Isolation and characterization of uterine leukocytes collected using a uterine swab technique.

PROBLEM: Leukocytes from the maternal-fetal interface are a valuable tool to study local changes in immune function during pregnancy; however, sampling can be challenging due to inadequate tissue availability and the invasive nature of placental bed biopsy. Here, we aim to purify and characterize leukocytes from paired peripheral and uterine blood samples to assess whether a less invasive method of uterine blood collection could yield a population of enriched uterine leukocytes suitable for ex vivo and in vitro analyses. METHOD OF STUDY: Human peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) expressed from surgical gauze post C-section were isolated, and immunophenotypic information was acquired by multi-parameter flow cytometry. PBMC and UBMC were stained for markers used to define T and B lymphocytes, macrophages, regulatory T (TReg ) cells, and natural killer (NK) cells. Prime flow was performed to check expression and analysis of CD16- CD56++ and CD16- CD56++ NK transcripts in PBMC and UBMC samples. RESULTS: Immunophenotyping revealed that over 95% of both live PBMC and UBMC consisted of CD45+ leukocytes. Higher percentages of CD16- CD56++ , characterized as uterine NK (uNK) cells, were observed in UBMC samples as compared to PBMC samples (18.41% of CD45+ CD3- vs. 2.73%, respectively), suggesting that CD16- CD56++ cells were enriched in these samples. In UBMC, 49.64% of CD3-negative cells were of peripheral NK phenotype (CD16+ CD56++ ), suggesting infiltration of maternal peripheral NK (pNK) cell in the uterine interface. CONCLUSION: Intrauterine leukocytes, especially CD16- CD56++ NK cells, can be collected in sufficient numbers with increased purity by sampling the uterine cavity postdelivery with surgical gauze. Our results suggest that this non-invasive protocol is a useful sampling technique for isolating CD16- CD56++ cells, however, due to peripheral blood contamination, the NK cell yield could be lower compared to actual decidual or endometrial samples post-partum which is more invasive.

Beneficial Effects of the Direct AMP-Kinase Activator PXL770 in In Vitro and In Vivo Models of X-Linked Adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a severe orphan disease caused by mutations in the peroxisomal ABCD1 transporter gene, leading to toxic accumulation of Very Long-Chain Fatty Acids (VLCFA - in particular C26:0) resulting in inflammation, mitochondrial dysfunction and demyelination. AMP-activated protein kinase (AMPK) is downregulated in ALD, and its activation is implicated as a therapeutic target. PXL770 is the first direct allosteric AMPK activator with established clinical efficacy and tolerability. Methods: We investigated its effects in ALD patient-derived fibroblasts/lymphocytes and Abcd1 KO mouse glial cells. Readouts included VLCFA levels, mitochondrial function and mRNA levels of proinflammatory genes and compensatory transporters (ABCD2-3). After PXL770 treatment in Abcd1 KO mice, we assessed VLCFA levels in tissues, sciatic nerve axonal morphology by electronic microscopy and locomotor function by open-field/balance-beam tests. Results: In patients' cells and Abcd1 KO glial cells, PXL770 substantially decreased C26:0 levels (by ∼90%), improved mitochondrial respiration, reduced expression of multiple inflammatory genes and induced expression of ABCD2-3 In Abcd1 KO mice, PXL770 treatment normalized VLCFA in plasma and significantly reduced elevated levels in brain (-25%) and spinal cord (-32%) versus untreated (P < 0.001). Abnormal sciatic nerve axonal morphology was also improved along with amelioration of locomotor function. Conclusion: Direct AMPK activation exerts beneficial effects on several hallmarks of pathology in multiple ALD models in vitro and in vivo, supporting clinical development of PXL770 for this disease. Further studies would be needed to overcome limitations including small sample size for some parameters, lack of additional in vivo biomarkers and incomplete pharmacokinetic characterization. SIGNIFICANCE STATEMENT: Adrenoleukodystrophy is a rare and debilitating condition with no approved therapies, caused by accumulation of very long-chain fatty acids. AMPK is downregulated in the disease and has been implicated as a potential therapeutic target. PXL770 is a novel clinical stage direct AMPK activator. In these studies, we used PXL770 to achieve preclinical validation of direct AMPK activation for this disease - based on correction of key biochemical and functional readouts in vitro and in vivo, thus supporting clinical development.

Endometriosis: Epidemiology, Diagnosis and Clinical Management.

PURPOSE OF REVIEW: Endometriosis is a disease of adolescents and reproductive-aged women characterized by the presence of endometrial tissue outside the uterine cavity and commonly associated with chronic pelvic pain and infertility. Here we review the epidemiology of endometriosis as well as potential biomarkers for detection and with the goal of highlighting risk factors that could be used in combination with biomarkers to identify and treat women with endometriosis earlier.. RECENT FINDINGS: Early age at menarche, shorter menstrual length, and taller height are associated with a higher risk of endometriosis while parity, higher body mass index (BMI) and smoking are associated with decreased risk. Endometriosis often presents as infertility or continued pelvic pain despite treatment with analgesics and cyclic oral contraceptive pills. SUMMARY: Despite a range of symptoms, diagnosis of endometriosis is often delayed due to lack of non-invasive, definitive and consistent biomarkers for diagnosis of endometriosis. Hormone therapy and analgesics are used for treatment of symptomatic endometriosis. However, the efficacy of these treatments are limited as endometriosis often recurs. In this review, we describe potential diagnostic biomarkers and risk factors that may be used as early non-invasive in vitro tools for identification of endometriosis to minimize diagnostic delay and improve reproductive health of patients.