SARS-CoV-2 Variant Spike and accessory gene mutations alter pathogenesis.

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against SARS-CoV-2, the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the Spike protein. Although these differences in Spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome which may affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other non-spike mutations on SARS-CoV-2 pathogenesis, we synthesized viruses where the WA1 Spike is replaced by each variant spike genes in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to the full variant viruses. Our work has revealed that non-spike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host and can lead to attenuating phenotypes in circulating variants of concern. This work suggests that while Spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may lead to less clinical disease, extended time toward knowing an infection exists in a person and thus increased time for transmission to occur. Significance: A hallmark of the COVID19 pandemic has been the emergence of SARS-CoV-2 variants that have increased transmission and immune evasion. Each variant has a set of mutations that can be tracked by sequencing but little is known about their affect on pathogenesis. In this work we first identify accessory genes that are responsible for pathogenesis in vivo as well as identify the role of variant spike genes on replication and disease in mice. Isolating the role of Spike mutations in variants identifies the non-Spike mutations as key drivers of disease for each variant leading to the hypothesis that viral fitness depends on balancing increased Spike binding and immuno-evasion with attenuating phenotypes in other genes in the SARS-CoV-2 genome.

Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice.

The involvement of cytokines in the pathogenesis of a generalized, Shwartzman-like lethal inflammatory response to bacterial lipopolysaccharides (LPS) was studied by testing the ability of cytokines or neutralizing anticytokine antibodies to modify the course of the syndrome. The reaction was elicitable in non-SPF NMRI mice by two consecutive injections of S. marcescens LPS: a first injection in the footpad, followed after 24 h by an intravenous dose; the size and route of the preparatory LPS dose were found to be critical. Treatment with mAbs against IFN-gamma was found to completely prevent the reaction. Treatment with IFN-gamma on the other hand, rendered the mice more sensitive to elicitation of the reaction. In contrast, systemic administration of IFN-alpha/beta exerted a desensitizing effect. The role of endogenous cytokines in the pathogenesis of this generalized Shwartzman reaction was also documented by a study of the cytokine levels in the serum of the mice. In comparisons between mice given lethal and nonlethal induction schedules, a good correlation was found between mortality rates and height of IFN or TNF levels, but no correlation was seen with IL-6 levels. Also, in mice that were protected by anti-IFN-gamma antibody, serum IFN and TNF were undetectable, whereas IL-6 levels were as high as in unprotected mice. These data provide evidence that among the cytokines that govern the inflammatory response to LPS, endogenous IFN-gamma occupies a key position. These findings therefore also open perspectives for clinical application of IFN-gamma antagonists.

Specimen Collection for Translational Studies in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disorder characterized by painful nodules, sinus tracts, and scars occurring predominantly in intertriginous regions. The prevalence of HS is currently 0.053-4%, with a predominance in African-American women and has been linked to low socioeconomic status. The majority of the reported literature is  retrospective, population based, epidemiologic studies. In this regard, there is a need to establish a repository of biospecimens, which represent appropriate gender and racial demographics amongst HS patients. These efforts will diminish knowledge gaps in understanding the disease pathophysiology. Hence, we sought to outline a step-by-step protocol detailing how we established our HS biobank to facilitate the formation of other HS tissue banks. Equipping researchers with carefully detailed processes for collection of HS specimens would accelerate the accumulation of well-organized human biological material. Over time, the scientific community will have access to a broad range of HS tissue biospecimens, ultimately leading to more rigorous basic and translational research. Moreover, an improved understanding of the pathophysiology is necessary for the discovery of novel therapies for this debilitating disease. We aim to provide high impact translational research methodology for cutaneous biology research and foster multidisciplinary collaboration and advancement of our understanding of cutaneous diseases.

Bimodal role of endogenous interleukin-6 in concanavalin A-induced hepatitis in mice.

Acute concanavalin A (Con A)-induced hepatitis in mice is an animal model for hepatic injury induced by activated T cells. The evolution of hepatic involvement can be followed from hour to hour by measuring serum transaminase levels. We investigated the possible role of endogenous interleukin-6 (IL-6) in this model. We found serum IL-6 levels and splenic IL-6 mRNA during Con A-induced hepatitis to be significantly lower in interferon-gamma (IFN-gamma)-deficient mice, which are resistant against the Con A-induced syndrome, than in wild-type ones, suggesting that systemic IL-6 production favors development of hepatic injury. However, IL-6-deficient mice proved to be more susceptible to the disease than wild-type mice, indicating that endogenous IL-6 plays a predominantly hepatoprotective role. Experiments in which wild-type mice were treated with anti-IL-6 antibodies, before or after Con A challenge, allowed us to reconcile these contrasting observations. The antibody injections resulted in a biphasic alteration of serum IL-6 levels, initial neutralization being followed by rebound increased levels due to accumulation of IL-6 in the form of antigen-antibody complexes. The effect of antibody on disease severity differed depending on the time of injection. Antibody injection at 2.5 h post Con A resulted in delayed disease manifestation, whereas treatment initiated before Con A resulted in accelerated disease. We conclude that endogenous IL-6 plays a bimodal role. IL-6 present before Con A challenge as well as that induced in the very early phase after Con A injection triggers hepatoprotective pathways. Continuation of IL-6 production beyond this early phase, by some other pathway, seems to be harmful to hepatocytes.

Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans.

We have studied the production of mouse tumor necrosis factor alpha (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the alpha-amylase-encoding gene (aml) of Streptomyces venezuelae ATCC15068 at 12 amino acids (aa) downstream from the signal-peptidase cleavage site so that the aa surrounding this processing site were conserved. S. lividans containing this construct secreted mTNF at moderately high levels (1-10 micrograms/ml) as a biologically active compound of high specific activity (1 x 10(8) units/mg protein). No unprocessed pre-protein and virtually no processed protein could be detected in the cell lysates. N-terminal aa sequence analysis indicated microheterogeneity (-3 to -6 forms) at the N-terminal site of secreted mTNF. It was demonstrated that this microheterogeneity was due to aminopeptidase activity.

In vivo neutrophil recruitment by granulocyte chemotactic protein-2 is assisted by gelatinase B/MMP-9 in the mouse.

Granulocyte chemotactic protein-2 (GCP-2) of the mouse is a potent neutrophil chemotactic and activating factor in vitro and in vivo. Gelatinase B/matrix metalloproteinase-9 is released from neutrophils within 1 h after stimulation with GCP-2. In vitro neutrophil chemotaxis by GCP-2 was not impaired by specific inhibitory monoclonal antibodies (mAb) against gelatinase B, indicating that gelatinase B is not involved in chemotaxis of neutrophils through polycarbonate filters. To investigate if gelatinase B degranulation is involved in in vivo cell migration toward GCP-2, experiments were performed with gelatinase B knockout mice. When mouse GCP-2 was injected intradermally in mice, a dose-dependent neutrophil chemotactic response was observed, and this cell migration was significantly impaired in young mice by genetic gelatinase B knockout. In adult vs. young gelatinase B-deficient mice, such compensatory mechanisms as higher basal neutrophil counts and less impairment of chemotaxis toward local GCP-2 injection were observed. These experiments prove the concept that gelatinase B release under pressure of GCP-2 is a relevant, but not exclusive, effector mechanism of neutrophil chemotaxis in vivo and that known mechanisms, other than the release of gelatinase B, allow for a full-blown chemotactic response and compensate for gelatinase B deficiency in adult life in the mouse.

Role of endogenous interleukin-12 (IL-12) in induced and spontaneous relapses of experimental autoimmune encephalomyelitis in mice.

Actively induced, chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied in SJL/J and in Biozzi ABH mice. In Biozzi ABH mice, relapses occurred spontaneously with high frequency. In SJL/J mice, spontaneous relapses occurred infrequently; however they could be induced reproducibly by reimmunization. In both models, moderately increased levels of serum IL-12(p40) were consistently found shortly before primary attacks, but irregularly at later times. Injections of anti-IL-12 antibody inhibited disease development in both SJL/J and in Biozzi ABH mice. The time window during which treatment needed to be initiated in order to be effective, ranged from before induction until shortly before the symptoms of primary attacks emerged. Such treatment inhibited not only the first attack but also the spontaneous or induced relapses. Most significantly, anti-IL-12 antibody given during remission of primary disease inhibited actively re-induced relapses in SJL/J, but not spontaneous relapses in Biozzi ABH mice. These results indicate that endogenous IL-12 favours EAE development by crucially affecting the active induction process, but that a second burst of IL-12 production may not be necessary for triggering spontaneous relapses.

[Treatment of young transsexuals in the Netherlands]. / De behandeling van jonge transseksuelen in Nederland.

For more than ten years transsexual adolescents have been diagnosed and treated psychologically at the department of Child and Adolescent Psychiatry, University Medical Centre in Utrecht, the Netherlands. The medical part of the treatment takes place at the Academic Hospital of the Free University of Amsterdam. Diagnosis is done in two phases: the first diagnostic phase and the 'real life test'. In this second phase the ability to live in the opposite gender role is tested. Gender dysphoric non-transsexual adolescents are offered psychological or psychiatric interventions. For transsexual adolescents with the express wish to undergo a sex change two types of hormones are prescribed. First, hormones which halt the own pubertal development, then cross-sex hormones with irreversible effects. Surgery for adolescents is not different from surgery for adults. Although the cause of transsexuality is probably impaired sexual differentiation at cerebral level, it appears that the risk of unjustified treatment is higher when the treatment is administered at an early age than in adults; justified treatment, however, has better results when it is administered at an early age.

Anti-interferon-gamma antibody protects mice against the generalized Shwartzman reaction.

A generalized Shwartzman reaction was found to occur in non-SPF-NMRI mice given a local injection of S. marcescens lipopolysaccharide (LPS) in the footpad, followed, with an interval of 24 h, by an i.v. injection. The reaction occurred several hours after the second injection. It was characterized by disseminated subcutaneous bleedings at the mouth, anus, conjuctiva, nose tip and tail end. Most mice died within 24-48 h. The size of the LPS dose given in the footpad (5 micrograms) was found to be critical in that the reaction failed to occur with higher as well as lower doses. The reaction was found to be completely absent in mice that had received one or more systemic injections of monoclonal antibodies with neutralizing potential for murine interferon-gamma (IFN-gamma). Mice treated with control preparations, including a monoclonal antibody with binding but no neutralizing activity for murine IFN-gamma, remained sensitive to induction of the reaction. These observations assign a crucial role to endogenous IFN-gamma in the elicitation of the generalized Shwartzman reaction and open perspectives for the prevention or therapy of clinical conditions related to the Shwartzman reaction by the use of antibodies to IFN-gamma.

Essential role for natural killer cells in the lethal lipopolysaccharide-induced Shwartzman-like reaction in mice.

Observations in our laboratory have provided evidence that interferon-gamma (IFN-gamma) is a key regulator of inflammatory responses to bacterial lipopolysaccharide (LPS) (Heremans et al., J. Exp. Med. 1990. 171: 1853): treatment of mice with neutralizing monoclonal antibody against IFN-gamma was found to completely prevent lethal shock reactions, in particular the generalized Shwartzman reaction, whereas treatment with IFN-gamma sensitized the mice to the development of such reactions. Since activated T cells and natural killer (NK) cells are the main if not the only potential source of LPS-induced IFN-gamma, we investigated the relative importance of these cells in the development of the generalized Shwartzman-like reaction in mice by depleting them selectively with relevant monoclonal antibodies. Treatment with antibodies directed against the CD4+ T cells subset was not effective in protecting mice. Anti-CD8 antibody did attenuate the reaction to some extent. However, markedly reduced mortality was seen in mice which were depleted of NK cells by systemic administration of polyclonal anti-asialo GM1 or monoclonal anti-NK1.1 antibodies. Failure of T cells to promote the Shwartzman reaction was also evidenced by the observation that thymus-less nude mice, which are deficient in T cells, were more rather than less sensitive to the reaction. Approximately 20 times less LPS was needed to induce the lethal reaction in these mice than in NMRI mice and 58 times more anti-IFN-gamma antibody was required to block mortality. Nu/nu mice reportedly have an over-active NK cell compartiment. IFN-gamma production by these cells in LPS-treated mice may account for the augmented sensitivity. Our data suggest that NK cells may be the most important source of endogenous IFN-gamma which mediates the LPS-induced lethal reactions in mice.

The role of cytokines in various animal models of inflammation.

Studies are reviewed in which the role of IFN-gamma in different models of inflammation in mice is examined: LPS-induced generalized Shwartzman reaction, experimental allergic encephalomyelitis (EAE) and experimental cerebral malaria (ECM). The particular role of the cytokine was studied by systemic administration and by blocking the endogenously produced cytokine by the use of neutralizing antibodies. IFN-gamma was found, depending on the model and circumstances, to exert an anti- or a pro-inflammatory effect. In the generalized Shwartzman reaction and ECM this cytokine has a disease promoting role. In EAE, on the contrary, endogenous as well as exogenous IFN-gamma exert a down-regulating effect.

Protective effect of anti-interleukin (IL)-6 antibody against endotoxin, associated with paradoxically increased IL-6 levels.

Two rat monoclonal antibodies (6B4 and 20F3) against mouse interleukin (IL)-6 were studied for their effects on the generalized Shwartzman reaction and on cytokine production elicited by endotoxin injections. Both antibodies were found to protect mice against the generalized Shwartzman reaction. Production of interferon and tumor necrosis factor in these animals, as assessed from serum levels, were not consistently affected by the antibody treatment, although rather increased levels were occasionally noted. Paradoxically, however, endotoxin-induced serum levels of IL-6 in anti-IL-6-treated mice were consistently found to be markedly increased and also to persist for longer time periods. The more vigorous and persistent response may have been due to slower elimination, increased synthesis, or a combination of both. Endogenous production of IL-6 in mice may be sufficiently large to supersede the neutralizing potential of an excess of antibody, as was evident from the fact that ascites fluid of the anti-IL-6-producing 6B4 hybridoma was biologically active in the IL-6 assay.

Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice: enhancement by monoclonal antibodies against interferon-gamma.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory and demyelinating disorder of the central nervous system. Depending on the experimental conditions, it takes an acute monophasic or a chronic relapsing-remitting course. We have previously reported that the incidence and severity of acute EAE in mice are reduced by administration of interferon (IFN)-gamma and augmented by treatment with neutralizing antibodies against IFN-gamma. Here, we investigated the role of IFN-gamma in chronic relapsing models of EAE (CREAE) in SJL/J and Biozzi ABH mice. Spontaneous relapses in Biozzi mice as well as induced relapses in SJL/J mice were facilitated by administration of neutralizing monoclonal antibody (mAb) against IFN-gamma in the disease-free interval. The enhancing effect of anti-IFN-gamma mAb given before and during the primary attack did not carry over to the relapses. However, early administration of IFN-gamma in Biozzi mice, which developed spontaneous relapses in a high proportion, provided partial protection not only against the first attack, but also against subsequent relapses. Administration of exogenous IFN-gamma during the remission phase provided some protection against subsequent relapses. These results indicate that in both types of relapses, IFN-gamma is produced and does provide a certain degree of protection against disease progression.

Increased circulating interleukin-6 (IL-6) activity in endotoxin-challenged mice pretreated with anti-IL-6 antibody is due to IL-6 accumulated in antigen-antibody complexes.

Mice pretreated with monoclonal anti-interleukin-6 (IL-6) antibody and then challenged with lipopolysaccharide (LPS), paradoxically develop higher levels of circulating biological IL-6 activity, as measured by the hybridoma growth promotion assay, than mice similarly challenged but not pretreated with antibody. Here we provide evidence that this increased biological activity was entirely accounted for by the presence of increased amounts of IL-6 protein, which could be isolated by immunoaffinity chromatography and subsequently visualized after gel electrophoresis. Chromatography on a protein G matrix and a sandwich ELISA allowed to demonstrate that all IL-6 present in the serum was in the form of antigen-antibody complexes. Serum samples of antibody-treated animals which contained the highest biological activity typically contained near equimolar concentrations of IL-6 and antibody. In vitro neutralization tests with pure antibody and IL-6 demonstrated that, with both antibodies tested, more than 1000-fold molar excess of antibody is needed for neutralization in the hybridoma growth assay. It is concluded that increased biological activity in serum of the anti-IL-6 antibody-treated mice is due to sequestration of the endogenous IL-6 in the form of antigen-antibody complexes which, due to the lack of sufficient antibody excess, produce nearly full activity in the hybridoma growth assay.

Modification of the anti-CD3-induced cytokine release syndrome by anti-interferon-gamma or anti-interleukin-6 antibody treatment: protective effects and biphasic changes in blood cytokine levels.

Anti-interferon-gamma (IFN-gamma) antibodies were found to protect mice against pathological changes induced by injection of anti-CD3 antibody: incidence of diarrhea, severity of hypothermia and mortality rates were dramatically reduced. In anti-IFN-gamma antibody-treated mice, IFN-gamma blood levels were significantly reduced at 1.5 h post anti-CD3 challenge, but more elevated levels were found from 4 to 24 h. This rebound-like IFN-gamma response coincided with more profound hypoglycemia. Tumor necrosis factor and interleukin (IL)-6 levels were not affected by anti-IFN-gamma treatment. Exogenous IFN-gamma, administered within 3 h (but not later) of the anti-CD3 challenge made the syndrome worse. Furthermore, inter-mouse strain differences in sensitivity to the anti-CD3 syndrome correlated with the ability of the strain to produce IFN-gamma. Anti-IL-6 antibodies provided only marginal protection against hypothermia and mortality, but did markedly reduce hypoglycemia. Levels of biologically active IL-6 in serum were not influenced by anti-IL-6 antibody treatment during the first few hours after anti-CD3 challenge, but were significantly increased at later times. The data provide evidence that endogenous IFN-gamma is a critical element in the early phase of the anti-CD3 syndrome; endogenous IL-6, while possibly being involved in hypoglycemia, seems of lesser importance for the outcome of the syndrome.

Role of interferon-gamma and nitric oxide in pulmonary edema and death induced by lipopolysaccharide.

Mice given lipopolysaccharide (LPS) intravenously developed lung edema, which was maximum after 6 h. Tumor necrosis factor, interleukin 12 (IL-12), IL-6, and interferon-gamma (IFN-gamma) appeared in the serum, and levels of nitrogen oxide (NO) derivatives were increased in serum and bronchoalveolar fluid. Mice pretreated with neutralizing anti-IFN-gamma antibodies had lower serum levels of IFN-gamma, and fewer died. However, levels of other cytokines and NO derivatives as well as lung edema were unchanged. If IFN-gamma and LPS were given together, pulmonary edema was less, but levels of cytokines and NO derivatives in serum were raised, and the mortality was greater. IFN-gamma receptor knockout mice had more edema after LPS, but were less sensitive to the lethal effects. Treatment with anti-IL-12 antibody inhibited IFN-gamma induction and reduced mortality, but had no effect on the lung edema; exogenous IL-12 also failed to affect edema, but boosted serum cytokine levels and increased the mortality. Aminoguanidine, an inhibitor of NO synthase, protected against pulmonary edema, but did not modify the lethal effects of LPS. Clearly, in this model, early pulmonary edema and lethality are not directly related, and induced IFN-gamma has no role in causing early lung edema, but augments other events that result in death.

Evaluation of a novel subtilisin inhibitor gene and mutant derivatives for the expression and secretion of mouse tumor necrosis factor alpha by Streptomyces lividans.

In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis factor alpha (mTNF) was chosen as a model protein. The mTNF cDNA was fused to the vsi signal sequence. The analysis of secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and biological activity measurements revealed an efficient translocation of mTNF. Up to 300 mg of secreted biologically active mTNF per liter could be obtained in shaken-flask cultures. By analyzing the effects of mutations in the N region of the VSI signal peptide on secretion, we found that decreasing the +3 charge of the wild-type protein to +2 resulted in a 3- to 10-fold increase in secretion.

Cholera and pertussis exotoxins protect mice against the lethal Schwartzman reaction and antagonize the effects of lipopolysaccharide on second messenger systems.

Pertussis toxin, and also cholera toxin are capable of inhibiting the effects of LPS in the elicitation of the generalized Schwartzman reaction. This is a potentially lethal generalized thrombo-haemorrhagic hypersensitivity and inflammatory-type response that occurs after two consecutive injections of LPS. The two exotoxins furnish significant protection against the lethal outcome of this reaction. It is known that the acute haematological and haemodynamic changes are accompanied by alterations in the levels of various endogenous mediators: glucocorticoid hormones, prostaglandins, arachidonic acid metabolites, cytokines and proteases. In vitro effects of LPS on murine leukocyte cell lines can be antagonized by pertussis toxin, implicating a Gi-like regulatory protein in the mediation of these effects. Experiments designed to study the involvement of particular second messenger systems (cAMP and phosphatidylinositol) used by LPS in vivo, revealed that the protective effects conferred by these exotoxins are associated with the antagonization of alterations caused by LPS. No correlation was found between the levels of IL-6 and the mortality rate in this experimental mouse model. The results indicate that G proteins play a role in the generation of the Schwartzman reaction and open a new approach for pharmacological intervention in endotoxemia and in clinical settings with Gram-negative sepsis.