Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaques.

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection.

The Safety and Efficacy of Resolving Burr Entrapment by the Ping-Pong and Mother-in-Child Techniques.

Burr entrapment is a serious risk when performing rotational atherectomy on specific anatomical features of lesions such as tortuosity, calcification, and acute angulation. This occurrence, known as the Kokeshi phenomenon in Japanese, is caused by the burr's proximal section being unable to ablate while pulling back the burr, leaving the distal end of the burr covered in diamond crumbs capable of lesion ablation following rotation. There are reports of different approaches used to retrieve an entrapped rotablator burr. In this case, we demonstrate that the ping-pong and mother-in-child techniques, which use separate guide catheters to engage the same coronary artery wiring across the lesion afterward and deep engagement of guide extension catheter manual traction, are highly effective and secure methods for retrieval.

SARS-CoV-2 induces inflammation and intracranial infection through the olfactory epithelium-olfactory bulb pathway in non-human primates.

We examined the histopathological changes in the olfactory mucosa of cynomolgus and rhesus macaque models of SARS-CoV-2 infection. SARS-CoV-2 infection induced severe inflammatory changes in the olfactory mucosa. A major histocompatibility complex (MHC) class II molecule, HLA-DR was expressed in macrophage and supporting cells, and melanocytes were increased in olfactory mucosa. Supporting cells and olfactory neurons were infected, and SARS-CoV-2 N protein was detected in the axons of olfactory neurons and in olfactory bulbs. Viral RNA was detected in olfactory bulbs and brain tissues. The olfactory epithelium-olfactory bulb pathway may be important as a route for intracranial infection by SARS-CoV-2.

MHC-DRB alleles with amino acids Val11, Phe13, and the shared epitopes promote collagen-induced arthritis and a rapid IgG1 response in Filipino cynomolgus macaques.

Macaques are useful animal models for studying the pathogenesis of rheumatoid arthritis (RA) and the development of anti-rheumatic drugs. The purpose of this study was to identify the major histocompatibility complex (MHC) polymorphisms associated with the pathology of collagen-induced arthritis (CIA) and anti-collagen IgG induction in a cynomolgus macaque model, as MHC polymorphisms affect the onset of CIA in other animal models. Nine female Filipino cynomolgus macaques were immunized with bovine type II collagen (b-CII) to induce CIA, which was diagnosed clinically by scoring the symptoms of joint swelling over 9 weeks. MHC polymorphisms and anti-b-CII antibody titers were compared between symptomatic and asymptomatic macaques. Four of 9 (44%) macaques were defined as the CIA-affected group. Anti-b-CII IgG in the affected group increased in titer approximately 3 weeks earlier compared with the asymptomatic group. The mean plasma IgG1 titer in the CIA-affected group was significantly higher (p < 0.05) than that of the asymptomatic group. Furthermore, the cynomolgus macaque MHC (Mafa)-DRB1*10:05 or Mafa-DRB1*10:07 alleles, which contain the well-documented RA-susceptibility five amino acid sequence known as the shared epitope (SE) in positions 70 to 74, with valine at position 11 (Val11, V11) and phenylalanine at position 13 (Phe13, F13), were detected in the affected group. In contrast, no MHC polymorphisms specific to the asymptomatic group were identified. In conclusion, the presence of V11 and F13 along with SE in the MHC-DRB1 alleles seems essential for the production of IgG1 and the rapid induction of severe CIA in female Filipino cynomolgus macaques.

The institutional experience of the implementing 4DCT in NSCLC radiotherapy planning.

Background: The study was to evaluate the effectiveness of dose distribution of four-dimensional computed tomography (4DCT) simulation. Materials and methods: The gross tumor volume (GTV) and clinical target volume (CTV) were contoured in all 10 respiratory phases of 4DCT in 30 patients with non-small cell lung cancer (NSCLC). Both 3D and 4D treatment plans were made individually for each patient using the planning volume (PTV). The PTV3D was taken from a single CTV plus the recommended margin, and the PTV4D was taken from the 4D internal target volume, including all 10 CTVs plus the setup margins. Results: The mean PTV was 460 ± 179 (69-820) cm3 for 3DCT and 401 ± 167 (127-854) cm3 for 4DCT (p = 0.0018). The dose distribution (DD) of organs at risk, especially the lungs, was lower for the 4DCT simulation. The V5%, V10%, and V20% of the total lung dose for 4DCT were significantly lower for the 3DCT. However, lung V30% the heart, esophagus, and spinal cord were not significantly different. In addition, the conformity index and the dose heterogeneity index of the PTV were not significantly different. The normal tissue complication probability (NTCP) of the lung and heart was significantly lower for 4DCT than for 3DCT. Conclusions: The 4DCT simulation gives better results on the NTCP. The organs at risk, especially the lungs, receive a significantly lower DD compared with the 3DCT. The conformity index (CI), heterogeneity index (HI) and the DD to the heart, spinal cord, and esophagus were not significantly different between the two techniques.

Complement factor D targeting protects endotheliopathy in organoid and monkey models of COVID-19.

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes.

Vertical Piezo-Optoelectronic Coupling in a 3C-SiC/Si Heterostructure for Self-Powered and Highly Sensitive Mechanical Sensing.

This paper presents a novel self-powered mechanical sensing based on the vertical piezo-optoelectronic coupling in a 3C-SiC/Si heterojunction. The vertical piezo-optoelectronic coupling refers to the change of photogenerated voltage across the 3C-SiC/Si heterojunction upon application of mechanical stress or strain. The effect is elucidated under different photoexcitation conditions and under varying tensile and compressive strains. Experimental results show that the relationship between the vertical photovoltage and applied strain is highly linear, increasing under the tensile strain while decreasing under the compressive strain. The highest sensitivities to tensile and compressive strains are 0.146 and 0.058 µV/ppm/µW, respectively, which are about 220 and 360 times larger than those of the lateral piezo-optoelectronic coupling reported in literatures. These extremely large changes in vertical photovoltages are explained by the alteration in effective mass, energy band shift, and repopulation of photogenerated holes in out-of-plane, in-plane longitudinal, and in-plane transverse directions when strains are exerted on the heterojunction. The significant enhancement of strain sensitivity will pave the way for development of ultrasensitive and self-powered mechanical sensors based on the proposed vertical piezo-optoelectronic coupling.

An attenuated vaccinia vaccine encoding the severe acute respiratory syndrome coronavirus-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human angiotensin-converting enzyme 2 transgenic mice from severe acute respiratory syndrome coronavirus-2 and its variants.

As long as the coronavirus disease-2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently required. We have developed a vaccine consisting of the attenuated vaccinia virus Dairen-I (DIs) strain platform carrying the SARS-CoV-2 S gene (rDIs-S). rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin-converting enzyme 2 (hACE2) transgenic mice, and the mouse model showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA.1 variant (TY38-873). Using a tandem mass tag (TMT)-based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that vaccination with rDIs-S maintains S protein-specific antibody titers for at least 6 months after a first vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current variants such as Omicron BA.1 and possibly future variants.

Potent SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from patients with COVID-19-convalescent, and identified antibodies that exhibited the comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced. Our antibodies showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

Effects of pretreatment and air drying temperature on Noni fruit powder.

The plant Morinda citrifolia L. (Noni) has been the subject of several recent research due to its positive impact on the treatment and prevention of a variety of diseases. Noni fruits contain a variety of phytochemicals, including flavonoid, polyphenol, and triterpenoid saponin. This study aimed to determine the best pre-treatment (including blanching, soaking in ascorbic acid solution and metabisulfite solution) and air-drying temperature (50, 60, 70, and 80 °C) to maximize the total polyphenol content (TPC), flavonoid content (TFC), and triterpenoid saponin contents (TSC) of the resultant Noni fruit powder. The results revealed that pre-soaked Noni fruit samples in ascorbic acid or metabisulfite solution before air-drying at 60 °C were beneficial in preserving TPC, TFC, and TSC. TPC, TFC, and TSC losses increased as drying temperatures (70 and 80 °C) rose. The optimum sample was held at five different relative humidity conditions until they attained weight equilibrium. The results indicated that the sorption isotherm curve of the Noni powder was the sigmoid shape and fitted with the BET and GAB models.

No Tumorigenicity of Allogeneic Induced Pluripotent Stem Cells in Major Histocompatibility Complex-matched Cynomolgus Macaques.

Tumorigenicity of induced pluripotent stem cells (iPSCs) is anticipated when cells derived from iPSCs are transplanted. It has been reported that iPSCs formed a teratoma in vivo in autologous transplantation in a nonhuman primate model without immunosuppression. However, there has been no study on tumorigenicity in major histocompatibility complex (MHC)-matched allogeneic iPSC transplantation with immune-competent hosts. To examine the tumorigenicity of allogeneic iPSCs, we generated four iPSC clones carrying a homozygous haplotype of the MHC. Two clones were derived from female fibroblasts by using a retrovirus and the other two clones were derived from male peripheral blood mononuclear cells by using Sendai virus (episomal approach). The iPSC clones were transplanted into allogenic MHC-matched immune-competent cynomolgus macaques. After transplantation of the iPSCs into subcutaneous tissue of an MHC-matched female macaque and into four testes of two MHC-matched male macaques, histological analysis showed no tumor, inflammation, or regenerative change in the excised tissues 3 months after transplantation, despite the results that iPSCs formed teratomas in immune-deficient mice and in autologous transplantation as previously reported. The results in the present study suggest that there is no tumorigenicity of iPSCs in MHC-matched allogeneic transplantation in clinical application.

Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques.

We examined the pathogenicity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in cynomolgus macaques for 28 days to establish an animal model of COVID-19 for the development of vaccines and antiviral drugs. Cynomolgus macaques infected with SARS-CoV-2 showed body temperature rises and X-ray radiographic pneumonia without life-threatening clinical signs of disease. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes producing interferon (IFN)-γ specifically for SARS-CoV-2 N-protein were detected on day 14 in one of three macaques with viral pneumonia. In the other two macaques, in which a neutralizing antibody was not detected, T-lymphocytes producing IFN-γ specifically for SARS-CoV-2 N protein increased on day 7 to day 14, suggesting that not only a neutralizing antibody but also cellular immunity has a role in the elimination of SARS-CoV-2. Thus, because of similar symptoms to approximately 80% of patients, cynomolgus macaques are appropriate to extrapolate the efficacy of vaccines and antiviral drugs for humans.

Efficacy of a Cap-Dependent Endonuclease Inhibitor and Neuraminidase Inhibitors against H7N9 Highly Pathogenic Avian Influenza Virus Causing Severe Viral Pneumonia in Cynomolgus Macaques.

H7N9 highly pathogenic avian influenza virus (HPAIV) infection in a human was first reported in 2017. A/duck/Japan/AQ-HE29-22/2017 (H7N9) (Dk/HE29-22), found in imported duck meat at an airport in Japan, possesses a hemagglutinin with a multibasic cleavage site, indicating high pathogenicity in chickens, as in the case of other H7 HPAIVs. In the present study, we examined the pathogenicity of Dk/HE29-22 and the effectiveness of a cap-dependent endonuclease inhibitor (baloxavir) and neuraminidase inhibitors (oseltamivir and zanamivir) against infection with this strain in a macaque model (n = 3 for each group). All of the macaques infected with Dk/HE29-22 showed severe signs of disease and pneumonia even after the virus had disappeared from lung samples. Virus titers in macaques treated with baloxavir were significantly lower than those in the other treated groups. After infection, levels of interferon alpha and beta (IFN-α and IFN-ß) in the blood of macaques in the baloxavir group were the highest among the groups, whereas levels of tumor necrosis factor alpha (TNF-α) and interleukin 13 (IL-13) were slightly increased in the untreated group. In addition, immune checkpoint proteins, including programmed death 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT), were expressed at high levels in the untreated group, especially in one macaque that showed severe signs of disease, indicating that negative feedback responses against vigorous inflammation may contribute to disease progression. In the group treated with baloxavir, the percentages of PD-1-, CTLA-4-, and TIGIT-positive T lymphocytes were lower than those in the untreated group, indicating that reduction in virus titers may prevent expression of immune checkpoint molecules from downregulation of T cell responses.

Efficacy of Neuraminidase Inhibitors against H5N6 Highly Pathogenic Avian Influenza Virus in a Nonhuman Primate Model.

Attention has been paid to H5N6 highly pathogenic avian influenza virus (HPAIV) because of its heavy burden on the poultry industry and human mortality. Since an influenza A virus carrying N6 neuraminidase (NA) has never spread in humans, the potential for H5N6 HPAIV to cause disease in humans and the efficacy of antiviral drugs against the virus need to be urgently assessed. We used nonhuman primates to elucidate the pathogenesis of H5N6 HPAIV as well as to determine the efficacy of antiviral drugs against the virus. H5N6 HPAIV infection led to high fever in cynomolgus macaques. The lung injury caused by the virus was severe, with diffuse alveolar damage and neutrophil infiltration. In addition, an increase in interferon alpha (IFN-α) showed an inverse correlation with virus titers during the infection process. Oseltamivir was effective for reducing H5N6 HPAIV propagation, and continuous treatment with peramivir reduced virus propagation and the severity of symptoms in the early stage. This study also showed pathologically severe lung injury states in cynomolgus macaques infected with H5N6 HPAIV, even in those that received early antiviral drug treatments, indicating the need for close monitoring and further studies on virus pathogenicity and new antiviral therapies.

Low replicative fitness of neuraminidase inhibitor-resistant H7N9 avian influenza a virus with R292K substitution in neuraminidase in cynomolgus macaques compared with I222T substitution.

Human cases of H7N9 influenza A virus infection have been increasing since 2013. The first choice of treatment for influenza is neuraminidase (NA) inhibitors (NAIs), but there is a concern that NAI-resistant viruses are selected in the presence of NAIs. In our previous study, an H7N9 virus carrying AA substitution of threonine (T) for isoleucine (I) at residue 222 in NA (NA222T, N2 numbering) and an H7N9 virus carrying AA substitution of lysine (K) for arginine (R) at residue 292 in NA (NA292K, N2 numbering) were found in different macaques that had been infected with A/Anhui/1/2013 (H7N9) and treated with NAIs. In the present study, the variant with NA292K showed not only resistance to NAIs but also lower replication activity in MDCK cells than did the virus with wild-type NA, whereas the variant with NA222T, which was less resistant to NAIs, showed replication activity similar to that of the wild-type virus. Next, we examined the pathogenicity of these H7N9 NAI-resistant viruses in macaques. The variants caused clinical signs similar to those caused by the wild-type virus with similar replication potency. However, the virus with NA292K was replaced within 7 days by that with NA292R (same as the wild-type) in nasal samples from macaques infected with the virus with NA292K, i.e. the so-called revertant (wild-type virus) became dominant in the population in the absence of an NAI. These results suggest that the clinical signs observed in macaques infected with the NA292K virus are caused by the NA292K virus and the NA292R virus and that the virus with NA292K may not replicate continuously in the upper respiratory tract of patients without treatment as effectively as the wild-type virus.