Aging dysregulates neutrophil extracellular trap formation in response to HIV in blood and genital tissues.

Women acquire HIV through sexual transmission, with increasing incidence in women >50 years old. Identifying protective mechanisms in the female genital tract (FGT) is important to prevent HIV-acquisition in women as they age. Human genital and blood neutrophils inactivate HIV by releasing neutrophil extracellular traps (NETs), an innate protective mechanism against HIV-infection. However, how NET formation is triggered by HIV in different tissues and whether this mechanism is affected by aging remain unknown. We demonstrate that the mechanisms that trigger NET release in response to HIV are different in blood and genital tissues, and that NET release decreases with aging. In blood neutrophils, HIV stimulation independently activated calcium pathways and endosomal TLR8, but aging reduced calcium responses, resulting in delayed NET release. In contrast, calcium responses were absent in genital neutrophils and NET release was triggered preferentially through TLR8 activation, but aging impaired this pathway. HIV induced NET formation through non-lytic pathways in blood and FGT neutrophils, except for a small subset of NETs that incorporated annexin V and lactoferrin predominantly in blood, suggesting proinflammatory and lytic NET release. Our findings demonstrate that blood neutrophils cannot model genital neutrophil responses which has important implications to understanding protection against HIV acquisition.

Aging modifies endometrial dendritic cell function and unconventional double negative T cells in the human genital mucosa.

BACKGROUND: Immune function in the genital mucosa balances reproduction with protection against pathogens. As women age, genital infections, and gynecological cancer risk increase, however, the mechanisms that regulate cell-mediated immune protection in the female genital tract and how they change with aging remain poorly understood. Unconventional double negative (DN) T cells (TCRαß + CD4-CD8-) are thought to play important roles in reproduction in mice but have yet to be characterized in the human female genital tract. Using genital tissues from women (27-77 years old), here we investigated the impact of aging on the induction, distribution, and function of DN T cells throughout the female genital tract. RESULTS: We discovered a novel site-specific regulation of dendritic cells (DCs) and unconventional DN T cells in the genital tract that changes with age. Human genital DCs, particularly CD1a + DCs, induced proliferation of DN T cells in a TFGß dependent manner. Importantly, induction of DN T cell proliferation, as well as specific changes in cytokine production, was enhanced in DCs from older women, indicating subset-specific regulation of DC function with increasing age. In human genital tissues, DN T cells represented a discrete T cell subset with distinct phenotypical and transcriptional profiles compared to CD4 + and CD8 + T cells. Single-cell RNA and oligo-tag antibody sequencing studies revealed that DN T cells represented a heterogeneous population with unique homeostatic, regulatory, cytotoxic, and antiviral functions. DN T cells showed relative to CD4 + and CD8 + T cells, enhanced expression of inhibitory checkpoint molecules and genes related to immune regulatory as well as innate-like anti-viral pathways. Flow cytometry analysis demonstrated that DN T cells express tissue residency markers and intracellular content of cytotoxic molecules. Interestingly, we demonstrate age-dependent and site-dependent redistribution and functional changes of genital DN T cells, with increased cytotoxic potential of endometrial DN T cells, but decreased cytotoxicity in the ectocervix as women age, with implications for reproductive failure and enhanced susceptibility to infections respectively. CONCLUSIONS: Our deep characterization of DN T cell induction and function in the female genital tract provides novel mechanistic avenues to improve reproductive outcomes, protection against infections and gynecological cancers as women age.

Nacer de una manera distinta: ¿son riesgosas las terapias de reproducción asistida? / To be born in a different way; are assisted reproductive therapies risky?

Revisión y comentario de varias publicaciones y meta-análisis en relación a las terapias de reproducción asistida (TRA) y el riesgo que el uso de ellas conlleva en cuanto a peores resultados perinatales y mayor cantidad de malformaciones en los niños concebidos de esta manera...

Review and comment on various publications and meta-analysis in relation to assisted reproductive therapy (ART) and the risk that it entails in terms of worse perinatal outcomes and increased number of malformations in children conceived this way...

Surgical privileging in gynecology: a Fellows' Pelvic Research Network study.

OBJECTIVES: This study aimed to describe the criteria used by US hospitals to grant surgical privileges for select gynecologic procedures and to compare the privileging processes between university-based and community-based hospitals. METHODS: We conducted a cross-sectional study from January 2011 to December 2012 that included institutions represented by Fellows' Pelvic Research Network members. A 5-page, anonymous survey was distributed to hospitals to determine the hospital criteria used for initial surgical privileges and for renewal of privileges for 13 gynecologic procedures. Information on training requirements, minimum number of supervised cases, and annual case number needed for maintenance was obtained. Criteria for privileging were described and compared between university-based and community-based hospitals. RESULTS: Of the 25 institutions that completed the surveys, 56% were university-based and 44% were community-based. Community hospitals differed significantly from university institutions with a larger portion of community hospitals requiring preceptorship for laparoscopic hysterectomy (70% vs 15%, P = 0.027), robotic hysterectomy (90% vs 25%, P = 0.012), robotic sacrocolpopexy (90% vs 20%, P = 0.009), and sacral neuromodulation (67% vs 0%, P = 0.004). CONCLUSIONS: Considerable variability exists in the criteria used by US hospitals for surgical privileging in gynecology. When compared to university centers, a higher proportion of community hospitals required preceptorship for minimally invasive hysterectomy, robotic sacrocolpopexy, and sacral neuromodulation.

Long-term efficacy of the pubovaginal Mersilene mesh sling.

OBJECTIVE: The objective of the study was to determine the efficacy of the pubovaginal Mersilene mesh sling (PVMMS) for complicated urodynamic stress incontinence (USI). STUDY DESIGN: Between 1990 and 2008, patients with USI plus an at-risk diagnosis underwent a PVMMS by a single surgeon. They were followed up with urodynamics (UDE) and Pelvic Floor Distress Inventory-short form 20 (PFDI-20). Stratification was in an at-risk hierarchy: intrinsic sphincter deficiency (ISD) greater than recurrent USI (RUSI) greater than USI with chronically increased intraabdominal pressure (CI-IAP). A cough stress test determined objective cure. PFDI question 17 assessed subjective cure. RESULTS: Three hundred six patients with ISD (43.5%), RUSI (26.8%), and CI-IAP (29.7%) had objective cure rates of 89.2% in the short term, 86.7% in the intermediate term, and 91.2% in the long term. A group of 48 patients with both short- and long-term UDEs showed cures of 100% and 91.7%. Long-term objective cure rates were: ISD, 90.5% (n = 21); RUSI, 84.2%, (n = 19); CI-IAP, 100% (n = 17). The mean score of postoperative PFDI question 17 was 0.57 (n = 119). Mean symptom improvement was -2.98 (n = 52; P < .0001). CONCLUSION: We demonstrated PVMMS to be subjectively and objectively effective in long-term treatment of complicated forms of USI.

p53-independent abrogation of a postmitotic checkpoint contributes to human papillomavirus E6-induced polyploidy.

Polyploidy is often an early event during cervical carcinogenesis, and it predisposes cells to aneuploidy, which is thought to play a causal role in tumorigenesis. Cervical and anogenital cancers are induced by the high-risk types of human papillomavirus (HPV). The HPV E6 oncoprotein induces polyploidy in human keratinocytes, yet the mechanism is not known. It was believed that E6 induces polyploidy by abrogating the spindle checkpoint after mitotic stress. We have tested this hypothesis using human keratinocytes in which E6 expression induces a significant amount of polyploidy. We found that E6 expression does not affect the spindle checkpoint. Instead, we provide direct evidence that E6 is capable of abrogating the subsequent G(1) arrest after adaptation of the mitotic stress. E6 targets p53 for degradation, and previous studies have shown an important role for p53 in modulation of the G(1) arrest after mitotic stress. Importantly, we have discovered that E6 mutants defective in p53 degradation also induce polyploidy, although with lower efficiency. These results suggest that E6 is able to induce polyploidy via both p53-dependent and p53-independent mechanisms. Therefore, our studies highlight a novel function of HPV E6 that may contribute to HPV-induced carcinogenesis and improve our understanding of the onset of the disease.

Verrucous carcinoma of the endometrium: case history, pathologic findings, brief review of literature and discussion.

BACKGROUND: Verrucous carcinoma is a rare condition. A defined disease of the oral cavity, larynx, esophagus, skin, vulva, vagina and cervix. But a verrucous carcinoma arising from the endometrium without evidence of cervical malignancy or endometrial adenocarcinoma is extremely rare. CASE: A 67-year-old G2P2 menopausal patient that was referred for consultation 1 year after presenting with vaginal bleeding to her gynecologist who subsequently underwent several endometrial biopsies where the pathological findings were repetitively similar: papillary squamous proliferation, cytologically bland with low mitotic activity but extensive proliferation. A hysterectomy with bilateral salpingo-oophorectomy was performed. The final histologic examination revealed a squamous cell carcinoma of endometrium, verrucous and well differentiated, and there was no cervical invasion identified. CONCLUSION: This is a rare form of endometrial cancer with apparent favorable prognosis that must be considered when squamous cells are identified on endometrial samplings.

Epstein-Barr virus nuclear protein 3A domains essential for growth of lymphoblasts: transcriptional regulation through RBP-Jkappa/CBF1 is critical.

Experimental reverse genetic replacement of Epstein-Barr virus nuclear antigen 3A (EBNA3A) with a conditional mutant EBNA3A revealed that EBNA3A is critical for continued lymphoblastoid cell (LCL) growth. Wild-type (wt) EBNA3A expressed in the LCLs specifically sustained growth under nonpermissive conditions, whereas EBNA3B or EBNA3C expression had no effect (S. Mauro, E. Johannsen, D. Illanes, A. Cooper, and E. Kieff, J. Virol. 77:10437-10447, 2003). This genetic system and related biochemical assays have now been used to discover that EBNA3A lacking amino acid residues 170 to 240 (delta170-240), TLGC202 to AAGA202, or delta300-386, which are deficient in repression of EBNA2 activation of an RBP-Jkappa/CBF1-dependent EBV Cp enhancer, are null mutations for LCL growth, whereas EBNA3A delta2-124, delta410-439, delta440-470, delta470-500, delta500-523, delta523-612, and delta620-820, which are wt in repression are wt for LCL growth. Thus, EBNA3A regulation of transcription through RBP-Jkappa/CBF1 is critical for LCL growth. EBNA3A mutants deleted of amino acid residues 240 to 300, 386 to 410, or 827 to 944 were intermediate, null, or intermediate, respectively, for LCL growth despite being wt for RBP-Jkappa association and repression. Amino acid residues 240 to 300, 386 to 410, and, particularly, C-terminal residues 827 to 944 are therefore likely to contribute to LCL growth through RBP-Jkappa-independent mechanisms.

Proteins of purified Epstein-Barr virus.

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.

EBNA3C coactivation with EBNA2 requires a SUMO homology domain.

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is critical for EBV immortalization of infected B lymphocytes and can coactivate the EBV LMP1 promoter with EBNA2. EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation and are required for SUMO-1 and SUMO-3 interaction. We found that EBNA3C but not EBNA3CDelta343-545 colocalized with SUMO-1 in nuclear bodies and was modified by SUMO-2, SUMO-3, and SUMO-1. EBNA3C amino acids 545 to 628 and amino acids 30 to 365 were also required for EBNA3C sumolation and nuclear body localization but were dispensable for coactivation, indicating that EBNA3C sumolation is not required for coactivation. Furthermore, EBNA3C amino acids 476 to 992 potently coactivated with EBNA2 but EBNA3C amino acids 516 to 922 lacked activity, indicating that amino acids 476 to 515 are critical for coactivation. EBNA3C amino acids 476 to 515 include DDDVIEV(507-513), which are similar to SUMO-1 EEDVIEV(84-90). EBNA3C m1 and m2 point mutations, DDD(507-509) mutated to AAA and DVIEVID(509-513) mutated to AVIAVIA, respectively, diminished SUMO-1 and SUMO-3 interaction in directed yeast two-hybrid and glutathione S-transferase pulldown assays. Furthermore, EBNA3C m1 and m2 did not coactivate the LMP1 promoter with EBNA2. Overexpression of wild-type SUMO-1, SUMO-3, and the SUMO-conjugating enzyme UBC9 coactivated the LMP1 promoter with EBNA2. Since EBNA2 activation is dependent on p300/CBP, the possible effect of EBNA3C on p300-mediated transcription was assayed. EBNA3C potentiated transcription of p300 fused to a heterologous DNA binding domain, whereas EBNA3C m1 and m2 did not. All of these data are consistent with a model in which EBNA3C upregulates EBNA2-mediated gene activation by binding to a sumolated repressor and inhibiting repressive effects on p300/CBP and other transcription factor(s) at EBNA2-regulated promoters.

Epstein-Barr Virus nuclear protein EBNA3A is critical for maintaining lymphoblastoid cell line growth.

To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.

EBNA3A association with RBP-Jkappa down-regulates c-myc and Epstein-Barr virus-transformed lymphoblast growth.

Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-Jkappa/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three- to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-Jkappa, did not change EBNA3C association with RBP-Jkappa or EBNA or LMP1 expression, decreased EBNA2 association with RBP-Jkappa, decreased c-myc expression, and caused G(0)/G(1) growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c-myc-induced apoptosis. In assays of EBNA2- and RBP-Jkappa-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-Jkappa binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c-myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-Jkappa binding domain. The dependence of EBNA3A effects on the core RBP-Jkappa interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-Jkappa association strongly implicates RBP-Jkappa in c-myc promoter regulation.