The immune landscape of the inflamed joint defined by spectral flow cytometry.

Cellular phenotype and function are altered in different microenvironments. For targeted therapies it is important to understand site-specific cellular adaptations. Juvenile Idiopathic Arthritis (JIA) is characterised by autoimmune joint inflammation, with frequent inadequate treatment responses. To comprehensively assess the inflammatory immune landscape, we designed a 37-parameter spectral flow cytometry panel delineating mononuclear cells from JIA synovial fluid (SF) of autoimmune inflamed joints, compared to JIA and healthy control blood. Synovial monocytes and NK cells (CD56bright) lack Fc-receptor CD16, suggesting antibody-mediated targeting may be ineffective. B cells and DCs, both in small frequencies in SF, undergo maturation with high 4-1BB, CD71, CD39 expression, supporting T cell activation. SF effector and regulatory T cells were highly active with newly described co-receptor combinations that may alter function, and suggestion of metabolic reprogramming via CD71, TNFR2 and PD-1. Most SF effector phenotypes, as well as an identified CD4-Foxp3+ T cell population, were restricted to the inflamed joint, yet specific SF-predominant CD4+Foxp3+ Treg subpopulations were increased in blood of active but not inactive JIA, suggesting possible recirculation and loss of immunoregulation at distal sites. This first comprehensive dataset of the site-specific inflammatory landscape at protein level will inform functional studies and the development of targeted therapeutics to restore immunoregulatory balance and achieve remission in JIA.

Single and multiple high-risk and low-risk Human Papillomavirus association with cervical lesions of 11,224 women in Jakarta.

PURPOSE: We sought to evaluate prevalence, age-adjusted distribution, and impact of single and multiple high- and low-risk human papillomavirus (HPV) subtypes and their associations with cervical lesions. METHODS: Data were extracted from 11,224 women who underwent routine screening of HPV genotyping and liquid-based cytology co-testing. Fifteen high-risk (HR) and six low-risk (LR) HPV types were genotyped. RESULTS: Overall HPV prevalence was 10.7 %, and young women (under 21 years old) harbored highest HPV infection rate (40.38 %). The rate declined in old women 9.49 % (age 30-49) and 6.89 % (age 50 and above). Normal cytology had lowest HPV (5.66 %) compared to low-grade (60.49 %), high-grade (71.96 %) squamous intraepithelial lesions (LSIL and HSIL) and squamous cell carcinoma SCC (86.9 %). LR HPV subtypes were absent in SCC and were consistently lower than HR HPV in LSIL (6.74 vs. 33.54 %) and HSIL (2.12 vs. 51.32 %). Multiple HPV infection was more frequent in young women under 30 years old (10 %) than older women (2 %) and in LSIL (20.2 %), HSIL (18.5 %) than SCC (4.4 %). HR HPV 52, 16, 18, and 58 were the most frequent subtypes in normal, LSIL, and HSIL. Greater or equal proportion of HPV 16, 18, 45, and 52 was found in SCC compared to normal cytology (SCC/normal ratios 4.8, 1.2, 1.6, and 1.7). While important in LSIL and HSIL, HPV58 was not detected in SCC. CONCLUSION: Taken together, identification of these HPV types, especially HPV 16, 18, 45, and 52, and their associated cervical lesions may improve cervical cancer preventive strategies in Indonesia.

ATM and ATR Signaling Regulate the Recruitment of Human Telomerase to Telomeres.

The yeast homologs of the ATM and ATR DNA damage response kinases play key roles in telomerase-mediated telomere maintenance, but the role of ATM/ATR in the mammalian telomerase pathway has been less clear. Here, we demonstrate the requirement for ATM and ATR in the localization of telomerase to telomeres and telomere elongation in immortal human cells. Stalled replication forks increased telomerase recruitment in an ATR-dependent manner. Furthermore, increased telomerase recruitment was observed upon phosphorylation of the shelterin component TRF1 at an ATM/ATR target site (S367). This phosphorylation leads to loss of TRF1 from telomeres and may therefore increase replication fork stalling. ATM and ATR depletion reduced assembly of the telomerase complex, and ATM was required for telomere elongation in cells expressing POT1ΔOB, an allele of POT1 that disrupts telomere-length homeostasis. These data establish that human telomerase recruitment and telomere elongation are modulated by DNA-damage-transducing kinases.

Inherited bone marrow failure associated with germline mutation of ACD, the gene encoding telomere protein TPP1.

Telomerase is a ribonucleoprotein enzyme that is necessary for overcoming telomere shortening in human germ and stem cells. Mutations in telomerase or other telomere-maintenance proteins can lead to diseases characterized by depletion of hematopoietic stem cells and bone marrow failure (BMF). Telomerase localization to telomeres requires an interaction with a region on the surface of the telomere-binding protein TPP1 known as the TEL patch. Here, we identify a family with aplastic anemia and other related hematopoietic disorders in which a 1-amino-acid deletion in the TEL patch of TPP1 (ΔK170) segregates with disease. All family members carrying this mutation, but not those with wild-type TPP1, have short telomeres. When introduced into 293T cells, TPP1 with the ΔK170 mutation is able to localize to telomeres but fails to recruit telomerase to telomeres, supporting a causal relationship between this TPP1 mutation and bone marrow disorders. ACD/TPP1 is thus a newly identified telomere-related gene in which mutations cause aplastic anemia and related BMF disorders.