Evaluation of a high-speed but low-throughput RT-qPCR system for detection of SARS-CoV-2.

With the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), a high-speed and convenient detection technology should be at the forefront of medical care worldwide. This study evaluated the usefulness of GeneSoC, a compact, high-speed reciprocal flow quantitative reverse transcription polymerase chain reaction system, for the detection of SARS-CoV-2. The results support the use of this system for the rapid identification of SARS-CoV-2. This approach can contribute to the strategic selection of initial management strategies for patients with COVID-19.

Unidirectional signal propagation in primary neurons micropatterned at a single-cell resolution.

The structure and connectivity of cultured neuronal networks can be controlled by using micropatterned surfaces. Here, we demonstrate that the direction of signal propagation can be precisely controlled at a single-cell resolution by growing primary neurons on micropatterns. To achieve this, we first examined the process by which axons develop and how synapses form in micropatterned primary neurons using immunocytochemistry. By aligning asymmetric micropatterns with a marginal gap, it was possible to pattern primary neurons with a directed polarization axis at the single-cell level. We then examined how synapses develop on micropatterned hippocampal neurons. Three types of micropatterns with different numbers of short paths for dendrite growth were compared. A normal development in synapse density was observed when micropatterns with three or more short paths were used. Finally, we performed double patch clamp recordings on micropatterned neurons to confirm that these synapses are indeed functional, and that the neuronal signal is transmitted unidirectionally in the intended orientation. This work provides a practical guideline for patterning single neurons to design functional neuronal networks in vitro with the direction of signal propagation being controlled.

An electrically resistive sheet of glial cells for amplifying signals of neuronal extracellular recordings.

Electrical signals of neuronal cells can be recorded non-invasively and with a high degree of temporal resolution using multielectrode arrays (MEAs). However, signals that are recorded with these devices are small, usually 0.01%-0.1% of intracellular recordings. Here, we show that the amplitude of neuronal signals recorded with MEA devices can be amplified by covering neuronal networks with an electrically resistive sheet. The resistive sheet used in this study is a monolayer of glial cells, supportive cells in the brain. The glial cells were grown on a collagen-gel film that is permeable to oxygen and other nutrients. The impedance of the glial sheet was measured by electrochemical impedance spectroscopy, and equivalent circuit simulations were performed to theoretically investigate the effect of covering the neurons with such a resistive sheet. Finally, the effect of the resistive glial sheet was confirmed experimentally, showing a 6-fold increase in neuronal signals. This technique feasibly amplifies signals of MEA recordings.

Comparative utility of interferon-γ release assay, QuantiFERON® TB-GIT and T-SPOT®.TB in rheumatoid arthritis.

SETTING: National hospital for tuberculosis (TB) and rheumatoid arthritis (RA) in Japan. OBJECTIVE: To compare two interferon-γ release assays (IGRAs), QuantiFERON®-TB Gold In-Tube (QFT) and T-SPOT®.TB (T-SPOT), in RA patients for detecting latent tuberculous infection (LTBI). DESIGN: QFT and T-SPOT were conducted concurrently in 230 prospectively enrolled RA patients. RESULTS: There were no active TB patients. The percentage of QFT- and T-SPOT-positive patients was respectively 8.3% and 5.7%. In patients aged ⩾60 years, these proportions were respectively 12.3% and 7.2%. The percentage of QFT positivity and T-SPOT positivity at age <60 years was respectively 2.2% and 3.3%. After multivariate logistic analysis for QFT positivity, age ⩾60 years and TB suspected based on chest X-ray were selected as independent factors, with adjusted odds ratios of respectively 4.73 and 3.25. No factors were selected for T-SPOT positivity. CONCLUSION: QFT had a higher positivity rate. In the light of the previous estimated rate of LTBI in Japan, both IGRAs underestimate LTBI, and neither IGRA has enough capability to detect LTBI.

Anti-tumor necrosis factor therapy in patients with difficult-to-treat lupus nephritis: a prospective series of nine patients.

OBJECTIVE: To clarify the efficacy and safety of anti-TNF-alpha therapy for intractable lupus nephritis. METHODS: In nine patients with systemic erythematosus who presented with lupus nephritis resistant to steroids and immunosuppressants, 200 mg/body of infliximab was drip-infused three times. No changes were made to other treatments for three months after the start of anti-TNF-alpha therapy, and urinary findings, renal function, serum complement, anti-DNA antibody, SLE activity, and adverse events were examined for six months after the start of anti-TNF-alpha therapy. RESULTS: One of the nine patients developed pyelonephritis after the first infliximab injection and received no further injections. The remaining eight patients received 3 infliximab injections. Of the eight patients, urinary protein decreased after anti-TNF-alpha therapy in six patients, and the SLEDAI improved in five patients. Urinary findings and/or SLE activity improved in six patients. Of the patients whose urinary protein levels decreased after anti-TNF-alpha therapy, proteinuria recurred six months after anti-TNF-alpha therapy in one patient. After anti-TNF-alpha therapy, proteinuria and the SLEDAI improved significantly. With respect to adverse events, therapy was discontinued in one patient who developed pyelonephritis, and one patient developed decreased blood pressure due to infusion reactions. In one patient in whom the steroid dosage was increased due to poor response to anti-TNF-alpha therapy, brainstem infarction occurred four months later. In one patient, anti-DNA antibody levels increased after therapy, but none of the patients had decreased serum complement levels or increased SLE activity. CONCLUSION: In intractable lupus nephritis, anti-TNF-alpha therapy improved urinary protein levels and SLE activity. Although adverse events must be monitored cautiously, it may be possible to use anti-TNF-alpha therapy as a third-line treatment.

IGF-I regulates pro-opiomelanocortin and GH gene expression in the mouse pituitary gland.

IGF-I is expressed in somatotrophs, and IGF-I receptors are expressed in most somatotrophs and some corticotrophs in the mouse pituitary gland. Our recent study demonstrated that IGF-I stimulates the proliferation of corticotrophs in the mouse pituitary. These results suggested that somatotrophs regulate corticotrophic functions as well as somatotrophic functions by the mediation of IGF-I molecules. The present study aimed to clarify factors regulating pituitary IGF-I expression and also the roles exerted by IGF-I within the mouse anterior pituitary gland. Mouse anterior pituitary cells were isolated and cultured under serum-free conditions. GH (0.5 or 1 microg/ml), ACTH (10(-8) or 10(-7) M), GH-releasing hormone (GHRH; 10(-8) or 10(-7) M), dexamethasone (DEX; 10(-8) or 10(-7) M) and estradiol-17beta (e2; 10(-11) or 10(-9) M) were given for 24 h. IGF-I mRNA levels were measured using competitive RT-PCR, and GH and pro-opiomelanocortin (POMC) mRNA levels were measured using Northern blotting analysis. GH treatment significantly increased IGF-I mRNA levels (1.5- or 2.1-fold). ACTH treatment did not alter GH and IGF-I mRNA levels. IGF-I treatment decreased GH mRNA levels (0.7- or 0.5-fold), but increased POMC mRNA levels (1.8-fold). GH treatment (4 or 8 microg/ml) for 4 days increased POMC mRNA levels. GHRH treatment increased GH mRNA levels (1.3-fold), but not IGF-I mRNA levels. DEX treatment significantly decreased IGF-I mRNA levels (0.8-fold). e2 treatment did not affect IGF-I mRNA levels. GH receptor mRNA, probably with GH-binding protein mRNA, was detected in somatotrophs, and some mammotrophs and gonadotrophs by in situ hybridization using GH receptor cDNA as a probe. These results suggested that IGF-I expression in somatotrophs is regulated by pituitary GH, and that IGF-I suppresses GH expression and stimulates POMC expression at the transcription level. Pituitary IGF-I produced in somatotrophs is probably involved in the regulation of somatotroph and corticotroph functions.

Frequency of spinocerebellar ataxia mutations in the Kinki district of Japan.

OBJECTIVES: To determine the frequencies of spinocerebellar ataxias (SCAs) in the Kinki district, the western part of the main island of Japan. MATERIAL AND METHODS: One hundred and forty-three families with dominantly inherited ataxia and 220 patients with apparently sporadic cerebellar ataxia were examined for the SCA1, SCA2, SCA3/Machado-Joseph disease (MJD), SCA6, SCA7, SCA8, SCA12 and dentatorubral-pallidoluysian atrophy (DRPLA) mutations. RESULTS: Among the dominant families, SCA1 accounted for 3%, SCA2 for 4%, SCA3/MJD for 24%, SCA6 for 31% and DRPLA for 12%. Neither SCA7 nor SCA12 mutations were detected. Among the apparently sporadic patients, 15% were found to have expanded triplet repeats. Of these, the SCA6 mutation was most frequently detected. CONCLUSION: SCA6 is the most common SCA in the Kinki district of Japan. Comparison of our results with those from other regions of Japan and different countries shows geographic and ethnic variation in the frequency of SCAs.

Expression of TNF-related apoptosis inducing ligand (TRAIL) on infiltrating cells and of TRAIL receptors on salivary glands in patients with Sjögren's syndrome.

BACKGROUND: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis of tumor cells but not normal cells; its role in normal non-transformed tissues is unknown. OBJECTIVE: To evaluate the role of apoptosis mediated by TRAIL and TRAIL-receptor (TRAIL-R) system in lymphocytic sialadenitis in patients with Sjögren's syndrome. METHODS: The expression of TRAIL and TRAIL-R1, 2, 3 and 4 in lymphocytic sialadenitis was examined by immunoperoxidase staining in patients with Sjögren's syndrome and in normal subjects. To elucidate the mechanism of de novo expression of TRAIL-R1 antigen, we quantitatively investigated its induction by cytokines in human salivary duct cell line (HSG) by cell enzyme-linked immunosorbent assay. In human salivary duct cells stimulated by cytokines, we investigated the induction of apoptotic cell death by recombinant TRAIL protein. RESULTS: In patients with massive mononuclear cell infiltration, some infiltrating cells showed TRAIL. In patients with severe lymphocytic sialadenitis, TRAIL-R1, TRAIL-R2, or both were strongly expressed on the ductal epithelial cells. Neither TRAIL-R3 nor R4 were observed on ductal epithelium. In contrast, TRAIL-R1 and R2 were not found in the minor salivary glands of normal subjects or patients with mild lymphocytic sialadenitis. Unstimulated HSG cells did not express TRAIL-R1. Interferon-gamma (IFN-gamma) consistently upregulated levels of TRAIL-R1. In contrast, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1 beta), IL-2, and IL-4 had no effect on TRAIL-R1 levels. HSG cells expressing TRAIL-R1 in response to IFN-gamma were susceptible to apoptosis by recombinant TRAIL protein. CONCLUSION: Our findings suggest that TRAIL and TRAIL-R system may play a role in the pathogenesis of lymphocytic sialadenitis in patients with Sjögren's syndrome.