Spontaneous adrenocorticotropic hormone (ACTH) normalisation due to tumour regression induced by metyrapone in a patient with ectopic ACTH syndrome: case report and literature review.

BACKGROUND: Ectopic adrenocorticotropic hormone (ACTH) syndrome (EAS) is caused by tumours releasing ACTH. Ectopic ACTH-producing tumour regression is rarely induced using steroidogenesis inhibitors. We presented a case of EAS in which ACTH production by a lung tumour was reduced by metyrapone (MTP) and also reviewed previous cases of ectopic ACTH production suppressed via steroidogenesis inhibition. CASE PRESENTATION: A 71-year-old female with general fatigue, central obesity and impaired glucose tolerance was diagnosed with Cushing's syndrome due to elevated ACTH (192.9 pg/mL; normal range, 7.2-63.3 pg/mL), cortisol (73.1 µg/dL; 6.4-21.0 µg/dL) and 24-h urinary free cortisol (UFC) (6160 µg/day; 11.2-80.3 µg/day) levels. Chest computed tomography identified a solid 26.6 × 22.9 × 30.0 mm tumour with a cavity in the upper lobe of the left lung. There was no adrenal gland enlargement. Tumour markers were not significantly elevated; ACTH levels were not suppressed by 8-mg dexamethasone. A corticotropin-releasing hormone stimulation test revealed blunted ACTH response (basal ACTH, 204.6 pg/mL; highest ACTH level during the 120-min stimulation test, 214.0 pg/mL). She was diagnosed with EAS due to a lung lesion. MTP treatment was started to reduce cortisol production. ACTH levels and cortisol and UFC levels were normalised and the ACTH-producing lung tumour was ablated after MTP treatment. In several reported cases, plasma ACTH levels reduced during steroidogenesis inhibitor treatment for EAS. Among the 10 patients, three cases of pheochromocytoma, one of thymic carcinoid and one of islet cell carcinoma were reported. In four cases, the tumour was not detected. In our case, the pathology of the lung tumour was unknown because of lack of tumour cells in biopsy. The patients were treated with ketoconazole (KTZ) and/or MTP and exhibited ACTH and cortisol/UFC suppression, but tumour regression was observed only in our case. CONCLUSION: MTP and/or KTZ may reduce ACTH and cortisol production. The tumour spontaneously regressed after MTP treatment, indicating that MTP may reduce the tumour size without surgery. The mechanisms of therapeutic effects of steroidogenesis inhibitors and prognosis of spontaneous remission should be elucidated further via molecular biology studies.

Reducing echocardiographic examination time through routine use of fully automated software: a comparative study of measurement and report creation time.

BACKGROUND: Manual interpretation of echocardiographic data is time-consuming and operator-dependent. With the advent of artificial intelligence (AI), there is a growing interest in its potential to streamline echocardiographic interpretation and reduce variability. This study aimed to compare the time taken for measurements by AI to that by human experts after converting the acquired dynamic images into DICOM data. METHODS: Twenty-three consecutive patients were examined by a single operator, with varying image quality and different medical conditions. Echocardiographic parameters were independently evaluated by human expert using the manual method and the fully automated US2.ai software. The automated processes facilitated by the US2.ai software encompass real-time processing of 2D and Doppler data, measurement of clinically important variables (such as LV function and geometry), automated parameter assessment, and report generation with findings and comments aligned with guidelines. We assessed the duration required for echocardiographic measurements and report creation. RESULTS: The AI significantly reduced the measurement time compared to the manual method (159 ± 66 vs. 325 ± 94 s, p < 0.01). In the report creation step, AI was also significantly faster compared to the manual method (71 ± 39 vs. 429 ± 128 s, p < 0.01). The incorporation of AI into echocardiographic analysis led to a 70% reduction in measurement and report creation time compared to manual methods. In cases with fair or poor image quality, AI required more corrections and extended measurement time than in cases of good image quality. Report creation time was longer in cases with increased report complexity due to human confirmation of AI-generated findings. CONCLUSIONS: This fully automated software has the potential to serve as an efficient tool for echocardiographic analysis, offering results that enhance clinical workflow by providing rapid, zero-click reports, thereby adding significant value.

Determination of Venetoclax Concentration in Plasma Using High-Performance Liquid Chromatography.

Venetoclax is an oral B-cell lymphoma-2 protein inhibitor. It is a key drug for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia. However, venetoclax is administered at a fixed dose, irrespective of body surface area or weight. Furthermore, the plasma concentration of venetoclax varies widely between individuals and is influenced by diet. Therefore, individualized dosing using therapeutic drug monitoring (TDM) may help to optimize treatment in clinical practice. In this study, we aimed to develop a simple method to determine venetoclax concentrations in plasma. The analysis required the extraction of a 50-µL plasma sample and precipitation of proteins using acetonitrile extraction. Venetoclax and the internal standard (12.5-µg/mL ibrutinib) were separated by high-performance liquid chromatography (HPLC). The calibration curve was linear over the plasma venetoclax concentration range 0.25-10 µg/mL with a coefficient of determination (r2) of 0.9999. The coefficients of intra-day and inter-day validation were 0.8-4.1% and 1.3-3.3%, respectively. The assay accuracy was -2.8 to 1.6%, and the recovery was >97.2%. These results demonstrate a very simple, novel and sensitive HPLC-UV-based method for determining the concentration of plasma venetoclax, and confirm its applicability to the TDM of venetoclax in a clinical setting.

High-Performance Liquid Chromatography for Ultra-Simple Determination of Plasma Voriconazole Concentration.

Voriconazole is an antifungal drug used to treat invasive aspergillosis. Voriconazole exhibits nonlinear behavior and considerable individual variability in its pharmacokinetic profile. Invasive aspergillosis has a poor prognosis, and failure of treatment owing to low voriconazole blood levels is undesirable. Thus, therapeutic drug monitoring (TDM) of voriconazole is recommended. However, plasma voriconazole concentration is rarely measured in hospitals, and the TDM of voriconazole is not widely practiced in Japan. We aimed to develop an ultra-simple method to measure plasma voriconazole concentration. Ten microliters of plasma sample was extracted, and proteins were precipitated using methanol extraction. Voriconazole and ketoconazole (internal standard) were separated using high-performance liquid chromatography. A calibration curve was prepared, which was linear over plasma voriconazole concentrations of 0.125−12.5 µg/mL, with a coefficient of determination of 0.9999. The intra-day and inter-day validation coefficients were 0.9−2.2% and 1.3−6.1%, respectively. The assay accuracy was −4.2% to 1.6%, and recovery was >97.8%. Our ultra-simple, sensitive, and inexpensive high-performance liquid chromatography ultraviolet method to determine plasma voriconazole concentration will help improve the voriconazole TDM implementation rate and contribute to effective and safe voriconazole use.

Irrespective of CD34 expression, lineage-committed cell fraction reconstitutes and re-establishes transformed Philadelphia chromosome-positive leukemia in NOD/SCID/IL-2Rgammac-/- mice.

Stem cells of acute myeloid leukemia (AML) have been identified as immunodeficient mouse-repopulating cells with a Lin(-)CD34(+)38(-) phenotype similar to normal hematopoietic stem cells. To identify the leukemia-propagating stem cell fraction of Philadelphia chromosome-positive (Ph(+)) leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n = 3) or Ph(+) acute lymphoblastic leukemia (n = 3) into NOD/SCID/IL-2Rgammac(-/-) mice. Engrafted cells were almost identical to the original leukemia cells as to phenotypes, IGH rearrangements, and karyotypes. CD34(+)CD38(-)CD19(+), CD34(+)38(+)CD19(+), and CD34(-)CD38(+)CD19(+) fractions could self-renew and transfer the leukemia, whereas the CD34(-)CD38(+)CD19(+) fraction did not stably propagate in NOD/SCID mice. These findings suggest that leukemia-repopulating cells in transformed Ph(+) leukemia are included in a lineage-committed but multilayered fraction, and that CD34(+) leukemia cells potentially emerge from CD34(-) populations.

BCR-ABL-independent and RAS / MAPK pathway-dependent form of imatinib resistance in Ph-positive acute lymphoblastic leukemia cell line with activation of EphB4.

OBJECTIVE: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) cell lines. METHODS: We established cell lines from a patient with Ph(+) ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. RESULTS: NPhA1 was sensitive to IM (IC(50) 0.05 microm) and NPhA2 showed mild IM resistance (IC(50) 0.3 microm). NPhA2/STIR could be maintained in the presence of 10 microm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. CONCLUSIONS: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.

ATP metabolizing enzymes ENPP1, 2 and 3 are localized in sensory neurons of rat dorsal root ganglion.

In dorsal root ganglion (DRG) neurons, ATP is an important neurotransmitter in nociceptive signaling through P2 receptors (P2Rs) such as P2X2/3R, and adenosine is also involved in anti-nociceptive signaling through adenosine A1R. Thus, the clearance system for adenine nucleotide/nucleoside plays a critical role in regulation of nociceptive signaling, but there is little information on it, especially ectoenzyme expression profiles in DRG. In this study, we examined expression and localization of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPs), by which ATP is metabolized to AMP, in rat DRG. The mRNA expression levels of ENPP2 were greater than those of ENPP1 and ENPP3 in rat DRGs. On immunohistochemical analysis, ENPP1, 2 and 3 were found in soma of DRG neurons. Immunopositive rate of ENPP3 was greater than that of ENPP1 and ENPP2 in all DRG neurons. ENPP3, as compared with ENPP1 and ENPP2, was expressed mainly by isolectin B4-positive cells, and slightly by neurofilament 200-positive ones. In this way, the expression profile of ENPP1, 2 and 3 was different in DRGs, and they were mainly expressed in small/medium-sized DRG neurons. Moreover, ENPP1-, 2- and 3-immunoreactivities were colocalized with P2X2R, P2X3R and prostatic acid phosphatase (PAP), as an ectoenzyme for metabolism from AMP to adenosine. Additionally, PAP-immunoreactivity was colocalized with equilibrative nucleoside transporter (ENT) 1, as an adenosine uptake system. These results suggest that the clearance system consisted of ENPPs, PAP and ENT1 plays an important role in regulation of nociceptive signaling in sensory neurons.

Hard-to-reach populations in Japan.

The objective of this study was to assess the trends of sampling locations and methods of studying hard-to-reach populations conducted in Japan. We accessed a Japanese medical database on 30 September 2005 to review 5 study types of hard-to-reach populations conducted in Japan: men who have sex with men, homeless, sex workers, undocumented migrants, and injecting drug users. We then categorized their sampling locations and methods. We found 298 articles on hard-to-reach populations published from 1983 to September 2005. Of the 285 studies sampled, approximately 92% were facility-based studies and the rest were community-based. This tendency was consistent in each subgroup; the majority of the studies were conducted among patients in medical facilities. Our study shows the majority of studies on hard-to-reach populations in Japan adopted a convenience sampling method and were facility-based. We suggest the utilization of comparatively valid techniques, such as time-location or respondent driven sampling to more clearly understand these populations.

Effect of myringotomy on prognosis in pediatric acute otitis media.

OBJECTIVE: In children with acute otitis media (AOM), we compared clinical outcomes between groups with and without myringotomy to elucidate the effect of this procedure on long-term clinical course and prognosis. METHODS: Fifty-nine children (29 male, 30 female) with tympanic membrane bulging or middle ear fluid (MEF) at initial presentation were assigned to one of two treatment groups. Group A received oral antibiotics and also underwent myringotomy at initial enrollment (36 cases), while group B received oral antibiotics without myringotomy (23 cases). Clinical outcomes were evaluated by otolaryngologic specialists using pneumatic otoscopy and tympanometry at 5, 10, 15, 30 days and 12 weeks and then every 2 weeks after the initial treatment. Otitis media with effusion (OME), early recurrence and recurrent AOM were used as the evaluation criteria for the prognosis. RESULTS: In group A, 6 children (16.7%) showed transition to OME, 11 (30.6%) showed early recurrence of AOM, and 9 (25.0%) developed recurrent AOM. In group B 10, 8, and 3 (43.5%, 34.8%, and 13.0%) showed these respective adverse outcomes. While early recurrence rates and recurrent AOM rates did not differ significantly between groups, progression of OME was significantly less frequent in group A than group B (P = 0.036). CONCLUSIONS: Lower rates of progression to OME in the group undergoing myringotomy suggested that myringotomy might be effective in preventing this outcome.

Expression, purification and characterization of methyl DNA binding protein from Bombyx mori.

A cDNA clone encoding methyl DNA binding domain-containing protein (bMBD2/3) was obtained by homology searches using a Bombyx mori fat body cDNA library. The cDNA encoded a polypeptide with 249 amino acids sharing 54% similarity with the methyl DNA binding protein from Drosophila melanogaster. To characterize the biochemical properties of bMBD2/3, the clone was expressed in Escherichia coli as His-tagged protein. The recombinant protein was purified to homogeneity using Ni-NTA superflow resin and heparin agarose. The protein showed specific methyl DNA binding activity and was phosphorylated by protein kinase in vitro. Immunoblotting using the purified antibody indicated that bMBD2/3 was expressed in almost all tissues. Using west-western blotting analysis, some proteins that interact with bMBD2/3 were identified in the brain. This is the first report that insect MBD is phosphorylated and is present in adult tissues. These results suggest that bMBD2/3 plays important roles in the DNA methylation-specific transcription of Bombyx mori.

A novel optical assay system for the quantitative measurement of chemotaxis.

We have developed an optically accessible, horizontal chemotaxis apparatus consisting of an etched silicon substrate and a flat glass plate, both of which form two compartments with a 5-microm-deep microchannel in between. The device is held together with a stainless steel holder with holes for injecting cells and a chemoattractant to the different compartments. Migration of cells in the channel is traced with time-lapse intervals using a CCD camera. By developing a method for aligning cells at the edge of the channel, we could successfully reduce the number of cells required for a chemotactic assay, depending on the experiment, to 100 or less. To prevent ceaseless flow of contents between the adjacent compartments via the communicating microchannel, a space at the top end of the holder was filled with medium after aligning the cells. By using a fluorescent probe, we demonstrated experimentally that a stable concentration gradient could be maintained. Furthermore, we determined theoretical details of the gradient established using a model chemokine and a computational fluid dynamics code. Reproducible kinetic results of cell migration were obtained using human neutrophils and IL-8 as a model. Migration of other cells such as eosinophils, basophils and Jurkat lymphocytes toward the appropriate chemokines were also demonstrated.

Multiplex nested reverse transcription-polymerase chain reaction for respiratory viruses in acute otitis media.

Because respiratory viruses play an important role in the causation and pathogenesis of acute otitis media (AOM), determining which virus has infected a child is important with respect to vaccines and antiviral drugs. In some instances, this information might be used to prevent the occurrence of AOM. We used a rapid, economical, and sensitive diagnostic system involving a multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) assay to detect various respiratory viruses in clinical specimens of middle ear fluid (MEF) from children with AOM in our hospital. Multiplex RT-PCR was completed on 40 MEF samples from 28 infants and children less than 6 years old with AOM. Viral RNA was detected in 17 MEF samples (43%). Respiratory syncytial virus type A was present in 12 samples, adenovirus in 3, rhinovirus in 2, and influenza A (H3N2) in 1. The multiplex RT-PCR assay is recommended to clinical laboratories that are considering adoption of a molecular technique for viral diagnosis.

Role of respiratory viruses in children with acute otitis media.

OBJECTIVE: The role of viral infection in acute otitis media (AOM) has not been fully elucidated. We determined the presence of various respiratory viruses in middle ear fluid (MEF) specimens from children with AOM in order to determine whether viral infection or combined effects of viral and bacterial infection enhance or prolong the inflammation in the middle ear, thus worsening clinical outcome. METHODS: Multiplex nested reverse transcription-polymerase chain reactions was carried out to detect influenza A and B viruses, respiratory syncytial virus (RSV) types A and B, parainfluenza virus types 1, 2, and 3; rhinovirus; and adenovirus in 93 MEF specimens from 79 children with AOM. And we examined whether viral infection with or without an identifiable bacterial infection affect clinical outcomes in AOM. We considered persistent MEF (fluid accumulation in the middle ear persisting up to 1 month after treatment), early recurrence of AOM (within 1 month after initial improvement), and recurrent AOM (more than three recurrences during 6 months of follow up) as indicators for evaluating clinical outcomes. RESULTS: One or more respiratory viruses were detected in 39 specimens (42%); a total of 42 viral infections identified (three specimens were infected by two viruses). Of the 42 infections, RSV type A was detected in 29, adenovirus in eight, rhinovirus in three, and influenza virus in two. RSV accounted for 73% of viral detections. In children younger than 2 years, RSV infection combined with Streptococcus pneumoniae or Hemophilus influenzae infection carried a higher risk for persistent middle ear effusion than infection with RSV infection alone or those bacterial infection alone. CONCLUSIONS: Accordingly, vaccination of young children against RSV as well as S. pneumoniae and H. influenzae is important in improving the prognosis in AOM.

Expression profile of vesicular nucleotide transporter (VNUT, SLC17A9) in subpopulations of rat dorsal root ganglion neurons.

ATP plays an important role in the signal transduction between sensory neurons and satellite cells in dorsal root ganglia (DRGs). In primary cultured DRG neurons, ATP is known to be stored in lysosomes via a vesicular nucleotide transporter (VNUT), and to be released into the intercellular space through exocytosis. DRGs consist of large-, medium- and small-sized neurons, which play different roles in sensory transmission, but there is no information on the expression profiles of VNUT in DRG subpopulations. Here, we obtained detailed expression profiles of VNUT in isolated rat DRG tissues. On immunohistochemical analysis, VNUT was found in DRG neurons, and was predominantly expressed by the small- and medium-sized DRG ones, as judged upon visual inspection, and this was compatible with the finding that the number of VNUT-positive DRG neurons in IB4-positive cells was greater than that in NF200-positive ones. These results suggest that VNUT play a role in ATP accumulation in DRG neurons, especially in small- and medium-sized ones, and might be involved in ATP-mediated nociceptive signaling in DRGs.

Promiscuous peptides on the nontypeable Haemophilus influenzae P6 outer membrane protein.

INTRODUCTION: P6 outer membrane protein is one of the candidates for a vaccine formulation against nontypeable Haemophilus influenzae (NTHi) infection. As otitis-prone children who have recurrent episodes of acute otitis media because of NTHi show an impaired immune response to P6, an innovative approach to vaccination is required to augment their immune response. RESULTS AND DISCUSSION: We previously identified human HLA-DR9-restricted T cell epitope peptide and highly immunogenic analog peptides on P6 for peptide vaccine candidates. To develop a vaccine formulation effective in the general population, we identified promiscuous T cell epitope peptides (p41-55, p71-85) on P6. In addition to stimulating with potentially promiscuous peptides (p30-44, p45-59) selected using a computer algorithm, we established peptide-specific T cell lines which respond to P6. CONCLUSION: Our present results indicate that these peptides would be candidates for a widely applicable peptide vaccine formulation.

Retention but significant reduction of BCR-ABL transcript in hematopoietic stem cells in chronic myelogenous leukemia after imatinib therapy.

Chronic myelogenous leukemia (CML) is effectively treated with imatinib mesylate (IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors in CML patients. While IM induces disease remission, it does not appear to eradicate BCR-ABL-positive stem cells. We investigated the residual CML cells in HSC and myeloid progenitors isolated using fluorescence-activated cell sorting after IM-therapy. Quantitative real-time polymerase chain reaction detecting BCR-ABL transcripts showed that CML progenitors were eradicated within 12 months while the BCR-ABL-positive HSC remained. However, IM-therapy continuation could significantly decrease the ratio of BCR-ABL to BCR also in the HSC population. Our results implicate that the sorted and purified stem cells are useful for more sensitive quantification of BCR-ABL-positive minimal residual disease.