Evaluation of sample treatments in a safe and straightforward procedure for the detection of SARS-CoV-2 in saliva.

OBJECTIVES: To evaluate four sample treatments in a safe and straightforward procedure to detect SARS-CoV-2 in saliva. METHODS: Four sample treatments were evaluated in a 3-step procedure to detect SARS-CoV-2 in saliva: 1) heating at 95 °C for 5 min for sample inactivation; 2) sample treatment; 3) analysis by reverse-transcription loop-mediated isothermal amplification (LAMP). Saliva samples used were from infected individuals or were spiked with known quantities of viral particles. RESULTS: Three treatments had a limit of detection (LOD) of 500.000 viral particles per ml of saliva and could be used to detect individuals with potential to transmit the disease. The treatment of phosphate buffer, dithiothreitol, ethylenediaminetetraacetic acid and proteinase K, with an additional 95 °C heating step, yielded a lower LOD of 95; its sensitivity ranged from 100% in patients with nasopharyngeal swab reverse-transcriptase polymerase chain reaction cycle threshold values <20 to 47.8% for values >30. CONCLUSIONS: This report highlights the importance of an adequate sample treatment for saliva to detect SARS-CoV-2 and describes a flexible procedure that can be adapted to point-of-care. Although its sensitivity when LAMP is used is lower than reverse-transcriptase polymerase chain reaction, this procedure can contribute to COVID-19 control by detecting individuals able to transmit the disease.

Activating Killer-Cell Immunoglobulin-Like Receptors Are Associated With the Severity of Coronavirus Disease 2019.

BACKGROUND: Etiopathogenesis of the clinical variability of the coronavirus disease 2019 (COVID-19) remains mostly unknown. In this study, we investigate the role of killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen class-I (HLA-I) interactions in the susceptibility and severity of COVID-19. METHODS: We performed KIR and HLA-I genotyping and natural killer cell (NKc) receptors immunophenotyping in 201 symptomatic patients and 210 noninfected controls. RESULTS: The NKcs with a distinctive immunophenotype, suggestive of recent activation (KIR2DS4low CD16low CD226low CD56high TIGIThigh NKG2Ahigh), expanded in patients with severe COVID-19. This was associated with a higher frequency of the functional A-telomeric activating KIR2DS4 in severe versus mild and/or moderate patients and controls (83.7%, 55.7% and 36.2%, P < 7.7 × 10-9). In patients with mild and/or moderate infection, HLA-B*15:01 was associated with higher frequencies of activating B-telomeric KIR3DS1 compared with patients with other HLA-B*15 subtypes and noninfected controls (90.9%, 42.9%, and 47.3%; P < .002; Pc = 0.022). This strongly suggests that HLA-B*15:01 specifically presenting severe acute respiratory syndrome coronavirus 2 peptides could form a neoligand interacting with KIR3DS1. Likewise, a putative neoligand for KIR2DS4 could arise from other HLA-I molecules presenting severe acute respiratory syndrome coronavirus 2 peptides expressed on infected an/or activated lung antigen-presenting cells. CONCLUSIONS: Our results support a crucial role of NKcs in the clinical variability of COVID-19 with specific KIR/ligand interactions associated with disease severity.

Qualitative assessment of SARS-CoV-2-specific antibody avidity by lateral flow immunochromatographic IgG/IgM antibody assay.

Knowledge of the precise timing of SARS-CoV-2 infection may be of clinical and epidemiological relevance. The presence of low-avidity IgGs has conventionally been considered an indicator of recent infection. Here, we carried out qualitative assessment of SARS-CoV-2-specific antibody avidity using an urea (6M) dissociation test performed on a lateral flow immunochromatographic IgG/IgM device. We included a total of 76 serum specimens collected from 57 COVID-19 patients, of which 39 tested positive for both IgG and IgM and 37 only for IgG. Sera losing IgG reactivity after urea treatment (n = 28) were drawn significantly earlier (P = .04) after onset of symptoms than those which preserved it (n = 48). This assay may be helpful to estimate the time of acquisition of infection in patients with mild to severe COVID-19.

T cell senescence predicts subclinical atherosclerosis in HIV-infected patients similarly to traditional cardiovascular risk factors.

The main objective of this study is to evaluate the predictive capacity of T cell activation/senescence in subclinical atherosclerosis (SCA) in a group of HIV-infected patients. So, a cross-sectional analysis was performed on 91 long-term triple-ART therapy HIV-infected patients from an observational and prospective cohort. Carotid Intima Media Thickness (cIMT) was measured. Binary logistic regression was used to evaluate independent variables associated with SCA. Compared to patients without SCA, patients with SCA (60.4%) were older (41.33 ±â€¯9.04 vs. 51.73 ±â€¯8.44 years old, p < 0.001) and showed Framingham risk score (2.63 ±â€¯3.127 vs. 7.66 ±â€¯5.84, p = 0.008), as well as higher numbers of CD4+CD8+ double positive T cells (0.50 ±â€¯0.42% vs. 0.81 ±â€¯0.79%, p = 0.037), CD8+CD28- T cells (41.70 ±â€¯16.96% vs. 50.22 ±â€¯16.15%, p = 0.018), higher expression of CD28 on CD8+CD28+ T cells (1865 ±â€¯789 vs. 2243 ±â€¯917 MFI, P = 0.046). In contrast, they showed lower expression of CD38 on CD19+ B cells (65.38 ±â€¯27.47% vs. 42.67 ±â€¯30.26%, P < 0.001). Logistic multivariable analysis showed that Framingham risk score >10% (OR = 14.84, CI95% 1.63-125; p = 0.016) and numbers of CD8+CD28- T cells (OR = 1.032, CI 95% 1-1.065; p = 0.045) were independent factors associated with SCA. Patients with CD8+CD28- T cells ≥59% compared to those <59% had higher risk of SCA (OR = 4, CI95% 1.19-13.3, p = 0.024). Interestingly, 27.4% of patients with low Framingham risk score had elevated levels of CD8+CD28- T cells. In conclusion, immune senescence represented by accumulation of CD8+CD28- T cells may contribute to improve the predictive capacity of the Framingham risk score, especially when the scores are low and can explain, at least in part, the higher prevalence of SCA observed in long-term ART-treated stable HIV infected patients.

Paracrine Anti-inflammatory Effects of Adipose Tissue-Derived Mesenchymal Stem Cells in Human Monocytes.

The inflammatory process is an essential phenomenon in the induction of immune responses. Monocytes are key effector cells during the inflammatory process. A wide range of evidence indicates that mesenchymal stem cells from adipose tissue (ASC) are endowed with immunomodulatory capacity. However, the interaction between ASC and monocytes in the innate immune response is not well understood. The aim of this work was to investigate the possible paracrine anti-inflammatory effects of ASC in human monocytes. Monocytes were isolated from buffy coats and ASC from fat of non-obese patients. Conditioned medium (CM) from ASC in primary culture was used. We have assessed the effects of CM on the production of inflammatory mediators, degranulation, migration, phagocytic activity, senescence, oxidative stress, mitochondrial membrane potential and macrophage polarization. We have shown that ASC exert paracrine anti-inflammatory actions on human monocytes. CM significantly reduced the production of TNFα, NO and PGE2 and the activation of NF-κB. In addition, we observed a significant reduction of degranulation, phagocytic activity and their migratory ability in the presence of the chemokine CCL2. The senescence process and the production of oxidative stress and mitochondrial dysfunction were inhibited by CM which also reduced the production of TNFα by M1 macrophages while enhanced TGFß1 and IL-10 release by M2 macrophages. This study have demonstrated relevant interactions of ASC with human monocytes and macrophages which are key players of the innate immune response. Our results indicate that ASC secretome mediates the anti-inflammatory actions of these cells. This paracrine mechanism would limit the duration and amplitude of the inflammatory response.

Haem oxygenase-1 down-regulates high mobility group box 1 and matrix metalloproteinases in osteoarthritic synoviocytes.

OBJECTIVES: Activation of osteoarthritic synoviocytes by pro-inflammatory cytokines results in the release of biochemical mediators such as MMPs and high mobility group box 1 (HMGB1). Extracellular HMGB1 can play an important role in joint diseases as a mediator of synovitis. We have shown previously that haem oxygenase-1 (HO-1) exerts protective effects during inflammatory responses. In this study, we have examined whether HO-1 induction would be an effective strategy to control MMP and HMGB1 production in osteoarthritic synoviocytes. METHODS: Osteoarthritic synoviocytes were obtained by digestion with collagenase and cultured until third passage. HO-1 was induced by cobalt protoporphyrin IX (CoPP). Lentiviral HO-1 vector (LV-HO-1) was also used for HO-1 overexpression. HO-1 gene silencing was achieved by using a specific small interfering RNA. Gene expression was analysed by quantitative PCR and protein expression by western blot, ELISA and IF. MMP activity was studied by fluorometric procedures. RESULTS: Induction of HO-1 by CoPP in the presence of IL-1beta decreased the expression of MMP-1 and -3, and MMP activity. IL-1beta stimulation of synoviocytes increased HMGB1 expression, its translocation into the cytoplasm and secretion. HO-1 induction exerted inhibitory effects on these processes. The consequences of HO-1 induction were counteracted by HO-1 gene silencing, whereas transfection with LV-HO-1 confirmed the effects of pharmacological HO-1 induction. CONCLUSIONS: We have provided direct evidence that HO-1 down-regulates MMP-1, -3 and HMGB1 in osteoarthritic synoviocytes. HO-1 may be a potential strategy to control inflammatory and degradative processes in the progression of OA.

COX-2 and sPLA2 inhibitory activity of aqueous extract and polyphenols of Rhizophora mangle (red mangrove).

The aqueous extract of Rhizophora mangle bark and its polyphenolic fractions showed remarkable in vitro antiinflammatory activity in a preliminary study. The low molecular weight fraction exhibited cyclooxygenase-2 inhibitory activity while the total aqueous extract and the low molecular weight fraction showed secretory phospholipase A(2) inhibitory activity.

Activity of lupane triterpenoids from Maytenus species as inhibitors of nitric oxide and prostaglandin E2.

In the present study, we report that three new lupane triterpenes (1-3), in addition to 16 known ones (4-19), were isolated from the root bark of Maytenus cuzcoina and the leaves of Maytenus chiapensis. Their structures were elucidated by spectral analysis, including homonuclear and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC, and HMBC). The natural compounds and derivatives 6a, 6b, 9a, and 9b have been tested for potential anti-inflammatory activity, and several compounds including 3-epicalenduladiol (2), 11alpha-hydroxy-glochidone (3), rigidenol (6), acetoxy-rigidenol (6a), 11alpha-acetoxy-30-chloro-3-oxo-lup-20(29)-ene (6b), betulin (9), 28-acetoxy-betulin (9a), epibetulin (12), epibetulinic acid (13), and betulonic acid (16) exhibited potent inhibitory effects on NO and prostaglandin E(2) production in mouse macrophages (RAW 264.7) stimulated with bacterial endotoxin. The structure-activity relationship is discussed in detail.

6-Dimethylamino 1H-pyrazolo[3,4-d]pyrimidine derivatives as new inhibitors of inflammatory mediators in intact cells.

The synthesis of 6-dimethylamino 1H-pyrazolo[3,4-d]pyrimidines substituted at positions 1 and 4, and their effects on murine macrophage and human neutrophil functions are described. Several compounds and especially 4b-6b are potent inhibitors of PGE(2) generation in murine macrophages. This action is related to a direct effect on COX-2 activity without affecting the enzyme expression. Some of these compounds also inhibited COX-1 and COX-2 in human monocytes and 4b showed selectivity for COX-2 inhibition.