Protective neutralizing influenza antibody response in the absence of T follicular helper cells.

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

Repression of the transcription factor Bach2 contributes to predisposition of IgG1 memory B cells toward plasma cell differentiation.

Memory B cells are essential for generating rapid and robust secondary antibody responses. It has been thought that the unique cytoplasmic domain of IgG causes the prompt activation of antigen-experienced IgG memory B cells. To assess this model, we have generated a mouse containing IgG1 B cells that have never encountered antigen. We found that, upon challenge, antigen-experienced IgG1 memory B cells rapidly differentiated into plasma cells, whereas nonexperienced IgG1 B cells did not, suggesting the importance of the stimulation history. In addition, our results suggest that repression of the Bach2 transcription factor, which results from antigen experience, contributes to predisposition of IgG1 memory B cells to differentiate into plasma cells.

Antenna-enhanced high harmonic generation in a wide-bandgap semiconductor ZnO.

High harmonic generation (HHG) in solids has great potential for coherent extreme ultraviolet (EUV) sources, all-optical band-structure reconstruction, and electron dynamics metrology. Solid HHG driven by plasmonic near-fields will open a new paradigm, enabling high repetition-rate HHG with a compact laser, HHG manipulation with meta-surfaces, and precise control over carrier trajectory. In this paper, we demonstrate antenna-enhanced HHG in a wide-bandgap semiconductor ZnO. By exploiting gold nano-antennas resonating at the driver wavelength of 2 µm, we successfully trigger HHG at input intensity of ~0.02 TW/cm2 and observe harmonic radiations up to 9th-order. Orders-of-magnitude enhanced conversion efficiency at the hot-spots brings about ten-fold enhancement in the total yield. The spectral selection rule is found to reflect crystal symmetry, suggesting the possibility of nano-scaled EUV sources and band-structure reconstruction.

Polarization-Resolved Study of High Harmonics from Bulk Semiconductors.

The polarization property of high harmonics from gallium selenide is investigated using linearly polarized midinfrared laser pulses. With a high electric field, the perpendicular polarization component of the odd harmonics emerges, which is not present with a low electric field and cannot be explained by the perturbative nonlinear optics. A two-dimensional single-band model is developed to show that the anisotropic curvature of an energy band of solids, which is pronounced in an outer part of the Brillouin zone, induces the generation of the perpendicular odd harmonics. This model is validated by three-dimensional quantum mechanical simulations, which reproduce the orientation dependence of the odd-order harmonics. The quantum mechanical simulations also reveal that the odd- and even-order harmonics are produced predominantly by the intraband current and interband polarization, respectively. These experimental and theoretical demonstrations clearly show a strong link between the band structure of a solid and the polarization property of the odd-order harmonics.

Prolonged maternal separation attenuates BDNF-ERK signaling correlated with spine formation in the hippocampus during early brain development.

Maternal separation (MS) is known to affect hippocampal function such as learning and memory, yet the molecular mechanism remains unknown. We hypothesized that these impairments are attributed to abnormities of neural circuit formation by MS, and focused on brain-derived neurotrophic factor (BDNF) as key factor because BDNF signaling has an essential role in synapse formation during early brain development. Using rat offspring exposed to MS for 6 h/day during postnatal days (PD) 2-20, we estimated BDNF signaling in the hippocampus during brain development. Our results show that MS attenuated BDNF expression and activation of extracellular signal-regulated kinase (ERK) around PD 7. Moreover, plasticity-related immediate early genes, which are transcriptionally regulated by BDNF-ERK signaling, were also reduced by MS around PD 7. Interestingly, detailed analysis revealed that MS particularly reduced expression of BDNF gene and immediate early genes in the cornu ammonis 1 (CA1) of hippocampus at PD 7. Considering that BDNF-ERK signaling is involved in spine formation, we next evaluated spine formation in the hippocampus during the weaning period. Our results show that MS particularly reduced mature spine density in proximal apical dendrites of CA1 pyramidal neurons at PD 21. These results suggest that MS could attenuate BDNF-ERK signaling during primary synaptogenesis with a region-specific manner, which is likely to lead to decreased spine formation and maturation observed in the hippocampal CA1 region. It is speculated that this incomplete spine formation during early brain development has an influence on learning capabilities throughout adulthood.

CD4 memory T cells develop and acquire functional competence by sequential cognate interactions and stepwise gene regulation.

Memory CD4(+) T cells promote protective humoral immunity; however, how memory T cells acquire this activity remains unclear. This study demonstrates that CD4(+) T cells develop into antigen-specific memory T cells that can promote the terminal differentiation of memory B cells far more effectively than their naive T-cell counterparts. Memory T cell development requires the transcription factor B-cell lymphoma 6 (Bcl6), which is known to direct T-follicular helper (Tfh) cell differentiation. However, unlike Tfh cells, memory T cell development did not require germinal center B cells. Curiously, memory T cells that develop in the absence of cognate B cells cannot promote memory B-cell recall responses and this defect was accompanied by down-regulation of genes associated with homeostasis and activation and up-regulation of genes inhibitory for T-cell responses. Although memory T cells display phenotypic and genetic signatures distinct from Tfh cells, both had in common the expression of a group of genes associated with metabolic pathways. This gene expression profile was not shared to any great extent with naive T cells and was not influenced by the absence of cognate B cells during memory T cell development. These results suggest that memory T cell development is programmed by stepwise expression of gatekeeper genes through serial interactions with different types of antigen-presenting cells, first licensing the memory lineage pathway and subsequently facilitating the functional development of memory T cells. Finally, we identified Gdpd3 as a candidate genetic marker for memory T cells.

Generation of memory B cells inside and outside germinal centers.

Germinal centers (GCs) are generally considered to be the sole site of memory B-cell generation. However, recent studies demonstrate that memory B cells can also develop in response to a T-cell dependent (TD) antigen before the onset, and independently of, the GC reaction. These two classes of memory cells persist equally over long periods of time and attain functional maturation through distinct but related transcriptional programs. Although the development of both memory B-cell types requires classical T-cell help, the generation of GC-dependent memory B cells requires TFH -cell help, while the generation of GC-independent memory cells does not. These findings led to the conclusion that B-cell memory is generated along two fundamentally distinct cellular differentiation pathways. In this review, we focus on the GC-independent pathway of memory B-cell development, and discuss how the unique features of memory B cells are maintained in the GC-independent pathway.

Both mutated and unmutated memory B cells accumulate mutations in the course of the secondary response and develop a new antibody repertoire optimally adapted to the secondary stimulus.

High-affinity memory B cells are preferentially selected during secondary responses and rapidly differentiate into antibody-producing cells. However, it remains unknown whether only high-affinity, mutated memory B cells simply expand to dominate the secondary response or if in fact memory B cells with a diverse VH repertoire, including those with no mutations, accumulate somatic mutations to create a new repertoire through the process of affinity maturation. In this report, we took a new approach to address this question by analyzing the VH gene repertoire of IgG1(+) memory B cells before and after antigen re-exposure in a host unable to generate IgG(+) B cells. We show here that both mutated and unmutated IgG1(+) memory B cells respond to secondary challenge and expand while accumulating somatic mutations in their VH genes in a stepwise manner. Both types of memory cells subsequently established a VH gene repertoire dominated by two major clonotypes, which are distinct from the original repertoire before antigen re-exposure. In addition, heavily mutated memory B cells were excluded from the secondary repertoire. Thus, both mutated and unmutated IgG1(+) memory cells equally contribute to establish a new antibody repertoire through a dynamic process of mutation and selection, becoming optimally adapted to the recall challenge.

Lyn signaling to upregulate GANP is critical for the survival of high-affinity B cells in germinal centers of lymphoid organs.

Signals through BCR and costimulatory molecules play essential roles in selecting high-affinity B cells with Ig V-region mutations in the germinal centers (GCs) of peripheral lymphoid organs. Lyn-deficient (lyn(-/-)) mice show impaired BCR signal triggering for cell proliferation and GC formation, causing hyper-IgM, and display autoimmunity after aging. In this study, we demonstrate that Lyn-mediated signaling to upregulate GANP is essential for the survival of mature GC-like (mGC) B cells with high-affinity type BCR mutations upon Ag immunization. Transgenic ganp expression into lyn(-/-) mice did not recover the Lyn-deficient phenotype with regard to B cell differentiation, serum Igs, and impaired GC formation in spleens after immunization with nitrophenyl-chicken γ-globulin, but it markedly rescued cell survival of mGC B cells by suppressing DNA damage, thereby increasing the frequency of the Trp(33)-to-Leu mutation in the IgV(H)-186.2 region and affinity maturation of nitrophenyl-binding B cells. GANP may play a critical role in Lyn-mediated signaling for the selection of high-affinity B cells in peripheral lymphoid organs.

Left Distal Transradial Approach for the Treatment of a Sacral Extradural Arteriovenous Fistula: A Technical Note and Literature Review.

BACKGROUND: Sacral extradural arteriovenous fistula (SEAVF) is relatively rare, and its etiology is unknown. They are mostly fed by the lateral sacral artery (LSA). For endovascular treatment, both the stability of the guiding catheter and accessibility of the microcatheter to the fistula, distal to the LSA are required for sufficient embolization of the fistulous point. Cannulation of these vessels requires either crossover at the aortic bifurcation or retrograde cannulation using the transfemoral approach. However, atherosclerotic femoral and tortuous aortoiliac vessels can make the procedure technically difficult. Although the right transradial approach (TRA) can reduce this difficulty by straightening the access route, a potential risk remains for cerebral embolism because it passes the aortic arch. Herein, we present a case of successful embolization of a SEAVF using a left distal TRA. METHODS: We report a case of a 47-year-old man with SEAVF treated with embolization using a left distal TRA. Lumbar spinal angiography showed a SEAVF with an intradural vein through the epidural venous plexus fed by the left LSA. A 6-French guiding sheath was cannulated into the internal iliac artery via the descending aorta using the left distal TRA. A microcatheter could be advanced into the extradural venous plexus over the fistula point from the intermediate catheter placed at the LSA. Embolization with coils and n-butyl cyanoacrylate was successfully performed. RESULTS: The SEAVF completely disappeared on neuroimaging, and the patient gradually recovered. CONCLUSIONS: Left distal TRA could be a useful, safe, and less invasive option for the embolization of SEAVF, especially for patients with high-risk factors for aortogenic embolism or puncture site complications.

HIV-1 Nef impairs multiple T-cell functions in antigen-specific immune response in mice.

The viral protein Nef is a key element for the progression of HIV disease. Previous in vitro studies suggested that Nef expression in T-cell lines enhanced TCR signaling pathways upon stimulation with TCR cross-linking, leading to the proposal that Nef lowers the threshold of T-cell activation, thus increasing susceptibility to viral replication in immune response. Likewise, the in vivo effects of Nef transgenic mouse models supported T-cell hyperresponse by Nef. However, the interpretation is complicated by Nef expression early in the development of T cells in these animal models. Here, we analyzed the consequence of Nef expression in ovalbumin-specific/CD4(+) peripheral T cells by using a novel mouse model and demonstrate that Nef inhibits antigen-specific T-cell proliferation and multiple functions required for immune response in vivo, which includes T-cell helper activity for the primary and memory B-cell response. However, Nef does not completely abrogate T-cell activity, as defined by low levels of cytokine production, which may afford the virus a replicative advantage. These results support a model, in which Nef expression does not cause T-cell hyperresponse in immune reaction, but instead reduces the T-cell activity, that may contribute to a low level of virus spread without viral cytopathic effects.

Status of In-Hospital Acute Ischemic Stroke Treated by Mechanical Thrombectomy.

Objective: To elucidate the current state of in-hospital acute ischemic stroke under the introduction of acute-phase mechanical thrombectomy. Methods: The study included 18 consecutive patients with in-hospital cerebral infarction who underwent thrombectomy between April 2014 and March 2020 at St. Marianna University School of Medicine Yokohama City Seibu Hospital. We analyzed the primary disease, department responsible for treatment, modified Rankin Scale (mRS) scores before onset and on discharge, status of onset, treatment course, and so on. Results: The mean age was 79.9 (66-93) years. There were nine females. The admission methods included scheduled admission in 5 patients and non-scheduled admission in 13 patients. The primary diseases consisted of malignant tumors in five patients and heart disease in four patients. The departments responsible for treatment consisted of the Department of Digestive Surgery for six patients and Department of Cardiology for three patients. The mRS score before admission was evaluated as 0-2 in 15 patients and 3-5 in 3 patients. The embolism was evaluated as cardiogenic in 14 patients. Antithrombotic therapy was discontinued before the onset of cerebral infarction in three patients. The mean interval from onset or last well known (LWK) until CT/MRI and puncture was 88.4 and 157.6 minutes. The median Alberta stroke program early CT score (ASPECTS; minimum-maximum) was 8 (2-10). Tissue plasminogen activator (t-PA) was administered to five patients. Concerning the degree of recanalization, the thrombolysis in cerebral infarction (TICI) grade was evaluated as 1 to 2a in 2 patients and 2b to 3 in 16. In the latter, the mean interval from onset or final onset-free confirmation until recanalization was 197.7 minutes. mRS score on discharge was evaluated as 0-2 in four patients, 3-5 in nine, and 6 in five patients. The mortality was related to a primary disease requiring admission in three patients. Conclusion: In-hospital onset cerebral infarction was markedly influenced by the primary disease requiring admission. Even when favorable recanalization was achieved, the number of patients with a favorable outcome was small.

Distinct Characteristics of Sweet's Syndrome of the Scrotum Caused by All-trans Retinoic Acid in a Patient with Acute Promyelocytic Leukemia.

Induction therapy with all-trans retinoic acid (ATRA) is effective for acute promyelocytic leukemia (APL). ATRA induces neutrophil differentiation and its associated side effects. The differentiation syndrome is the most characterized ATRA-induced adverse effect. Sweet's syndrome, also known as neutrophilic dermatosis, is another form of ATRA-associated disease characterized by neutrophil infiltrating erythema that develops with fever. This is a case of a 34-year-old Japanese man diagnosed with APL. At the onset, the patient did not have skin involvement of APL cells. He was treated with ATRA and induction chemotherapy with idarubicin and cytarabine. Scrotal skin rash occurred at day 14, which developed into scrotal ulceration up to day 28 even after eliminating APL cells in his peripheral blood. Sweet's syndrome is a pathological diagnosis of scrotal skin ulceration representing neutrophil infiltration. The infiltrating neutrophils showed PML-RARα rearrangement. The patient was diagnosed with ATRA-associated Sweet's syndrome with skin ulcer. His cutaneous lesion did not respond to intravenous prednisolone therapy; thereby, ATRA was discontinued. After the cessation of ATRA, the skin lesion improved in the next week. We confirmed he achieved a complete response after induction chemotherapy. In our observation, ATRA-associated Sweet's syndrome is characterized by the following clinical manifestations: preferable occurrence in the scrota, tend to progress into skin ulcer, and pathogenicity associated with PML-RARα-positive matured neutrophils. The etiology, pathogenesis, and risk factors of ATRA-associated scrotal ulceration were discussed in the literature review.

Characterization of cereblon-dependent targeted protein degrader by visualizing the spatiotemporal ternary complex formation in cells.

Targeted protein degradation (TPD) through a proteasome-dependent pathway induced by heterofunctional small molecules is initiated by the formation of a ternary complex with recruited E3 ligases. This complex formation affects the degradation ability of TPD molecules, and thus we tested for visualization of the intracellular dynamics of ternary complex formation. In this study, we applied the fluorescent-based technology detecting protein-protein interaction (Fluoppi) system, in which detectable fluorescent foci are formed when ternary complex formation induced by TPD molecules occurs in cells. We show here that cells coexpressing BRD4 and cereblon (CRBN) tagged with the Fluoppi system formed detectable foci in both live and fixed cells only when treated with BRD4-targeting degraders utilizing CRBN as an E3 ligase in dose- and time-dependent manners. Notably, the maintenance and efficacy of TPD molecule-induced foci formation correlated with the ability to degrade target proteins. Furthermore, we demonstrated that BRD4-targeting and FKBP12F36V-targeting degraders formed ternary complexes mainly in the nucleus and cytoplasm, respectively, suggesting that TPD molecules utilize the proteasome to degrade target proteins in their corresponding localized region. Our results also suggest that the Fluoppi system is a powerful tool for characterizing TPD molecules by visualizing the spatiotemporal formation of ternary complex.

Risk Factors of Cerebellar Microembolic Infarctions After Carotid Artery Stenting.

BACKGROUND: This study analyzes the incidence of microembolic infarctions (MIs) in the cerebellum after carotid artery stenting (CAS) to determine the risk factors. METHODS: From 2012 to 2019, 162 CASs in 155 patients were performed at our hospital. Fifty-seven patients (35.7%) showing new MIs on diffusion-weighted imaging after CAS were enrolled. Patients were assigned to either the cerebellar group (n = 14, 8.8%) if their MIs were in the cerebellum and/or cerebrum or the cerebral group (n = 43, 26.9%) if their MIs were only in the cerebrum. Patient characteristics, anatomic features, and clinical data were retrospectively compared between the 2 groups. RESULTS: Advanced age, right-sided carotid stenosis, severe calcification of aortic arch and brachiocephalic trunk, and vertebral artery narrowing with intraprocedural hemodynamic depression (IHD) significantly increased the development of cerebellar MIs. On multivariate analysis, advanced age, right-sided carotid stenosis, and vertebral artery narrowing with IHD were independent predictors of developing new cerebellar MIs. Cerebellar MIs after CAS were not uncommon. CONCLUSIONS: Catheter maneuvering in the aortic arch or the brachiocephalic trunk could be the main cause of thromboemboli in cerebellar MIs. Careful attention should be paid to catheter maneuvering, especially in older patients with right-sided carotid lesions. In addition, cerebellar hypoperfusion caused by vertebral artery narrowing with IHD might reduce washout of debris, a cause of cerebellar MIs.

Layilin enhances the invasive ability of malignant glioma cells via SNAI1 signaling.

BACKGROUND: Malignant gliomas are characterized by high invasive ability. In this study, we investigated roles of layilin, a C-type lectin-homologous protein, in the invasive ability of malignant glioma cells. METHODS: Expression of layilin was investigated by western blotting in the malignant glioma cell lines of U251-MG, A172, and T98G and in astrocytes. The effects of layilin-knockdown on the expression and protein levels of snail family transcriptional repressor 1 (SNAI1), a transcriptional factor involved in the acquisition and enhancement of invasive ability in malignant gliomas, and on the expression of its target genes, matrix metalloproteinase 2 (MMP2), MMP9, and collagen type I alpha 1 chain (COL1A1), were investigated by qPCR and/or western blotting. Furthermore, the effects of layilin-knockdown on the expression and protein levels of metastasis associated 1 family member 3 (MTA3), a transcriptional repressor of SNAI1, were also investigated by qPCR and western blotting. Finally, the effects of layilin-knockdown on the invasive ability of the cells were investigated by a wound healing assay. RESULTS: All the tested malignant glioma cells highly expressed layilin, compared to astrocytes, one of representative glial cell types. Layilin-knockdown reduced SNAI1 both at the mRNA and protein levels in A172 cells, and consequently mRNA levels of MMP2, MMP9, and COL1A1 were also reduced. Furthermore, layilin-knockdown increased nuclear protein levels of MTA3 in A172 cells. Notably, layilin-knockdown suppressed the invasive ability of the cells. CONCLUSION: Layilin up-regulates the expression of SNAI1 via down-regulation of MTA3. This process enhances the invasive ability of malignant glioma cells.

Genome-wide identification and characterization of transcripts translationally regulated by bacterial lipopolysaccharide in macrophage-like J774.1 cells.

Although Escherichia coli LPS is known to elicit various proinflammatory responses in macrophages, its effect on the translational states of transcripts has not yet been explored on a genome-wide scale. To address this, we investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total cellular, polysomal, and free mRNP fractions enabled us to identify transcripts that were modulated at the translational level by LPS: among 19,791 transcripts, 115 and 418 were up- and downregulated at 1, 2, or 4 h after LPS stimulation (100 ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based analysis suggested that translation-dependent downregulated genes frequently include those encoding proteins in the mitochondrial respiratory chain. In fact, the mRNA levels of some transcripts for complexes I, IV, and V in the mitochondrial respiratory chain were translationally downregulated, eventually contributing to the decline of their protein levels. Moreover, the amount of metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased without any change of its mRNA level in total cellular fraction after LPS stimulation. Consistently, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, and the attenuation was independent of nitric oxide. These results demonstrated that translational suppression may play a critical role in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric oxide-independent manner in J774.1 cells.

Formalin-treated UV-inactivated SARS coronavirus vaccine retains its immunogenicity and promotes Th2-type immune responses.

The demand for rapid and simple development of a vaccine against a newly emerging infectious disease is increasing worldwide. We previously revealed that UV-inactivated severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) virions (UV-V) elicited high levels of humoral immunity and a weak Th0 response in mice immunized subcutaneously. To ensure the safety of such a whole inactivated SARS-CoV vaccine, we additionally treated the UV-V vaccine with formalin, resulting in the UV-F-V vaccine. Analysis of the immunogenicity of the UV-F-V+alum vaccine in mice revealed that it generated comparable neutralizing serum anti-SARS-CoV IgG antibody levels as the UV-V+alum vaccine. Moreover, both vaccines induced similar frequencies of anti-SARS-CoV IgG antibody-producing cells in bone marrow. Interestingly, the UV-F-V vaccine induced fewer IgG(2a) subtype antibodies and higher interleukin-4 production in vaccinated mice than did UV-V. Thus, UV-F-V imposes a Th2-type bias on the immune response, unlike UV-V. We propose here that doubly-inactivated SARS-CoV virions by UV and formalin constitute a safe vaccine that may effectively induce neutralizing antibodies in humans.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.