1-positive ALL.

Lifetime treatment of mice with azidothymidine (AZT) produces myelodysplasia.

AZT has induced a macrocytic anemia in AIDS patients on long term AZT therapy. It is generally assumed that DNA elongation is stopped by the insertion of AZT into the chain in place of thymidine thus preventing the phosphate hydroxyl linkages and therefore suppresses hemopoietic progenitor cell proliferation in an early stage of differentiation. CBA/Ca male mice started on AZT 0.75 mg/ml H2O at 84 days of age and kept on it for 687 days when dosage reduced to 0.5 mg/ml H2O for a group, another group removed from AZT to see recovery, and third group remained on 0.75 mg. At 687 days mice that had been on 0.75 mg had average platelet counts of 2.5 x 10(6). Histological examination on 9 of 10 mice with such thrombocytopenia showed changes compatible with myelodysplastic syndrome (MDS). A variety of histological patterns was observed. There were two cases of hypocellular myelodysplasia, two cases of hypersegmented myelodysplastic granulocytosis, two cases of hypercellular marrow with abnormal megakaryocytes with bizarre nuclei, one case of megakaryocytic myelosis associated with a hyperplastic marrow, dysmyelopoiesis and a hypocellular marrow and two cases of myelodysplasia with dyserythropoiesis, hemosiderosis and a hypocellular marrow. Above mentioned AZT incorporation may have induced an ineffective hemopoiesis in the primitive hemopoietic progenitor cells, which is known to be seen commonly in the myelodysplastic syndrome.