Sprouting angiogenesis is the formation of new capillaries from existing vessels in response to tissue hypoxia due to growth/development, repair/healing, and also chronic inflammation. In this study, we aimed to elucidate the effect of IL-6, a pleiotropic cytokine with both pro-inflammatory and anti-inflammatory functions, in regulating the sprouting angiogenic response of endothelial cells (ECs). We found that activation of IL-6 trans-signaling inhibited the migration, proliferation, and tube formation ability of ECs. In addition, inhibition of the autocrine IL-6 classic-signaling by depleting endogenous IL-6 from ECs impaired their tube formation ability. At the molecular level, we found that IL-6 trans-signaling in ECs upregulated established endogenous anti-angiogenic factors such as CXCL10 and SERPINF1 while at the same time downregulated known endogenous pro-angiogenic factors such as cKIT and CXCL8. Furthermore, prior activation of ECs by IL-6 trans-signaling alters their response to vascular endothelial growth factor-A (VEGF-A), causing an increased p38, but decreased Erk1/2 phosphorylation. Collectively, our data demonstrated the dual facets of IL-6 in regulating the sprouting angiogenic function of ECs. In addition, we shed light on molecular mechanisms behind the IL-6 trans-signaling mediated impairment of endothelial sprouting angiogenic response.

Rapid quantification of Mycobacterium tuberculosis (Mtb) is important for monitoring bacterial load during treatment and in clinical studies. The molecular bacterial load assay detecting Mtb 16S ribosomal RNA (TB-MBLA) can quantify mycobacterial load within hours. As the current TB-MBLA protocol includes highly toxic chemicals and manual RNA extraction, we aimed to optimize TB-MBLA for routine clinical laboratories. We compared several strategies to inactivate Mtb in sputum samples, which would facilitate analysis outside a BSL3 laboratory. Heating at 80°C for 20 min was the most efficient inactivation strategy, whereas GTC-DTT and 1 M NaOH failed to inactivate Mtb sufficiently. We used Mtb-inoculated sputum samples to compare manual phenol-chloroform RNA extraction, as recommended by the TB-MBLA protocol, to an automated magnetic bead-based extraction. Automated RNA extraction reduced total analysis time by 2 h and performed equally to phenol-chloroform extraction, with a detection limit of close to 100 CFU in 10-fold dilution series (mean Ct value: 36.0 ± 1.4 automated versus 33.5 ± 3.6 manual, n = 6). Freezing samples in lysis buffer up to 14 days did not affect TB-MBLA performance (mean Ct value: 22.6 ± 0.3 fresh versus 22.6 ± 0.6 frozen, n = 6). The final TB-MBLA protocol quantified bacterial load up to 11 days faster than time-to-positive culture (TTP) (mean TTP: 11 days, 2–24 days, n = 9) in sputum from patients with pulmonary TB. We adapted the TB-MBLA method for faster throughput in a routine clinical laboratory by using inactivation at 80°C for 20 min, followed by automated RNA extraction, achieving equal sensitivity compared to the original TB-MBLA method, and avoiding manual extraction and toxic reagents. IMPORTANCE: The current tuberculosis molecular bacterial load assay (TB-MBLA) protocol relies on toxic chemicals and manual RNA extraction, making the procedure both time‑consuming and labor‑intensive. We optimized and validated a faster TB-MBLA strategy, which can be adapted for routine clinical laboratories, including inactivation by heating, to facilitate implementation in settings without a BSL3 laboratory.

Topically applied TH1579 alleviated the psoriatic phenotype in the imiquimod-induced psoriasis mouse model by decreasing CD45⁺, Ly6b⁺, and CD3⁺ cell infiltration and downregulating the expression of the proliferation marker PCNA. Moreover, TH1579 strongly suppressed IL-17 expression in mouse skin, accompanied by reduced infiltration of IL-17-producing γδ-T cells. Furthermore, TH1579 decreased keratinocyte viability and proliferation. Mass spectrometry data analysis revealed the enrichment of proteins associated with nucleotide excision repair and cell cycle regulation. The key cell cycle regulatory protein F-box protein S-phase kinase-associated protein 2 (SKP2) was significantly downregulated, along with the psoriasis-associated proliferation marker WNT5a, identified as a SKP2 downstream target. The downregulation of SKP2 and WNT5a was confirmed in MTH1i-treated mouse skin. Our findings support the topical administration of MTH1i TH1579 as a psoriasis treatment. The therapeutic effects depended on the SKP2/WNT5a pathway, which mediates psoriatic hyperproliferation. This study introduces a conceptually innovative topical treatment for psoriasis patients with mild-to-moderate disease who have limited therapeutic options.