scholarly journals Artemisia Naphta: A novel oil extract for sensitive and acne prone skin

2021 ◽  
Vol 5 (1) ◽  
pp. 022-029
Author(s):  
Tao Kan ◽  
Guo Lili ◽  
Fernandez Jose ◽  
Webb Corey ◽  
Liu Junfeng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Artemisia Annua ◽  
Bioactive Molecules ◽  
Gas Chromatography Mass Spectrometry ◽  
Rt Pcr ◽  
Potential Benefits ◽  
Oil Extract ◽  
Skin Conditions ◽  
Pro Inflammatory Cytokines

Background: The plant Artemisia annua has been used in traditional Chinese medicine for many years. Rich in bioactive molecules, the A. annua plant is used to extract the anti-malaria compound artemisinin (< 1%), which results in most of the plant being unutilized. One byproduct of artemisinin extraction is artemisia naphtha (AN), which has yet to be studied extensively. Aims: Study the activity of a novel AN oil extract against microbes, pro-inflammatory cytokines, and dermatological endpoints that are key for eczema and acne pathogenesis to determine if an effective A. annua extract for these skin conditions can be developed. Methods: Gas chromatography-mass spectrometry was performed to determine the composition of AN oil. P. acnes, S. aureus, M. furfur, and C. albicans were cultured to determine minimal inhibitory concentration. in vitro studies utilizing keratinocytes and macrophages were treated with AN oil and gene expression measured by quantitative RT-PCR. A 13-subject clinical trial was performed with 1% AN oil Gel to assess its potential benefits for sensitive and acne prone skin. Results: AN oil upregulates filaggrin gene expression and possesses antimicrobial and anti-inflammatory activity inhibiting LPS, S. aureus and "Th2 induced" pro-inflammatory mediator release (IL-6, IL-8 and TSLP). Clinical assessment of 1% AN Gel shows it reduces acne blemishes and the appearance of redness. Conclusion: Previously an underutilized and unpurified byproduct, AN is now the source to develop the first topical AN oil for cosmetic use with an activity profile that suggests it is effective for those with sensitive and/or acne prone skin.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

In vitro evaluation of potential benefits of a silica-rich thermal water (Monfortinho Thermal Water) in hyperkeratotic skin conditions

2020 ◽  
Vol 64 (11) ◽  
pp. 1957-1968
Author(s):  
Ana Sofia Oliveira ◽  
Cátia Vicente Vaz ◽  
Ana Silva ◽  
Sara Correia ◽  
Raquel Ferreira ◽  
...  
Keyword(s):  
Thermal Water ◽  
In Vitro Evaluation ◽  
Potential Benefits ◽  
Skin Conditions

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

The Outgrowth of CLL Nurselike Cells in Vitro, and Its Relation to Clinical and Hematological Parameters.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2360-2360
Author(s):  
Agata A Filip ◽  
Dorota Koczkodaj ◽  
Tomasz Kubiatowski ◽  
Ewa Wasik-Szczepanek ◽  
Anna Dmoszynska
Keyword(s):  
Gene Expression ◽  
Serum Level ◽  
Ex Vivo ◽  
Hematological Parameters ◽  
Tp53 Gene ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Gene Status

Abstract Abstract 2360 Poster Board II-337 Introduction: Despite their longevity in vivo, CLL lymphocytes die rapidly when put to in vitro cultures, what proves that the resistance to apoptosis is not an intrinsic feature of leukemic cells, but depends on environmental signals. Recently it was shown that mononuclear cells from peripheral blood of CLL patients differentiate in vitro into large, adherent cells that grow in close contact with CLL lymphocytes. They were termed “nurselike cells” (NLCs), because they support leukemic lymphocyte survival in culture. The presence of the cells morphologically and phenotypically similar to NLCs was demonstrated in peripheral lymphatic organs of CLL patients. It may suggest their role in CLL lymphocytes protection in vivo and, as a consequence, point the new target in CLL treatment. Patients and Methods: The study included the group of 65 previously untreated CLL patients, 24 women and 41 men, aged from 36 to 86 yrs. 12 patients (18%) were diagnosed with stage 0 according to Rai, 15 patients (23%) with stage I, 30 patients (46%) with stage II, 5 patients (8%) with stage III and 3 patients (5%) with stage IV. Peripheral blood lymphocytes ex vivo were examined for CD14, CD38, BCL2 and ZAP70 expression by flow cytometry and for BCL2, SURVIVIN and ZAP70 gene expression by RT-PCR. TP53 gene status was assessed by FISH. Lymphocytes of 20 patients were assayed for apoptosis-related gene expression by means of cDNA macroarrays (Clontech). To generate NLCs, PB leukemic cells were cultured in vitro for 14 days on standard medium (RPMI 1640 with L-glutamine, 15% FCS, antibiotics/antimycotics; cell density 3 × 106/ml) and the outgrowth and number of NLCs was assessed in relation to clinical and hematological parameters. NLCs were identified morphologically and by CD31/VIMENTIN protein expression. Results: In 58 cases (89%) the outgrowth of NLCs was observed, while their number differed in cultures of the cells of different patients: in 49 cultures (84.5%) there were over 20 NLCs/mm2 (up to 52 NLCs/mm2), and in 9 cases (15.5%) less than 20 NLCs/mm2. Positive correlation was shown between NLC number and B2M serum level (p=0.044) and absolute monocyte count (p=0.019). Significantly higher NLC number was observed in case of patients with higher CD14+ cell number (p<0.0001) and higher SURVIVIN gene expression assessed by RT-PCR (p<0.0001) and macroarrays (p=0.013). We found no statistically significant relation of NLCs number and: the Rai stage of the disease, WBC, lymphocyte count, LDH serum level, BCL2, CD38 and ZAP70 expression and TP53 gene status. During the follow-up period of 6 years we observed the tendency for longer overall survival in patients that produce less than 20 NLCs/mm2 (fig. 1), but it was not statistically significant. Conclusions: The number of NLC cells obtained in vitro from PBL of CLL patients correlates with B2M serum level and SURVIVIN gene expression in CLL cells ex vivo. High B2M level is a marker of poor prognosis. SURVIVIN represents a family of IAP (Inhibitor of APoptosis) proteins. While rare in PBL of CLL patients, its expression is typical for proliferating leukemic cells pool in pseudofollicle microenvironment. SURVIVIN inhibits apoptosis by blocking caspase-3 and -7. Considering the protective role of NLC cells towards CLL lymphocytes in vitro, these results altogether with observed tendency to shorter survival of patients generating high NLCs number may prove the presence of supportive mechanisms exerted by NLCs in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The PI3KCA and AKT Inhibitory Activities of Litsea Cubeba Lour. Fruits and Heartwoods Towards Hela Cells

2019 ◽  
Vol 7 (9) ◽  
pp. 1422-1424
Author(s):  
Aminah Dalimunthe ◽  
Poppy Anjelisa Zaitun Hasibuan ◽  
Denny Satria
Keyword(s):  
Gene Expression ◽  
Cervical Cancer ◽  
Cell Culture ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Litsea Cubeba ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Inhibitory Activities

AIM: To investigated the activities of chloroform fractions at pH 7 of Litsea cubeba Lour. Fruits and heartwoods (CF-7F and CF-7H) in decrease expression of PI3KCA, Akt-1 and Akt-2 genes towards cervical cancer cell culture (HeLa) experiments in vitro. MATERIAL AND METHODS: CF-7F and CF-7H (12.5 and 25 µg/mL) were tested for its potential inhibition on gene expression of PI3KCA, Akt-1 and Akt-2 genes by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. RESULT: CF-7F and CF-7H were showed the activity to reduce the expression of PI3KCA, Akt-1 and Akt-2 genes. CONCLUSION: Our results suggest that CF-7F and CF-7H significantly inhibit the expression of PI3KCA, Akt-1 and Akt-2 genes.

Abstract P007: Cardiomyocyte Damage-Released Extracellular S100A1 Protein Promotes Regeneration of Infarcted Myocardium by Modulating Cardiac Fibroblast Function

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
David Rohde ◽  
Gang Qiu ◽  
Nicole Herzog ◽  
Hugo A Katus ◽  
Angelika Bierhaus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Gene Expression Pattern ◽  
Left Ventricular ◽  
Apical Region ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Cardiomyocytes

Background: Similar to heart muscle-specific creatinkinase (CK-MB), S100A1 protein is released from damaged human cardiomyocytes in response to myocardial infarction (MI). Since S100A1-knock out (SKO) mice display rapid post-MI onset of adverse myocardial remodeling and accelerated transition to heart failure, we assessed the hypothesis that ischemia-related release of S100A1 protein modulates myocardial regeneration. Methods and Results: After LAD ligation in C57/B6 mice, S100A1 serum levels peaked at 10 µg/ml 8 hours post-MI, precisely mirroring the time course previously observed in MI patients. RT-PCR analyses in post-MI whole heart samples revealed significantly lower I-CAM (−50%) and IL-10 (−75%) mRNA abundance as well as heightened Collagen-1 (+40%) and VEGF (+80%) expression in SKO vs. WT mice (p<0.05, n=6 in each group). Interestingly, injection of an S100A1-neutralizing antibody prior to MI in WT mice mimicked the abnormalities observed in post-ischemic SKO animals. To further elucidate extracellular S100A1 biological activity, cardiomyocytes, cardiac fibroblasts (CF), endothelial and smooth muscle cells were exposed to S100A1 in vitro . A rapid internalization of S100A1 was exclusively found in CF, resulting in a phosphorylation of ERK1/2, JNK, and p38 with subsequent activation of NF-kappaB as assessed by Western Blot (WB) and EMSA. RT-PCR and WB analyses revealed significant alterations in CF gene expression in response to S100A1, including an increase in I-CAM (3,5-fold) and IL-10 (20-fold) mRNA levels and diminished Col-1 (−80%) expression. Similar effects were observed after direct injection of S100A1 protein into the left ventricular apical region of WT mice in vivo (S100A1- vs. PBS-injection, n=6). In SKO mice, intraperitoneal application of S100A1 prior to MI largely normalized the adverse gene expression pattern towards WT animals. Conclusions: Our study provides first evidence for cardiomyocyte damage-released S100A1 to act as an endogenous mediator of post-MI inflammation and tissue repair. Considering today's unability to manipulate these molecular mechanisms, extracellular S100A1 might represent a promising target for future therapies of MI.

Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4255-4255
Author(s):  
Ewa Carrier ◽  
Shermila Kausal ◽  
Anand S. Srivastava
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Growth Factors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Globin Genes ◽  
Rt Pcr ◽  
Expression Studies ◽  
Gene Expression Studies

Abstract We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.

Defibrotide Counteracts the Modifications of Anti-Thrombotic Phenotype of Endothelial Cells Induced by Thalidomide.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2502-2502 ◽  
Author(s):  
Cinara Echart ◽  
Barbara Graziadio ◽  
Cinzia Repice ◽  
Mario Boccadoro ◽  
Antonio Palumbo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fibrin Clot ◽  
Rt Pcr ◽  
Plasminogen Activator Inhibitor 1 ◽  
Adhesive Properties ◽  
Pai 1

Abstract Introduction: Patients with Multiple myeloma are at relatively high risk of developing thromboembolic events, usually deep vein thromboses (DVT). There are numerous contributing factors, including therapy, such as thalidomide, where DVT has been identified as a major toxicity, especially when thalidomide is used in combination with other treatments such as dexamethasone. The mechanisms by which thalidomide predisposes to thrombosis are not well understood. Defibrotide (DF) is an orally biovailable polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic and anti-adhesive properties. Previously, DF has been shown to dose-dependently counteracted the increase in Plasminogen Activator Inhibitor-1 (PAI-1) expression and decrease on tissue plasminogen activator (t-PA) activity after lipopolysaccharide (LPS) stimulation of endothelial cells in vitro. Methods and Results: We have conducted in vitro studies using human microvascular endothelial cells (HMEC) in order to investigate the effect of different doses of thalidomide on various fibrinolytic factors. In addition, we evaluated whether DF modulates changes of fibrinolysis induced by thalidomide. HMEC were treated with 50 and 100μg/ml of thalidomide for 24 hours in presence and absence of DF (at a dose of 150μg/ml). t-PA and PAI-1 gene expression were evaluated through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Release of t-PA and PAI-1 were evaluated by imunoenzymatic assay (ELISA). Furthermore, we evaluated the fibrinolytic activity of cell surpernatant using a fibrin clot plate assay. In this method the fibrin clot was formed by mixing fibrinogen, plasminogen and thrombin. The plasmin generated by the cell surpernatant was able to digest fibrin and also hydrolyzed the chromogenic substrate S-2251. The RT-PCR results showed that thalidomide reduces t-PA (2.2 fold) and increases PAI-1 gene expression (4.0 fold) in HMEC cells, whereas DF was able to counteract this effect by up-regulating the t-PA and down-regulating PAI-1 gene expression induced by thalidomide (8.8 and 2.0 fold, respectivielly). Similar results was observed analyzing t-PA release by HMEC cells treated with different concentrations of thalidomide with and without DF. Thalidomide significantly reduces the t-PA released in both concentrations (p&lt;0.001) and DF significantly increase the release of t-PA reduced by thalidomide (p&lt;0.01). The changes of fibrinolytic activity in HMEC by thalidomide and the capacity of DF to restore the fibrinolysis was confirmed by analyzing the lyses of fibrin clots with endothelial cell surpernatant (p&lt;0.01). Conclusions: These results show that DF is able to counteract the alterations of fibrinolytic factors in HMEC treated with thalidomide. Whilst further studies in preclinical MM models are underway, these data suggest a potential role for DF in the prevention of DVT induced by thalidomide and support ongoing clinical trials of DF in combination with thalidomide-based treatment.

scholarly journals The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

2005 ◽  
Vol 73 (7) ◽  
pp. 4281-4287 ◽  
Author(s):  
Sarika Agarwal ◽  
Carol A. King ◽  
Ellen K. Klein ◽  
David E. Soper ◽  
Peter A. Rice ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Iron Concentration ◽  
Pcr Analysis ◽  
Positive Trend ◽  
Gonococcal Infection ◽  
Rt Pcr ◽  
Male Subjects

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

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