Cellular events of acute, resolving or progressive COVID-19 in SARS-CoV-2 infected non-human primates.

Understanding SARS-CoV-2 associated immune pathology is crucial to develop pan-effective vaccines and treatments. Here we investigate the immune events from the acute state up to four weeks post SARS-CoV-2 infection, in non-human primates (NHP) with heterogeneous pulmonary pathology. We show a robust migration of CD16 expressing monocytes to the lungs occurring during the acute phase, and we describe two subsets of interstitial macrophages (HLA-DR+CD206-): a transitional CD11c+CD16+ cell population directly associated with IL-6 levels in plasma, and a long-lasting CD11b+CD16+ cell population. Trafficking of monocytes is mediated by TARC (CCL17) and associates with viral load measured in bronchial brushes. We also describe associations between disease outcomes and high levels of cell infiltration in lungs including CD11b+CD16hi macrophages and CD11b+ neutrophils. Accumulation of macrophages is long-lasting and detectable even in animals with mild or no signs of disease. Interestingly, animals with anti-inflammatory responses including high IL-10:IL-6 and kynurenine to tryptophan ratios show less severe illness. Our results unravel cellular mechanisms of COVID-19 and suggest that NHP may be appropriate models to test immune therapies.

Two distinct GUCY2C circuits with PMV (hypothalamic) and SN/VTA (midbrain) origin.

Guanylyl cyclase C (GUCY2C) is the afferent central receptor in the gut-brain endocrine axis regulated by the anorexigenic intestinal hormone uroguanylin. GUCY2C mRNA and protein are produced in the hypothalamus, a major center regulating appetite and metabolic homeostasis. Further, GUCY2C mRNA and protein are expressed in the ventral midbrain, a principal structure regulating hedonic reward from behaviors including eating. While GUCY2C is expressed in hypothalamus and midbrain, its precise neuroanatomical organization and relationship with circuits regulating satiety remain unknown. Here, we reveal that hypothalamic GUCY2C mRNA is confined to the ventral premammillary nucleus (PMV), while in midbrain it is produced by neurons in the ventral tegmental area (VTA) and substantia nigra (SN). GUCY2C in the PMV is produced by 46% of neurons expressing anorexigenic leptin receptors, while in the VTA/SN it is produced in most tyrosine hydroxylase-immunoreactive neurons. In contrast to mRNA, GUCY2C protein is widely distributed throughout the brain in canonical sites of PMV and VTA/SN axonal projections. Selective stereotaxic ablation of PMV or VTA/SN neurons eliminated GUCY2C only in their respective canonical projection sites. Conversely, specific anterograde tracer analyses of PMV or VTA/SN neurons confirmed distinct GUCY2C-immunoreactive axons projecting to those canonical locations. Together, these findings reveal two discrete neuronal circuits expressing GUCY2C originating in the PMV in the hypothalamus and in the VTA/SN in midbrain, which separately project to other sites throughout the brain. They suggest a structural basis for a role for the GUCY2C-uroguanylin gut-brain endocrine axis in regulating homeostatic and behavioral components contributing to satiety.

The effect of addition of liraglutide to high-dose intensive insulin therapy: a randomized prospective trial.

AIMS: Patients with type 2 diabetes and insulin resistance may require high insulin doses to control hyperglycaemia. The addition of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) to basal insulin therapy has been shown to reduce insulin requirement while reducing insulin-associated weight gain [1,2]. The effect of GLP-1 RA therapy added to intensive (basal/bolus) insulin therapy has not been studied in a prospective trial. This trial evaluated the effect of the addition of liraglutide to high-dose intensive insulin therapy compared with standard insulin up-titration in obese insulin-resistant patients with type 2 diabetes requiring high-dose insulin therapy. METHODS: Thirty-seven subjects with type 2 diabetes requiring >100 units of insulin daily administered either by continuous subcutaneous insulin infusion (CSII) or by multiple daily injections (MDIs) with or without metformin were randomized to receive either liraglutide plus insulin (LIRA) or intensive insulin only (controls). Liraglutide was initiated at 0.6 mg subcutaneously (sq) per day and increased to either 1.2 or 1.8 mg daily in combination with intensive insulin therapy. Controls received intensive insulin up-titration only. RESULTS: At 6 months, subjects receiving liraglutide plus insulin experienced statistically significant reductions in HbA1c, weight, insulin dose and glycaemic variability (GV) by continuous glucose monitor (CGM) compared with the control group receiving insulin only. CONCLUSIONS: Adding liraglutide to intensive high-dose (basal/bolus) insulin therapy results in greater improvement in glycaemic control than insulin therapy alone, with additional benefits of weight loss and reduced GV.

HIV and complement: hijacking an immune defense.

The complement system is a central player of the innate immune system. Activation of the complement system protects the host against pathogens. However, uncontrolled synthesis can be detrimental to host. This concise review summarizes the current understanding of the mechanism(s) of complement activation, the mechanism of C3 regulation, and the role of complement in human immunodeficiency virus (HIV) pathogenesis with emphasis on the cross-talk between HIV and complement system in NeuroAIDS and HIV-associated nephropathy (HIVAN).

p27(SJ), a novel protein in St John's Wort, that suppresses expression of HIV-1 genome.

Transcription of the HIV-1 genome is controlled by the cooperation of viral regulatory proteins and several host factors which bind to specific DNA sequences within the viral promoter spanning the long terminal repeat, (LTR). Here, we describe the identification of a novel protein, p27(SJ), present in a laboratory callus culture of Hypericum perforatum (St John's Wort) that suppresses transcription of the HIV-1 genome in several human cell types including primary culture of microglia and astrocytes. p27(SJ) associates with C/EBPbeta, a transcription factor that regulates expression of the HIV-1 genome in macrophages and monocytic cells, and the viral transactivator, Tat. The association of p27(SJ) with C/EBPbeta and Tat alters their subcellular localization, causing their accumulation in the perinuclear cytoplasmic compartment of the cells. Fusion of a nuclear localization signal to p27(SJ) forces its entry into the nucleus and diminishes the capacity of p27(SJ) to suppress Tat activity, but does not alter its ability to suppress C/EBPbeta activation of the LTR. Results from binding assays showed the inhibitory effect of p27(SJ) on C/EBPbeta interaction with DNA. Finally, our results demonstrate that expression of p27(SJ) decreases the level of viral replication in HIV-1-infected cells. These observations suggest the potential for the development of a therapeutic advance based on p27(SJ) protein to control HIV-1 transcription and replication in cells associated with HIV-1 infection in the brain.

Genomic analysis of Fas and FasL genes and absence of correlation with disease progression in AIDS.

Apoptosis has been suggested as a major mechanism for the CD4(+) T-lymphocyte depletion observed in patients infected with human immunodeficiency virus 1 (HIV-1). To evaluate the impact of genetic variations to apoptosis during progression of acquired immunodeficiency syndrome (AIDS), we have performed an extensive genetic analysis of Fas and Fas ligand ( FasL) genes. The coding regions and promoters of these genes were resequenced in a cohort of 212 HIV-1-seropositive patients presenting extreme disease phenotypes and 155 healthy controls of Caucasian origin. Overall, 33 single nucleotide polymorphisms (SNPs) with an allele frequency >1% were identified and evaluated for their association with disease progression. Among them, 14 polymorphisms were newly characterized. We did not find any statistically significant association of Fas and FasL polymorphisms and haplotypes with AIDS progression.

Genomic analysis of Th1-Th2 cytokine genes in an AIDS cohort: identification of IL4 and IL10 haplotypes associated with the disease progression.

Polymorphisms of Th1-Th2 cytokine genes have previously been implicated in the rate of progression to AIDS in seropositive patients. To evaluate further the impact of these genes in the development of AIDS, we have performed an extensive genetic analysis of IL2, IL4, IL6, IL10, IL12p35 and p40, IL13 and IFNgamma. The coding regions and promoters of these genes were sequenced in a Caucasian cohort of 337 HIV-1 seropositive extreme patients (the GRIV cohort) consisting of patients with slow progression and rapid progression, and up to 470 healthy controls. In all, 64 single nucleotide polymorphisms (SNPs) and four deleterious polymorphisms with frequency >1% were identified and evaluated for their association with disease. Statistically significant associations were observed with haplotypes of the IL4 and IL10 genes, but no relation was found with variants of other genes. The catalogue of SNP and haplotypes presented here will facilitate further genetic investigations of Th1-Th2 cytokines in AIDS and other immune-related disorders.

CNS invasion by CD14+/CD16+ peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection.

Increases in circulating CD14+/CD16+ monocytes have been associated with HIV dementia; trafficking of these cells into the CNS has been proposed to play an important role in the pathogenesis of HIV-induced neurological disorders. This model suggests that events outside the CNS leading to monocyte activation initiate the process leading to HIV dementia. To investigate the role of this activated monocyte subset in the pathogenesis of HIV dementia, we examined brain specimens from patients with HIV encephalopathy (HIVE), HIV without encephalopathy, and seronegative controls. An accumulation of perivascular macrophages was observed in HIVE. The majority of these cells identified in microglial nodules and in the perivascular infiltrate were CD14+/CD16+. P24 antigen colocalized with both CD14 and CD16 suggesting that the CD14+/CD16+ macrophage is a major reservoir of HIV-1 infection in CNS. Using CD45/LCA staining, the perivascular macrophage was distinguished from resident microglia. In addition to perivascular and nodular localizations, CD16 also stained ramified cells throughout the white matter. These cells were more ramified and abundant than cells positive for CD14 in white matter. Double staining for p24 and CD16 suggests that these cells were often infected with HIV-1. The prominent distribution of CD14+ cells in HIVE prompted our analysis of soluble CD14 levels in cerebrospinal fluid. Higher levels of soluble CD14 (sCD14) were observed in patients with moderate-to-severe HIV dementia, suggesting the utility of sCD14 as a surrogate marker. CD14+/CD16+ monocytes may play a role in other neurological disorders and sCD14 may be useful for evaluating these conditions.

Validation of genetic case-control studies in AIDS and application to the CX3CR1 polymorphism.

New polymorphisms have been recently identified in CX3CR1, a coreceptor for some HIV-1 strains, one of which was associated with a strong acceleration of HIV disease progression. This effect was observed both by a case-control study involving 63 nonprogressors (NP) from the asymptomatic long-term (ALT) cohort and Kaplan-Meier analysis of 426 French seroconverters (SEROCO cohort). These results prompted us to analyze these polymorphisms in 244 nonprogressors (NPs) and 80 rapid progressors (RPs) from the largest case-control cohort known to date, the GRIV cohort. Surprisingly, the genetic frequencies found were identical for both groups under all genetic models (p >.8). The discrepancy with the previous work stemmed only from the difference between GRIV NPs versus ALT NPs. We hypothesized this might be due to the limited number of NPs in ALT (n = 63) and in this line we reanalyzed the data previously collected on GRIV for over 100 different genetic polymorphisms: we effectively observed that the genetic frequencies of some polymorphisms could vary by as much as 10% (absolute percentage) when computing them on the first 50 NP subjects enrolled, on the first 100, or on all the NPs tested (240 study subjects). This observation emphasizes the need for caution in case-control studies involving small numbers of subjects: p values should be low or other control groups should be used.However, the association of the CX3CR1 polymorphism with progression seems quite significant in the Kaplan-Meier analysis of the SEROCO cohort (426 individuals), and the difference observed with GRIV might be explained by a delayed effect of the polymorphism on disease. Further studies on other seroconverter cohorts are needed to confirm the reported association with disease progression.

Autoantibodies to TNFalpha in HIV-1 infection: prospects for anti-cytokine vaccine therapy.

Tumor necrosis factor alpha (TNFalpha) is a proinflammatory cytokine principally involved in the activation of lymphocytes in response to viral infection. TNFalpha also stimulates the production of other cytokines, activates NK cells and potentiates cell death and/or lysis in certain models of viral infection. Although TNFalpha might be expected to be a protective component of an antiviral immune response, several lines of evidence suggest that TNFalpha and other virally-induced cytokines actually may contribute to the pathogenesis of HIV infection. Based on the activation of HIV replication in response to TNFalpha, HIV appears to have evolved to take advantage of host cytokine activation pathways. Antibodies to TNFalpha are present in the serum of normal individuals as well as in certain autoimmune disorders, and may modulate disease progression in the setting of HIV infection. We examined TNFalpha-specific antibodies in HIV-infected non-progressors and healthy seronegatives; anti-TNFalpha antibody levels are significantly higher in GRIV seropositive slow/non-progressors (N = 120, mean = 0.24), compared to seronegative controls (N= 12, mean = 0.11). TNFalpha antibodies correlated positively with viral load, (P = 0.013, r = 0.282), and CD8+ cell count (P = 0.03, r = 0.258), and inversely with CD4+ cell count (P = 0.003, r = - 0.246), percent CD4+ cells (P = 0.008, r = -0.306), and CD4 :CD8 ratio (P = 0.033, r = - 0.251). TNFalpha antibodies also correlated positively with antibodies to peptides corresponding to the CD4 binding site of gp160 (P = 0.001, r = 0.384), the CD4 identity region (P = 0.016, r = 0.29), the V3 loop (P = 0.005, r = 0.34), and the amino terminus of Tat (P = 0.001, r = 0.395); TNFalpha antibodies also correlated positively with antibodies to Nef protein (P = 0.008, r = 0.302). The production of anti-TNFalpha antibodies appears to be an adaptive response to HIV infection and suggests the potential utility of modified cytokine vaccines in the treatment of HIV infections as well as AIDS-related and unrelated autoimmune and CNS disorders.

Clinical applications of a finite-element model of the human middle ear.

Computer-generated models are increasingly being used in otolaryngology for teaching purposes, preoperative planning, and clinical simulations, especially when dealing with small, complex areas such as the middle ear. One technique used to analyze the mechanics of complex models is the finite-element method, whereby the system of interest is divided into a large number of small, simple elements. The mechanical properties and applied forces are represented by functions defined over each element, and the mechanical response of the whole system can then be computed. We present a unique three-dimensional finite-element model of the human eardrum and middle ear. Our model takes advantage of phase-shift moiré shape measurements to precisely define the shape of the eardrum. The middle ear geometry is derived from histologic serial sections and from high-resolution magnetic resonance microscopy of the human ear. We discuss the importance of this model in terms of understanding and teaching the mechanics of the human middle ear, simulating various pathologic conditions, and designing ossicular prostheses.

Hearing preservation in acoustic neuroma surgery.

This article reviews the Montreal experience of hearing preservation in acoustic neuroma surgery. The medical records since 1995 of 36 patients who underwent acoustic neuroma extirpation with the intent to preserve hearing were examined. Intraoperative monitoring was conducted using auditory brainstem response measurement with electrocochleography via a transtympanic electrode. The role of intraoperative monitoring in guiding surgical technique and its correlation with postoperative hearing outcome are discussed. A review of the literature regarding hearing preservation in acoustic neuroma surgery is included.

Detection of HIV-1 Tat and JCV capsid protein, VP1, in AIDS brain with progressive multifocal leukoencephalopathy.

HIV-1 infection can lead to severe central nervous system (CNS) clinical syndromes in more than 50% of HIV-1 positive individuals. Progressive multifocal leukoencephalopathy (PML) is the frequent opportunistic infection of the CNS which is seen in as high as 5% of AIDS patients. Results from previous cell culture studies showed that the HIV-1 regulatory protein, Tat can potentiate transcription of the human neurotropic virus, JCV, the causative agent for PML in cells derived from the human CNS. In this communication we examine the presence of the HIV-1 regulatory protein, Tat, as well as the HIV-1 and JCV structural proteins, p24 and VP1, respectively in AIDS/PML clinical samples. We demonstrate high level expression of the JCV capsid protein, VP1, in oligodendrocytes and to some degree in astrocytes of AIDS with PML. In HIV-1+ samples expression of HIV-1 core protein, p24 was detected in perivascular monocytic cells and to a lesser extent in astrocytes and endothelial cells. A lack of p24 expression in oligodendrocytes suggested no infection of these cells with HIV-1. Interestingly, HIV-1 Tat was detected in various infected cells as well as in uninfected oligodendrocytes from HIV-1+ tissue, supporting the earlier in vitro findings that secreted Tat from the infected cells can be localized in the neighboring uninfected cells. The presence of Tat in oligodendrocytes was particularly interesting as this protein can up-modulate JCV gene transcription and several key cell cycle regulatory proteins including cyclin E, Cdk2, and pRb. The data presented here provide in vivo evidence for a role of HIV-1 Tat in the pathogenesis of AIDS/PML by acting as a positive regulatory protein that affects the expression of JCV and other cell regulatory proteins in the CNS.

1999 Seymour B. Sarason Award. Community narratives: tales of terror and joy.

Inspired by a literary-feminist reading of biblical texts, it is suggested that the mission of community psychology/social science can be understood as a calling to use our tools (research methods, critical analysis and observation, scholarship, social influence) to assist others in the job of turning tales of terror into tales of joy. Such work is the essence of personal and social change. These are not things we can do alone; they require collaboration between us and the people of concern. Applying concepts from narrative theory, including description and critical analysis of community and setting narratives, dominant cultural narratives, and personal stories, we can learn from our own communities and we can use our resources to help make known (perhaps even help to imagine new) tales of joy. Some of these themes are illustrated in three very different contexts: a religious community that has made itself inclusive of gay and lesbian people, a mutual help organization that offers a sense of community and hope for the future to people with a history of serious mental illness, and a public elementary school.

Transdominant activity of human immunodeficiency virus type 1 Vpr with a mutation at residue R73.

The 96-amino-acid-long human immunodeficiency virus type 1 virion-encoded accessory protein Vpr is of particular interest, as this protein, which is found in association with viral particles, can exert a regulatory effect on both virus replication and host cell function. Evidently, Vpr, through interaction with several host regulatory proteins, can modulate transcription from the viral long terminal repeat promoter. Expression of Vpr in cells results in deregulation of cell proliferation during the cell cycle pathway at the G(2) stage. Vpr has unique structural features consisting of multiple functional domains. In this study, we have focused on the leucine/isoleucine-rich domain near the carboxyl terminus of Vpr at residue 73 (arginine) and have demonstrated that alterations at this residue result in ablation of transcriptional activity of Vpr and its ability to block cell cycle events at the G(2) stage. Interestingly, substitution mutations at R73 have resulted in a peptide with dominant negative activities on wild-type functions in transcription and host proliferation events. Results from in vitro and in vivo protein-protein interaction studies have revealed that functionally inactive mutant Vpr can be associated with wild-type protein, presumably through the N-terminal regions of the protein which have been shown to be important for Vpr oligomerization. Thus, it is likely that complexation of the mutant Vpr with wild-type protein functionally inactivates Vpr. The importance of these findings in light of the development of therapeutic strategies is discussed.

Analysis of telomere length and thymic output in fast and slow/non-progressors with HIV infection.

There are two models for CD4+ T-cell depletion leading to AIDS: a kinetic model and an immune suppression model. In the kinetic model, direct cell killing due to viral replication results in a continuous demand for CD4+ T-cells, which eventually exhausts their capacity for renewal by proliferative mechanisms. In the immune suppression model, CD4+ T-cell decline is due to an indirect global inhibitory effect of the virus on uninfected immune cell function. In order to address differences in the two models, we investigated proliferative history and thymic output in PBMC from the GRIV cohort of fast (FP) and slow/non-progressors (S/NP), and uninfected controls. Proliferative history and thymic output were assessed by measurement of mean telomeric restriction fragment (TRF) length and T-cell receptor Rearrangement Excision Circles (TREC) levels, respectively. Mean TRF lengths were significantly shorter in S/NP (n = 93, 7.59 +/- 0.11 kb) and FP (n = 42, 7.25 +/- 0.15 kb) compared to controls (n = 35, 9.17 +/- 0.19 kb). Mean TRF length in PBMC (n = 9, 7.32 +/- 0.31 kb) and CD4+ enriched fractions (n = 9, 7.41 +/- 0.30 kb) from a subset of non-GRIV HIV-1 infected samples were also significantly smaller than PBMC (n = 8, 9.77 +/- 0.33 kb) and CD4+ fractions (n = 8, 9.41 +/- 0.32 kb) from uninfected controls. Rates of telomeric shortening, however, were similar among S/NP (n = 93, -45 +/- 20 bp/yr), FP (n = 42, -41 +/- 14 bp/yr) and controls (-29 +/- 17 bp/yr). Paralleling differences observed in mean TRF length, TREC levels were significantly reduced in S/NP (n = 10, 3,433 +/- 843 mol/mu and FP (n = 8, 1,193 +/- 413) compared to controls (n = 15, 22,706 +/- 5,089), indicative of a defect in thymopoiesis in HIV-1 infection. When evaluated in the context of reduced thymopoiesis, the difference in mean TRF length between S/NP and controls (1.58 +/- 0.30 kb) is similar to that observed between memory and naïve T-cells (1.4 +/- 0.1 kb), and may reflect perturbations in the peripheral T-cell population due to a decline in thymic output of naïve T-cells rather than increased turnover. Based on the different clinical criteria used to select S/NP and FP, the sight difference in TREC between these two groups suggests the threshold for pathogenesis as a result of naïve T-cell depletion may be quite low, and incremental increases in thymic output may yield substantial clinical results. Future studies regarding therapeutic vaccination, specifically with HIV-1 Tat targeted anti-immunosuppressive vaccines, should address the defect in thymic output in HIV-1 infection by using TREC analysis as a rapid method for biological evaluation.

Role of HIV-1 Tat and CC chemokine MIP-1alpha in the pathogenesis of HIV associated central nervous system disorders.

Two syndromes affecting cognitive and motor function in the setting of AIDS have been described as HIV encephalopathy (HIVE) and progressive multifocal leukoencephalopathy (PML). HIVE is characterized by the presence of microglial nodules with accompanying astrocytosis. PML is a fatal demyelinating disease of the white matter induced by the human papovavirus JCV which causes cytolytic destruction of glial cells. In addition to the effect of HIV-1 induced immune suppression, HIV may act directly as a co-factor for stimulation of JCV replication in AIDS patients, in part due to Tat-induced activation of JCV gene transcription. Since Tat has been implicated in CNS pathogenesis, we examined its localization in CNS specimens from HIV infected patients with HIVE and PML as well as controls. Based on the observation of CC chemokine induction in monocytes by Tat, we also examined the cellular localization of the CC chemokine Macrophage Inflammatory Protein-1alpha (MIP-1alpha) and its cognate receptor CCR-5 in these samples. In HIVE, Tat was primarily localized in astrocytes and microglia, within the nodular lesions. In PML, a marked increase in the number of Tat positive astrocytes was observed. In both HIVE and PML, prominent expression of MIP-1alpha and CCR-5 was found within areas containing histopathological lesions. CCR-5 positivity of microglia was localized primarily to nodular lesions in HIVE. In PML, increased numbers of cells with monocyte/microglial morphology were observed relative to HIVE. The increased MIP-1 alpha positivity, and potentially other chemokines, may contribute to the pathogenesis of PML in the setting of HIV infection. Tat may play an important role in the pathogenesis of both HIV associated CNS disease states, acting indirectly through cytokine and chemokine dysregulation.