TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

Cytotoxic T Cells from Human Immunodeficiency Virus Type 2-Infected Patients Frequently Cross-React with Different Human Immunodeficiency Virus Type 1 Clades

Knowledge of immune mechanisms responsible for the cross-protection between highly divergent viruses such as human immunodeficiency virus type 1 (HIV-1) and HIV-2 may contribute to an understanding of whether virus variability may be overcome in the design of vaccine candidates which are broadly protective across the HIV subtypes. We demonstrate that despite the significant difference in virus amino acid sequence, the majority of HIV-2-infected individuals with different HLA molecules possess a dominant cytotoxic T-cell response which is able to recognize HIV-1 Gag protein. Furthermore, HLA-B5801-positive subjects show broad cross-recognition of HIV-1 subtypes since they mounted a T-cell response that tolerated extensive amino acid substitutions within HLA-B5801-restricted HIV-1 and HIV-2 epitopes. These results suggests that HLA-B5801-positive HIV-2-infected individuals have an enhanced ability to react with HIV-1 that could play a role in cross-protection.Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are related human retroviruses that show various biological and structural differences. HIV-2 is found mainly in West Africa, whereas HIV-1 is spreading throughout the world. HIV-2 is less transmissible, and HIV-2-positive patients exhibit longer clinical latency periods than individuals infected with HIV-1 (23). A recent report has also shown that the mortality in HIV-2-infected individuals is only twice as high as in the uninfected population and, in the majority of adults, survival is not affected by HIV-2 status (31).Although the two viruses are similar in genomic organization, various genetic and enzymatic differences have been found at many stages of the retroviral life cycle. They differ significantly in terms of amino acid sequence, the more conserved being the Pol and Gag sequences, which exhibit less than 60% homology (17).Despite these differences, epidemiological data and animal studies have shown some evidence of cross-protection between the two viral infections. Travers et al. reported that HIV-2-infected women had a lower incidence of HIV-1 infection than did HIV-seronegative women in a cohort of commercial sexual workers in Dakar (37), and rhesus macaques immunized with a recombinant HIV-1 poxvirus vaccine are protected against HIV-2 challenge (2). These studies, though not conclusive (1, 6), suggest that differences in the virus may not necessarily preclude the development of defensive immunity to a subsequent pathogenic infection, an old-fashioned concept pioneered by Jenner, who used cowpox to vaccinate against human smallpox.The immunological basis of cross-protection is largely unknown, and a clear understanding of the role played by the humoral or cell-mediated immune response in HIV protection is still lacking. However, mounting evidence suggests that cytotoxic T-lymphocyte (CTL) response could be the key element. Indeed, the protection afforded in animal models against simian (13) and feline (12) immunodeficiency virus infections is closely correlated with the induction of specific CTL response, and HIV-1 and HIV-2 HLA-B35-restricted cross-reactive CTLs have been postulated to confer protection against repeated HIV exposure (33).CTLs recognize short viral peptides, 8 to 11 amino acids long, that are generated by the intracellular processing of endogenously synthesized viral antigens within the infected cells, which are expressed at the cell surface in the binding groove of HLA class I molecules. The specificity of the T-cell response is determined by the interaction of the antigen-specific T-cell receptor (TCR) with the peptide-HLA complex, and this interaction, together with non-antigen-specific signals, activates the CTLs (15).The presence of cross-reactive CTLs able to lyse HIV-1- or HIV-2-infected cells should be dependent on the extent of conservation between the two viruses within the epitopes selected by particular HLA class I molecules. It is well known that amino acid substitutions within the epitopes can abrogate the CTL response by inhibiting either HLA binding or TCR recognition (32). However, a number of recent studies have shown that T cells can recognize apparently unrelated peptides (10, 41), and crystallographic data have shown physical limits to the TCR epitope specificity due to the limited size of contact between the TCR and the peptide (14), suggesting a flexibility in T-cell recognition of antigen (19).Some individuals with a particular HLA profile which is responsible for presentation of the viral antigen and for selection of the T-cell repertoire may possess a CTL response not affected by mutations within the epitope, as has been demonstrated in subjects with HLA alleles B27 (28) and B35 (33). In these cases, amino acid substitutions within the HIV-1 and -2 epitopes were tolerated by the CTLs.In this study, we have investigated the extent of cross-reacting CTLs between HIV-2 and HIV-1 in a group of HIV-2-infected subjects with different HLA class I types. We have shown that despite differences in amino acid sequence between the two viruses, the majority of HIV-2-positive subjects possess CTLs which are able to recognize HIV-1 Gag protein.Furthermore, analysis of HLA profiles and the fine specificity of the cytotoxic response demonstrated that HLA-B5801-positive subjects show broad cross-recognition of HIV-1 isolates. These subjects mounted a CTL response that tolerated extensive amino acid substitutions within an HLA-B5801-restricted HIV-1 epitope.     

Determination of acetylcholine by on-line microdialysis coupled with pre- and post-microbore column enzyme reactors with electrochemical detection

A sensitive procedure consisting of a pre- and post-microbore column reactor sequence of a LC-electrochemical detection system coupled with on-line microdialysis system is described in the present study to measure endogenous acetylcholine concentration in freely moving rats. The pre-column packed, with immobilized choline oxidase and catalase, was used to remove choline, whereas the post-column, packed with immobilized acetylcholine oxidase and choline oxidase, was used to measure acetylcholine selectively. The detection limit of acetylcholine was found to be 5 fmol/μl (50 fmol/10 μl). The usefulness of the described methodology was evaluated by examining the change in the striatal acetylcholine concentration of freely moving rats after physostigmine (0.5 mg/kg, s.c.) administration.     

The effect of viewing eccentricity on enumeration

Visual acuity and contrast sensitivity progressively diminish with increasing viewing eccentricity. Here we evaluated how visual enumeration is affected by visual eccentricity, and whether subitizing capacity, the accurate enumeration of a small number (～3) of items, decreases with more eccentric viewing. Participants enumerated gratings whose (1) stimulus size was constant across eccentricity, and (2) whose stimulus size scaled by a cortical magnification factor across eccentricity. While we found that enumeration accuracy and precision decreased with increasing eccentricity, cortical magnification scaling of size neutralized the deleterious effects of increasing eccentricity. We found that size scaling did not affect subitizing capacities, which were nearly constant across all eccentricities. We also found that size scaling modulated the variation coefficients, a normalized metric of enumeration precision, defined as the standard deviation divided by the mean response. Our results show that the inaccuracy and imprecision associated with increasing viewing eccentricity is due to limitations in spatial resolution. Moreover, our results also support the notion that the precise number system is restricted to small numerosities (represented by the subitizing limit), while the approximate number system extends across both small and large numerosities (indexed by variation coefficients) at large eccentricities.     

Role of the hypothalamic PVN in the reflex reduction in mesenteric blood flow elicited by hyperthermia

The hypothalamic paraventricular nucleus (PVN) is an important integrative center in the brain. In the present study, we investigated whether the PVN is a key region in the mesenteric vasoconstriction that normally accompanies an increase in core body temperature. Anesthetized rats were monitored for blood pressure, heart rate, mesenteric blood flow, and vascular conductance. In control rats, elevation of core body temperature to 41 degrees C had no significant effect on blood pressure, increased heart rate, and reduced mesenteric blood flow by 21%. In a separate group of rats, muscimol was microinjected bilaterally (1 nmol/side) into the PVN. Compared with the control group, there was no significant difference in the blood pressure and heart rate responses elicited by the increase in core body temperature. In contrast to control animals, however, mesenteric blood flow did not fall in the muscimol-treated rats in response to the elevation in core body temperature. In a separate group, in which muscimol was microinjected into regions outside the PVN, elevating core body temperature elicited the normal reduction in mesenteric blood flow. The results suggest that the PVN may play a key role in the reflex decrease in mesenteric blood flow elicited by hyperthermia.     

In vitro partial relipidation of apolipoproteins in plasma.

In vitro recombination of lipids with apolipoproteins is achieved when a concentrated solution of plasma lipids in petroleum ether is mixed with delipidated plasma. Combination of phospholipids and unesterified fatty acids are observed in amounts comparable with those originally present in the native unextracted plasma; triglycerides combine partially and cholesterol only slightly. On agarose gel immunoelectrophoresis, a component in the delipidated plasma which is reactive with high density lipoprotein antibodies migrates more slowly than high density lipoprotein in the undelipidated control plasma. However, the component reacting with low density lipoprotein antibodies in the delipidated plasma moves more rapidly than low density lipoprotein in the native plasma. These changes are reversed by recombination of lipid with delipidated plasma. All lipids present in the plasma phase after relipidation travel with the lipoproteins during zonal electrophoresis. The apparent concentrations of proteins reacting with high density and low density lipoprotein antibodies decrease when no lipid is present in plasma on assay by single radial immunodiffusion and immunoelectrophoresis, using commercially available lipoprotein antibodies. On relipidation, full immunochemical properties of high density lipoprotein are restored, but relipidated low density lipoprotein exhibits only partial immunochemical restoration.