TB Antigen-Stimulated CXCR3 Ligand Assay for Diagnosis of Tuberculous Lymphadenitis

The diagnosis of tuberculous lymphadenitis (TB-LAP) is challenging. We evaluated the role of blood CXC chemokine receptor 3 (CXCR3) ligands in its diagnosis. A total of 65 lymphadenopathy patients were enrolled and lymph node sampling was performed. We also recruited 113 control subjects, consisting of 27 with positive results and 86 with negative results, in the interferon (IFN)-γ release assay (IGRA). In all study subjects, whole-blood samples were collected using the IGRA methodology. After incubation, plasma levels of IFN-γ and two CXCR3 ligands, IFN-inducible T-cell a chemoattractant (I-TAC) and monokine induced by IFN-γ (MIG), were measured using immunoassay. Fifty-three TB-LAP patients were enrolled. TB antigen-stimulated IFN-γ, I-TAC, and MIG levels were all significantly higher in the TB-LAP patients than in the controls and non-TB-LAP patients. The levels of I-TAC and MIG, but not IFN-γ, showed significant differences between the TB-LAP patients and IGRA-positive controls. Area under the receiver operating characteristic curves (AUROCs) of IFN-γ, I-TAC, and MIG were 0.955, 0.958, and 0.959, respectively, for differentiating TB-LAP from control group, and were 0.912, 0.956, and 0.936, respectively, for differentiating TB-LAP from non-TB-LAP. In conclusion, the TB antigen-stimulated MIG and I-TAC could be useful biomarkers in the diagnosis of TB-LAP.

Rapid and sustained decline in CXCL-10 (IP-10) annotates clinical outcomes following TNF-α antagonist therapy in hospitalized patients with severe and critical COVID-19 respiratory failure

Background: A feed-forward pathological signaling loop generated by TNFα and IFN-γ in inflamed lung tissue, driving CXCL-10 (IP-10) and CXCL-9 chemokine-mediated activated T-cell and monocyte/macrophage tissue recruitment, may define, sustain and amplify the inflammatory biology of lethal COVID-19 respiratory failure. Methods: To assess TNFα-antagonist therapy, 18 hospitalized adults with hypoxic respiratory failure and COVID-19 pneumonia received single-dose infliximab-abda therapy 5mg/kg intravenously between April and December 2020. The primary endpoint was time to increase in oxygen saturation to fraction of inspired oxygen ratio (SpO2/FiO2) by >50 compared to baseline and sustained for 48 hours. Secondary endpoints included 28-day mortality, dynamic cytokine profiles (Human Cytokine 48-Plex Discovery Assay, Eve Technologies), secondary infections, duration of supplemental oxygen support and hospitalization. Findings: Patients were predominantly in critical respiratory failure (15/18, 83%), male (14/18, 78%), above 60 years (median 63 yrs, range 31-80), race-ethnic minorities (13/18, 72%), lymphopenic (13/18, 72%), steroid-treated (17/18, 94%), with a median ferritin of 1953ng/ml. Sixteen patients (89%) met the primary endpoint within a median of 4 days, 15/18 (83%) recovered from respiratory failure, and 14/18 (78%) were discharged in a median of 8 days and were alive at 28-day follow-up. Deaths among three patients > 65yrs age with pre-existing lung disease or multiple comorbidities were attributed to secondary lung infection. Mean plasma IP-10 levels declined sharply from 9183 pg/ml to 483 pg/ml at Day 3 and further to 146 pg/ml at Day 14/discharge. Significant declines in IFN-γ, TNFα, IL-27, CRP and ferritin were specifically observed at Day 3 whereas other cytokines were unmodified. IL-6 levels declined sharply among patients with baseline levels >10 pg/ml. Among 13 lymphopenic patients, six (46%) had resolution of lymphopenia by day 3, and 11 by day 14. CXCR3-ligand (IP-10 and CXCL-9) declines were strongly correlated among patients with lymphopenia reversal (Day 3, Pearson r: 0.98, p-value: 0.0006). Interpretation: Consistent with a pathophysiological role of TNFα, the clinical and cytokine data indicate that infliximab-abda may rapidly abrogate pathological inflammatory signaling to facilitate clinical recovery in severe and critical COVID-19. Randomized studies are required to formally assess mortality outcomes. Funding: National Center for Advancing Translational Sciences

İnsan Kemokin Reseptörü CXCR3'ün N-Terminal Bölgesinin (1-53) Moleküler Dinamik Simülasyon Yöntemi İle Modellenmesi ve Yapısal Analizi

G protein eşlikli reseptör (GPER) ailesinden olan Kemokin reseptörlerinin birçok ilaç için hedef bölgesi teşkil ettikleri ve farklı hücre tiplerini aktive ederek çeşitli hastalıkların oluşmasında ve tedavisinde önemli roller oynadıkları bilinmektedir. Bu reseptörlerden biri olan CXCR3 kemokin reseptörünün kristalografik yapısının elde edilmiş olmaması CXCR3-ligand etkileşimlerinin açıklanmasını ve bu reseptöre yönelik ilaçların geliştirilmesini zorlaştırmaktadır. Yapılan çalışmalar, bu reseptörlerin özellikle hücre dışında bulunan N-terminal bölgesinin, bağlanma afinitesi ve reseptör seçiciliğinin belirlenmesinin yanı sıra sinyalleme faaliyetlerinin düzenlenmesinde de kritik roller oynadığını göstermiştir. Bu bölgenin doğal ligandlarıyla etkileşimi hakkında birçok çalışma olmasına rağmen, oldukça esnek bir yapıya sahip olan CXCR3’ün N-terminal bölgesinin katlanma mekanizması ve yapısal özellikleri henüz tam olarak analiz edilmemiştir. Bu nedenle, farklı konformasyonlar gösterebilen bu esnek yapının dinamik davranışlarını incelemek için bölgenin aminoasit sekanslarından yola çıkarak bilgisayarlı yöntemler ile modellenmesi büyük önem taşımaktadır. Bu amaçla çalışmamızda Moleküler Dinamik simülasyon yöntemiyle CXCR3’ün N-terminal bölgesinin aminoasit kompozisyonu modellenerek yapının 300 K sıcaklıkta sulu çözelti içindeki kararlılığı ve dinamik davranışları incelenmiştir. Sonuç olarak modellenen yapının iyi bir şekilde katlanarak kompakt bir form oluşturduğu ve bu yapısal oluşumda hidrojen bağlarının önemli rol oynadığı görülmüştür. Elde edilen bulguların gelecekteki muhtemel ilaç tasarım ve hedefleme çalışmalarına rehberlik etmesi beklenmektedir.

Mapping Out Receptors: High Throughput Screening of Endothelial Monocyte Activating Polypeptide II (EMAPII) Using PRESTO-TANGO

Background: Secreted endothelial monocyte activating polypeptide II (EMAPII/AIMP1) is a pro-apoptotic, pro-inflammatory ligand implicated in diseases such as colorectal cancer, cardiovascular disease, and emphysema. Thus, EMAPII has been shown to induce apoptosis through CXCR3 receptor binding. However, not all EMAPII functions have been attributed to CXCR3. Discovery of new receptors interacting with EMAPII could lead to development of new therapies blocking cognate ligand-receptor binding. We hypothesize the existence of at least one unknown secondary receptor for EMAPII. Methods: The PRESTO-TANGO assay, a construct which converts G-protein coupled receptor (GPCR) ligand binding into luciferase activity measurable by luminometer, was validated using transfection with TANGO-modified CXCR3 and S1P1R as test receptors in HTLA cells. Protocols for cell transfection, adherence, and cultivation were optimized with IP10, EMAPII, and S1P as test ligands. Results: The assay was successfully validated using several GPCR activation readouts. Binding curves were generated for S1P/S1P1 (EC50= 569nM), IP10/CXCR3 (EC50= 47.1 nM), and EMAPII/CXCR3 (EC50= 628 nM). Conditions for the PRESTO-TANGO assay were further refined for maximal signal-to-noise ratio and robust inter-assay reproducibility in preparation for high-throughput screening. We are currently testing 314 TANGO-modified GPCRs for EMAPII affinity. Conclusion: We have validated the Tango assay for the known EMAPII-CXCR3 ligand-receptor system, a valuable tool for evaluation of anti-EMAPII therapeutics. Discovery of a novel EMAPII receptor would allow for the development of therapies including neutralizing antibodies (analogous to the PD1 receptor antibody Pembrolizumab for solid tumors) in diseases such as colorectal cancer, cardiovascular disease, and emphysema.