Coronavirus disease 2019 , an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now spread globally. Some patients develop severe complications including multiple organ failure. It has been suggested that excessive inflammation associated with the disease plays major role in the severity and mortality of COVID-19. To elucidate the inflammatory mechanisms involved in COVID-19, we examined the effects of SARS-CoV-2 spike protein S1 subunit (hereafter S1) on the pro-inflammatory responses in murine and human macrophages. Murine peritoneal exudate macrophages produced pro-inflammatory mediators in response to S1 exposure. Exposure to S1 also activated nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling pathways. Pro-inflammatory cytokine induction by S1 was suppressed by selective inhibitors of NF-κB and JNK pathways. Treatment of murine peritoneal exudate macrophages and human THP-1 cell-derived macrophages with a toll-like receptor 4 (TLR4) antagonist attenuated pro-inflammatory cytokine induction and the activation of intracellular signaling by S1 and lipopolysaccharide. Similar results were obtained in experiments using TLR4 siRNA-transfected murine RAW264.7 macrophages. In contrast, TLR2 neutralizing antibodies could not abrogate the S1-induced pro-inflammatory cytokine induction in either RAW264.7 or THP-1 cellderived macrophages. These results suggest that SARS-CoV-2 spike protein S1 subunit activates TLR4 signaling to induce pro-inflammatory responses in murine and human macrophages. Therefore, TLR4 signaling in macrophages may be a potential target for regulating excessive inflammation in COVID-19 patients.

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ObjectiveApproximately 8–10% of newborns with asymptomatic congenital cytomegalovirus (cCMV) infection develop sensorineural hearing loss (SNHL). However, the relationship between CMV load, SNHL and central nervous system (CNS) damage in cCMV infection remains unclear. This study aimed to examine the relationship between urinary CMV load, SNHL and CNS damage in newborns with cCMV infection.Study designThe study included 23 368 newborns from two maternity hospitals in Saitama Prefecture, Japan. Urine screening for cCMV infection (quantitative real-time PCR) and newborn hearing screening (automated auditory brainstem response (AABR) testing) were conducted within 5 days of birth to examine the incidence of cCMV infection and SNHL, respectively. CNS damage was assessed by MRI of cCMV-infected newborns.ResultsThe incidence of cCMV infection was 60/23 368 (0.257%; 95% CI 0.192% to 0.322%). The geometric mean urinary CMV DNA copy number in newborns with cCMV was 1.79×106 copies/mL (95% CI 7.97×105 to 4.02×106). AABR testing revealed abnormalities in 171 of the 22 229 (0.769%) newborns whose parents approved hearing screening. Of these 171 newborns, 22 had SNHL (12.9%), and 5 of these 22 were infected with cCMV (22.7%). Newborns with both cCMV and SNHL had a higher urinary CMV DNA copy number than newborns with cCMV without SNHL (p=0.036). MRI revealed CNS damage, including white matter abnormalities, in 83.0% of newborns with cCMV. Moreover, newborns with CNS damage had a significantly greater urinary CMV load than newborns without CNS damage (p=0.013).ConclusionsWe determined the incidence of cCMV infection and urinary CMV DNA copy number in seemingly healthy newborns from two hospitals in Saitama Prefecture. SNHL and CNS damage were associated with urinary CMV DNA copy number. Quantification of urinary CMV load may effectively predict the incidence of late-onset SNHL and neurodevelopmental disorders.

Prolonged inactivity is known to induce changes in responses of many physiological defense systems such as the hypothalamo-hypophyseal-adrenocortical axis, the sympathetic nervous system, and immuno-responsive systems. However, effects of various types of inactivity on immuno-responsive systems are still unknown. Therefore, the effects of two types of inactivity (immobilization: IMM and whole body suspension: WBS) on the number of white blood cells were studied in rats. Rats were divided into the control group and each inactivity group to compare the number of total white blood cells, lymphocytes, monocyte, neutrophil, eosinophil, and basophil during the experimental periods. Both IMM and WBS were maintained for 11 days. IMM markedly increased the number of total white blood cells, monocyte, neutrophil, and eosinophil in the 1st to 10th day. However, the number of total white blood cells, monocyte, neutrophil, and eosinophil during the experiment of WBS were characterized by the presence of a lag phase followed by the significant increased actions. IMM did not change the number of basophil during the experimental period. However, WBS increased the number of basophil in the 1st to 8th day to 2.8-4.8 times, compared with the values of the control. Both IMM and WBS did not change the number of lymphocytes. From these results, WBS increases the number of natural immunity cells without changing acquired immunity cells, and there are different responses in the number of total white blood cells, monocyte, neutrophil, eosinophil, and basophil between IMM and WBS.

Abstract. Clenbuterol [CLE: 4-amino-α(t-butyl-amino)methyl-3,5-dichlorobenzyl alcohol] is well known as a potent β 2 -adrenergic agonist and non-steroidal anabolic drug, and thus it is generally used for sports doping and asthma therapy. Although the functions of immune cells such as white blood cells (WBCs) have shown to be modulated through β 2 -adrenoceptors, the effects of CLE on immune-responsive systems have not been elucidated systematically. Therefore, the effects of CLE on the number of WBCs were studied in rats. Male adult rats were divided into CLE-administered group and the control group to compare the number of total WBCs, neutrophils, monocytes, lymphocytes, eosinophils, and basophils. The administration (dose = 1.0 mg ⋅ kg −1 body weight ⋅ day −1 , s.c.) of CLE was maintained for 30 days. CLE did not change the number of total WBCs during the experimental period. However, CLE increased significantly the number of neutrophils and monocytes, while CLE decreased drastically the number of lymphocytes and eosinophils. There was no significant change in the number of basophils between both groups. These results suggest that the administration of CLE induces drastic redistribution of WBCs in circulation without changing the number of total WBCs, and these responses of WBCs during the administration of CLE are sustained for at least 30 days.