Project description:In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. Here we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner’s seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the peri-ovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. One pair of first biopsy and second biopsy RNA samples from each treatment group were reverse transcribed into cDNA and hybridized to Affymetrix Human Gene 1.0 ST arrays. A total of 713 probe sets were identified as differentially expressed (fold change >2) between first and second biopsies after unprotected coitus, with 436 genes upregulated and 277 genes downregulated. Ingenuity Pathway Analysis revealed that gene pathways including inflammatory response, immune response, immune cell trafficking, cellular movement and antigen presentation were significantly affected by seminal fluid exposure. Amongst these were genes encoding several chemokines which target granulocytes, monocyte/macrophages, dendritic cells and lymphocytes, proinflammatory cytokines and regulators of cytokine synthesis, prostaglandin pathway gene including PTGS2; COX-2) and several matrix metalloproteinases (MMPs). Of these genes, no change or a substantially smaller change was seen between first and second biopsies obtained after coitus with condom use, or abstinence. An increase in CSF2, IL6, IL8 and IL1A expression was confirmed by qRT-PCR in larger sets of duplicate biopsies (n=6-7 per group). We conclude that seminal fluid introduced at intercourse elicits expression of pro-inflammatory cytokines and chemokines which underpins the accompanying recruitment of macrophages, dendritic cells and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens, and potentially susceptibility to cervical metaplasia. Women aged 18-40 years, with regular menstrual cycles and a current sexual relationship with a proven fertile regular partner, volunteered for the study. All women had previously undergone tubal ligation and none of the women used steroidal contraceptives or an intrauterine device for a minimum of three months preceding the study. Subjects were screened to exclude bacterial or viral infection. A negative test result was a prerequisite for inclusion in the study. The study was approved by the local ethics committee of Karolinska University Hospital, Stockholm, Sweden.
Project description:In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. Here we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner’s seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the peri-ovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. One pair of first biopsy and second biopsy RNA samples from each treatment group were reverse transcribed into cDNA and hybridized to Affymetrix Human Gene 1.0 ST arrays. A total of 713 probe sets were identified as differentially expressed (fold change >2) between first and second biopsies after unprotected coitus, with 436 genes upregulated and 277 genes downregulated. Ingenuity Pathway Analysis revealed that gene pathways including inflammatory response, immune response, immune cell trafficking, cellular movement and antigen presentation were significantly affected by seminal fluid exposure. Amongst these were genes encoding several chemokines which target granulocytes, monocyte/macrophages, dendritic cells and lymphocytes, proinflammatory cytokines and regulators of cytokine synthesis, prostaglandin pathway gene including PTGS2; COX-2) and several matrix metalloproteinases (MMPs). Of these genes, no change or a substantially smaller change was seen between first and second biopsies obtained after coitus with condom use, or abstinence. An increase in CSF2, IL6, IL8 and IL1A expression was confirmed by qRT-PCR in larger sets of duplicate biopsies (n=6-7 per group). We conclude that seminal fluid introduced at intercourse elicits expression of pro-inflammatory cytokines and chemokines which underpins the accompanying recruitment of macrophages, dendritic cells and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens, and potentially susceptibility to cervical metaplasia. Women aged 18-40 years, with regular menstrual cycles and a current sexual relationship with a proven fertile regular partner, volunteered for the study. All women had previously undergone tubal ligation and none of the women used steroidal contraceptives or an intrauterine device for a minimum of three months preceding the study. Subjects were screened to exclude bacterial or viral infection. A negative test result was a prerequisite for inclusion in the study. The study was approved by the local ethics committee of Karolinska University Hospital, Stockholm, Sweden. RNA from each of six cervical biopsies was analysed. The six tissues comprised three pairs of cervical biopsies (a ‘first’ and a ‘second’ biopsy); one pair from each of three women assigned to three different treatment protocols. Treatments were (1) no coitus (control); (2) coitus with a condom (condom), or (3) unprotected coitus (coitus). To synchronise the timing of the biopsies, all subjects determined their LH peak in urine samples taken twice daily from approximately day 10 after the onset of menstruation to the time at which an LH increase was detected (LH0-LH+1), using a rapid self-test (Clearplan, Searle Unipath Ltd., Bedford, UK). The first biopsy was recovered during the peri-ovulatory stage of the menstrual cycle (at 0900 h - 1200 h on day LH0 to LH+1), and the second biopsy was recovered 48 h later (at 0900 h - 1200 h on day LH+2 to LH+3). Small needle biopsies (approximately 50-100 mg tissue) were taken from the ectocervix, approximately 1 cm from the transformation zone. Women then abstained from intercourse for 36 hours to enable haemostasis and healing of the biopsy site. Women allocated to groups (1) and (2) had intercourse, without or with condom use respectively, on one occasion approximately 36 hours after the first biopsy and 12 hours prior to the second biopsy. The abstinent woman did not have intercourse between the two biopsies. Before all biopsies, the cervix was washed with 10 ml of saline solution to clear mucous and debris. All cervical washings were collected and examined microscopically for the presence of sperm to confirm compliance.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:This dataset contains small RNA sequencing data and mRNA capture sequencing data from 20 different human biofluids (amniotic fluid, aqueous humor, ascites, bile, bronchial lavage fluid, breast milk, cerebrospinal fluid, colostrum, gastric fluid, pancreatic cyst fluid, plasma, saliva, seminal fluid, serum, sputum, stool, synovial fluid, sweat, tear fluid and urine). In total, 180 samples were sequenced. Files are provided in fastQ format. Samples were sequenced on a NextSeq 500.
Project description:Honey bee queens undergo dramatic behavioral (e.g., reduced sexual receptivity), physiological (e.g., ovary activation, ovulation, and modulation of pheromone production) and molecular changes after they complete mating. To elucidate how queen post-mating changes are influenced by seminal fluid, a non-spermatozoa-containing component of semen, we injected queens with semen or seminal fluid alone. We assessed queen sexual receptivity, ovary development, worker retinue response (which is influenced by queen pheromone production), and transcriptional changes in queen abdominal fat body and brain tissues. Injection with either seminal fluid or semen resulted in decreased sexual receptivity, increased attractiveness of queens to workers, and altered expression of several genes that are also regulated in naturally mated queens. The post-mating and transcriptional changes of queens receiving seminal fluid were not significantly different from queens treated with seminal fluid, suggesting that components in seminal fluid, such as seminal fluid proteins, are largely responsible for stimulating post-mating changes in queens.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:Seminal fluid factors modulate the female immune response at conception to facilitate embryo implantation and reproductive success. Whether sperm affect this response has not been clear. We evaluated global gene expression by microarray in the mouse uterus after mating with intact or vasectomized males. Intact males induced greater changes in gene transcription, prominently affecting pro-inflammatory cytokine and immune regulatory genes, with TLR4 signaling identified as a top-ranked upstream driver. Recruitment of neutrophils and expansion of peripheral regulatory T cells were elevated by seminal fluid of intact males. In vitro, epididymal sperm induced IL6, CXCL2, and CSF3 in uterine epithelial cells of wild-type, but not Tlr4 null females. Collectively these experiments show that sperm assist in promoting female immune tolerance by eliciting uterine cytokine expression through TLR4-dependent signaling. The findings indicate a biological role for sperm beyond oocyte fertilization, in modulating immune mechanisms involved in female control of reproductive investment. We used microarrays to detail the global programme of gene expression in mice following exposure to different components of seminal fluid