Use of Immunohistochemistry to Demonstrate In Vivo Expression of the Burkholderia mallei Virulence Factor BpaB During Experimental Glanders.

Burkholderia mallei causes the highly contagious and debilitating zoonosis glanders, which infects via inhalation or percutaneous inoculation and often culminates in life-threatening pneumonia and sepsis. In humans, glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. No vaccine exists to protect against B. mallei, and there is concern regarding its use as a bioweapon. The authors previously identified the protein BpaB as a potential target for devising therapies due to its role in adherence to host cells and the formation of biofilms in vitro and its contribution to pathogenicity in a mouse model of glanders. In the present study, the authors developed an immunostaining approach to probe tissues of experimentally infected animals and demonstrated that BpaB is produced exclusively in vivo by wild-type B. mallei in target organs from mice and marmosets. They detected the expression of BpaB by B. mallei both extracellularly and within macrophages, neutrophils, and epithelial cells in respiratory tissues (7/10 marmoset; 2/2 mouse). The authors also noted the intracellular expression of BpaB by B. mallei in macrophages in the regional lymph nodes of mice (2/2 tissues) and MALT of marmosets (4/5 tissues). It is interesting that B. mallei bacteria infecting distal organs did not express BpaB (2/2 mice; 3/3 marmosets), suggesting that the protein is not necessary for bacterial fitness in these anatomic locations. These findings underscore the value of BpaB as a target for developing medical countermeasures and provide insight into its role in pathogenesis.

Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

Infection of mice, ferrets, and rhesus macaques with a clinical mumps virus isolate.

In recent years, many mumps outbreaks have occurred in vaccinated populations worldwide. The reasons for these outbreaks are not clear. Animal models are needed to investigate the causes of outbreaks and to understand the pathogenesis of mumps virus (MuV). In this study, we have examined the infection of three animal models with an isolate of mumps virus from a recent outbreak (MuV-IA). We have found that while both ferrets and mice generated humoral and cellular immune responses to MuV-IA infection, no obvious signs of illness were observed in these animals; rhesus macaques were the most susceptible to MuV-IA infection. Infection of rhesus macaques via both intranasal and intratracheal routes with MuV-IA led to the typical clinical signs of mumps 2 weeks to 4 weeks postinfection. However, none of the infected macaques showed any fever or neurologic signs during the experimental period. Mumps viral antigen was detected in parotid glands by immunohistochemistry (IHC). Rhesus macaques represent the best animal model for the study of mumps virus pathogenesis.

Characterization of an autotransporter adhesin protein shared by Burkholderia mallei and Burkholderia pseudomallei.

BACKGROUND: Autotransporters form a large family of outer membrane proteins specifying diverse biological traits of Gram-negative bacteria. In this study, we report the identification and characterization of a novel autotransporter gene product of Burkholderia mallei (locus tag BMA1027 in strain ATCC 23344). RESULTS: Database searches identified the gene in at least seven B. mallei isolates and the encoded proteins were found to be 84% identical. Inactivation of the gene encoding the autotransporter in the genome of strain ATCC 23344 substantially reduces adherence to monolayers of HEp-2 laryngeal cells and A549 type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, expression of the autotransporter on the surface of recombinant E. coli bacteria increases adherence to these cell types by 5-7 fold. The gene specifying the autotransporter was identified in the genome of 29 B. pseudomallei isolates and disruption of the gene in strain DD503 reduced adherence to NHBE cultures by 61%. Unlike B. mallei, the mutation did not impair binding of B. pseudomallei to A549 or HEp-2 cells. Analysis of sera from mice infected via the aerosol route with B. mallei and B. pseudomallei revealed that animals inoculated with as few as 10 organisms produce antibodies against the autotransporter, therefore indicating expression in vivo. CONCLUSIONS: Our data demonstrate that we have identified an autotransporter protein common to the pathogenic species B. mallei and B. pseudomallei which mediates adherence to respiratory epithelial cells and is expressed in vivo during the course of aerosol infection.

Evaluations of the 2019 Annual Statistics Under the Cervical Cytology Quality Assurance Agreement: 2019 Annual Statistics for Cervical Cytology from 15608413 Women.

Background: Cervical cancer screening, which was introduced into the programme of medical care covered by statutory health insurance in Germany in 1971 and has since been constantly updated through quality assurance measures, was fundamentally revised and developed in 2008 through the Cervical Cytology Quality Assurance Agreement pursuant to Section 135(2) of the German Social Code Book V [SGB V]. Since 2015 it has been mandatory for cytology facilities to record annual statistics based on the Munich Nomenclature III. The aim of this article is to present the results of the annual statistics for 2019, which was the last year before the introduction of the cervical cancer screening programme in accordance with the Federal Joint Committee's guideline on organised cancer screening programmes 1 . Materials and Methods: The annual statistics of the laboratories, including histology analyses performed up until 30 June the following year, are reported to the Regional Associations of Statutory Health Insurance Physicians. The laboratories receive benchmark reports from their Regional Associations of Statutory Health Insurance Physicians, and these statistics are transmitted anonymously to the National Association of Statutory Health Insurance Physicians (KBV). Results: In 2019, 17609082 smears from 15608413 women were examined in Germany. Of these smears, 97.49% were normal and 2.51% showed atypical or suspicious changes, consisting mostly of minor squamous epithelial changes in groups II-p (0.81%) and IIID1 (0.735%).Histology specimens are available for "Dysplasia findings with higher probability of regression" in group IIID1 (4.89% of initial IIID1 cytology findings), group IIID2 (18.60%), "unclear or doubtful findings" in group III-p to x (20.7%), and "immediate precursors to cervical carcinoma" in group IV (83.1%) and group V (77.19%).In the cytology findings for group IVa-p, which corresponds to CIN 3 target lesions, the cytology correlated with the histology finding in 80.48% of cases.Lesions found in 2019: 23463 CIN 3 lesions, 668 adenocarcinomas in situ, 3891 malignant tumours, including 2342 cervical carcinomas of which 1743 were squamous cell carcinomas and 599 were cervical adenocarcinomas (25.57%); 1549 endometrial carcinomas and other malignancies. Inference/Conclusion: The data demonstrate the good practicability of cervical cancer screening in 2019. Higher grade lesions were reliably clarified histologically.

Characterizing the mechanical parameters of forward and backward falls as experienced in snowboarding.

Wrist injuries are frequently observed after falls in snowboarding. In this study, laboratory experiments mimicking forward and backward falls were analysed. In six different falling scenarios, participants self-initiated falls from a static initial position. Eighteen volunteers conducted a total of 741 trials. Measurements were taken for basic parameters describing the kinematics as well as the biomechanical loading during impact, such as impact force, impact acceleration, and velocity. The effective mass affecting the wrist in a fall also was determined. The elbow angle at impact showed a more extended arm in backward falls compared to forward falls, whereas the wrist angle at impact remained similar in forward and backward falls. The study results suggest a new performance standard for wrist guards, indicating the following parameters to characterize an impact: an effective mass acting on one wrist of 3-5 kg, an impact angle of 75 degrees of the forearm relative to the ground, and an impact velocity of 3 m/s.

Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents.

Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.

Characterization of conformation-specific monoclonal antibodies against rabies virus nucleoprotein.

Three anti-rabies virus (RABV) nucleoprotein (N) monoclonal antibodies (Mab) were characterized by immunofluorescence assays, western blotting, and immunohistochemistry. One of these Mabs recognized the antigen by all of the assays, while the other two recognized N only in the native form in the immunofluorescence assay. These data, together with epitope mapping studies, suggest that two anti-N Mabs recognize conformational epitopes located within the N-terminal region of the RABV N protein. The availability of Mabs specific for both linear and epitope-specific antibodies should prove valuable for rabies diagnosis as well as for RABV N protein structure-function studies.

Production of H5-specific monoclonal antibodies and the development of a competitive enzyme-linked immunosorbent assay for detection of H5 antibodies in multiple species.

The hemagglutinin gene of an avian influenza virus (AIV) A/duck/NC/674964/07 (H5N2) was cloned and expressed in a baculovirus system (H5-Bac). In parallel, a recombinant hemagglutinin of A/Vietnam/1203/04 (H5N1) was expressed in mammalian cells, purified, and used for generation of H5-specific monoclonal antibodies (MAb). The purified H5-Bac was used to develop a competitive enzyme-linked immunosorbent assay (cELISA) to detect H5 antibodies in a species-independent approach using one of the established H5-specific MAbs as the competitor antibody. The cELISA performed with influenza antibody-free sera or with sera of animals infected with other than H5-encoding AIV showed no significant inhibition of H5-MAb binding, indicating high test specificity. In contrast, sera of poultry (chickens, turkeys, ducks) experimentally infected with H5-encoding AIV were able to significantly inhibit the binding of the MAb in a species-independent approach. Comparison of the results of the cELISA with results obtained by a hemagglutination inhibition assay showed a gradient of the sensitivity (turkeys > ducks > chicken). The described results show that H5-specific antibodies in sera can be detected in a species-independent approach by using a recombinant protein.

Are current back protectors suitable to prevent spinal injury in recreational snowboarders?

OBJECTIVE: Back protectors for snowboarders were analysed with respect to their potential to prevent spinal injury. DESIGN: In 20 Swiss skiing resorts, athletes were interviewed on the slope. In addition, an online survey was conducted. The performance of 12 commercially available back protectors was investigated by means of mechanical testing. A currently used drop test according to standard EN1621 (motorcycle protectors), testing energy damping was supplemented by penetration tests according to standard EN1077, which reflects snowsport safety concerns. RESULTS: 6 out of 12 back protectors fulfilled the higher safety level defined in EN1621. Protectors making use of energy-absorbing layers performed particularly well. In contrast, hard shell protectors exhibited a higher potential to withstand the penetration test. The surveys confirmed that approximately 40-50% of snowboarders use a back protector. A large majority of users expect protection from severe spinal injury such as vertebral fractures or spinal cord injury. CONCLUSIONS: The currently used test standards are fulfilled by many back protectors. Users, however, expect protectors to be efficient in impact scenarios that result in spinal injury, which are more severe than impacts as addressed in the current standards. This study highlights that there is a mismatch between the capabilities of current back protectors to prevent spinal injury in snowboarding and the expectations users have of these protectors.

The Peptidoglycan-associated lipoprotein Pal contributes to the virulence of Burkholderia mallei and provides protection against lethal aerosol challenge.

BURKHOLDERIA MALLEI: is a highly pathogenic bacterium that causes the fatal zoonosis glanders. The organism specifies multiple membrane proteins, which represent prime targets for the development of countermeasures given their location at the host-pathogen interface. We investigated one of these proteins, Pal, and discovered that it is involved in the ability of B. mallei to resist complement-mediated killing and replicate inside host cells in vitro, is expressed in vivo and induces antibodies during the course of infection, and contributes to virulence in a mouse model of aerosol infection. A mutant in the pal gene of the B. mallei wild-type strain ATCC 23344 was found to be especially attenuated, as BALB/c mice challenged with the equivalent of 5,350 LD50 completely cleared infection. Based on these findings, we tested the hypothesis that a vaccine containing the Pal protein elicits protective immunity against aerosol challenge. To achieve this, the pal gene was cloned in the vaccine vector Parainfluenza Virus 5 (PIV5) and mice immunized with the virus were infected with a lethal dose of B. mallei. These experiments revealed that a single dose of PIV5 expressing Pal provided 80% survival over a period of 40 days post-challenge. In contrast, only 10% of mice vaccinated with a PIV5 control virus construct survived infection. Taken together, our data establish that the Peptidoglycan-associated lipoprotein Pal is a critical virulence determinant of B. mallei and effective target for developing a glanders vaccine.

The autotransporter protein BatA is a protective antigen against lethal aerosol infection with Burkholderia mallei and Burkholderia pseudomallei.

BACKGROUND: Burkholderia mallei and Burkholderia pseudomallei are the causative agents of glanders and melioidosis, respectively. There is no vaccine to protect against these highly-pathogenic and intrinsically antibiotic-resistant bacteria, and there is concern regarding their use as biological warfare agents. For these reasons, B. mallei and B. pseudomallei are classified as Tier 1 organisms by the U.S. Federal Select Agent Program and the availability of effective countermeasures represents a critical unmet need. METHODS: Vaccines (subunit and vectored) containing the surface-exposed passenger domain of the conserved Burkholderia autotransporter protein BatA were administered to BALB/c mice and the vaccinated animals were challenged with lethal doses of wild-type B. mallei and B. pseudomallei strains via the aerosol route. Mice were monitored for signs of illness for a period of up to 40 days post-challenge and tissues from surviving animals were analyzed for bacterial burden at study end-points. RESULTS: A single dose of recombinant Parainfluenza Virus 5 (PIV5) expressing BatA provided 74% and 60% survival in mice infected with B. mallei and B. pseudomallei, respectively. Vaccination with PIV5-BatA also resulted in complete bacterial clearance from the lungs and spleen of 78% and 44% of animals surviving lethal challenge with B. pseudomallei, respectively. In contrast, all control animals vaccinated with a PIV5 construct expressing an irrelevant antigen and infected with B. pseudomallei were colonized in those tissues. CONCLUSION: Our study indicates that the autotransporter BatA is a valuable target for developing countermeasures against B. mallei and B. pseudomallei and demonstrates the utility of the PIV5 viral vaccine delivery platform to elicit cross-protective immunity against the organisms.

Listener-based analysis of surface importance for acoustic metrics.

Acoustic quality in room acoustics is measured by well defined quantities, like definition, which can be derived from simulated impulse response filters or measured values. These take into account the intensity and phase shift of multiple reflections due to a wave front emanating from a sound source. Definition (D50) and clarity (C50) for example correspond to the fraction of the energy received in total to the energy received in the first 50 ms at a certain listener position. Unfortunately, the impulse response measured at a single point does not provide any information about the direction of reflections, and about the reflection surfaces which contribute to this measure. For the visualization of room acoustics, however, this information is very useful since it allows to discover regions with high contribution and provides insight into the influence of all reflecting surfaces to the quality measure. We use the phonon tracing method to calculate the contribution of the reflection surfaces to the impulse response for different listener positions. This data is used to compute importance values for the geometry taking a certain acoustic metric into account. To get a visual insight into the directional aspect, we map the importance to the reflecting surfaces of the geometry. This visualization indicates which parts of the surfaces need to be changed to enhance the chosen acoustic quality measure. We apply our method to the acoustic improvement of a lecture hall by means of enhancing the overall speech comprehensibility (clarity) and evaluate the results using glyphs to visualize the clarity (C50) values at listener positions throughout the room.

Peroxisome proliferator-activated receptor (PPAR) expression in cultured bovine endometrial cells and response to omega-3 fatty acid, growth hormone and agonist stimulation in relation to series 2 prostaglandin production.

The peroxisome proliferator-activated receptors (PPARs) are a family of nuclear transcription factors thought to act as receptors for polyunsaturated fatty acids and to reduce production of series 2 prostaglandins (PG). The objectives of the current study were to characterize PPAR expression and the prostaglandin synthetic activity of cultured bovine endometrial cells in response to known PPAR ligands, as well as to key stimulators and inhibitors of series 2 prostaglandin secretion. PPARalpha and PPARdelta, but not PPARgamma, mRNAs are expressed in the BEND cell line regardless of treatment. Under resting conditions, PPARalpha mRNA levels increase in response to growth hormone (P < 0.05). In cells stimulated with PdBu, growth hormone depresses PPARalpha mRNA levels, regardless of whether cells also are treated with IFNtau. In contrast, PPARdelta mRNA levels are increased by exposure to PdBu, eicosapentanoic acid and IFNtau, and these effects are additive. PPAR mRNA levels are not predictive of prostaglandin accumulation. Agonist activation of PPARalpha, PPARdelta or PPARgamma augments PdBu-induced increases in prostaglandin H synthase-2 mRNA and media accumulation of prostaglandins F2alpha and E2. Treatment with the PPARalpha/delta agonist carbaprostacyclin, but not the PPARalpha agonist Wy14643 or PPARgamma agonist ciglitazone, completely reverses the IFNtau suppression of prostaglandin synthesis. In conclusion, PPARalpha and PPARdelta function in the response of bovine endometrium to growth hormone and long chain omega-3 polyunsaturated fatty acids.

Comparative visualization for wave-based and geometric acoustics.

We present a comparative visualization of the acoustic simulation results obtained by two different approaches that were combined into a single simulation algorithm. The first method solves the wave equation on a volume grid based on finite elements. The second method, phonon tracing, is a geometric approach that we have previously developed for interactive simulation, visualization and modeling of room acoustics. Geometric approaches of this kind are more efficient than FEM in the high and medium frequency range. For low frequencies they fail to represent diffraction, which on the other hand can be simulated properly by means of FEM. When combining both methods we need to calibrate them properly and estimate in which frequency range they provide comparable results. For this purpose we use an acoustic metric called gain and display the resulting error. Furthermore we visualize interference patterns, since these depend not only on diffraction, but also exhibit phase-dependent amplification and neutralization effects.

The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection.

Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥ 300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

Use of the common marmoset to study Burkholderia mallei infection.

Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets (Callithrix jacchus) were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 10(4) to 2.5 X 10(5) bacteria developed acute lethal infection within 3-4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 10(3) bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 10(3) organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B. mallei.

Direct interaction of the Krüppel-like family (KLF) member, BTEB1, and PR mediates progesterone-responsive gene expression in endometrial epithelial cells.

The present study was undertaken to evaluate the underlying mechanism(s) by which PR and a Krüppel-like family member, basic transcription element binding protein (BTEB1), mediate endometrial epithelial expression of pregnancy-associated genes. Human endometrial carcinoma cell lines (Hec-1-A) expressing high and low levels of BTEB1 were transiently transfected with a human PR isoform (PR-B) expression construct and a luciferase reporter gene driven by the uteroferrin gene promoter that is responsive to both BTEB1 and the PR ligand progesterone. Unliganded PR inhibited luciferase activity in low and high BTEB backgrounds, and this effect was reversed by the synthetic progestin R5020 in both lines. Transactivation by PR of uteroferrin promoter activity (approximately 4-fold) was maximal at lower R5020 concentrations (10 nM) in endometrial cells with higher BTEB1 expression, suggesting that nuclear BTEB1content influenced target gene promoter sensitivity to progesterone. BTEB1 and PR-B were found to physically interact in a progesterone-independent manner, using a coimmunoprecipitation assay that employed antibodies specific to either protein. Moreover, the formation of the BTEB1/PR complex, independent of progesterone, occurred within the context of uterine endometrial proteins and was diminished in late-pregnancy endometrium. Mammalian two-hybrid assays using the entire open reading frame of BTEB1 and/or PR-B fused to either the GAL4 DNA-binding domain or VP16 activation domain and a reporter gene (pG5CAT) under the control of GAL4-binding sites were used to evaluate the formation of functional PR-B/BTEB1 dimer in Cos-1 cells. GAL4/PR-B and VP16/PR-B induced ( approximately 3- to 4-fold) chloramphenicol acetyltransferase (CAT) activity in a progesterone-dependent manner, suggesting PR-dimer formation. By contrast, VP16/PR-B and GAL4/BTEB1 had no effect on basal CAT activity. The combination of VP16- and GAL4-PR-B fusion proteins with the BTEB1 expression construct, pCDNA3-BTEB1 enhanced ligand-bound PR-mediated CAT activity by approximately 3-fold. In transient cotransfection assays using the CAT reporter gene driven by the mouse mammary tumor virus-long terminal repeat promoter, which is responsive to ligand-bound PR but not BTEB1, BTEB1 increased PR-B-mediated CAT activity in a progesterone-dependent manner, consistent with a BTEB1/PR-dimer complex occurring independent of BTEB1 binding to DNA. Unliganded PR-B disrupted the DNA-binding activity of BTEB1 in gel retardation assays, and this effect was enhanced by the presence of PR ligand. Together, these findings support the conclusion that BTEB1 and PR-B are coregulatory proteins that mediate progesterone responsiveness of target genes by direct interactions, leading to the formation of a functional BTEB1/PR-dimer complex.

Molecular markers of endometrial epithelial cell mitogenesis mediated by the Sp/Krüppel-like factor BTEB1.

Basic transcription element binding (BTEB1) protein is one of at least 20 Sp/KLF family members that function as transcriptional activators or repressors by binding to GC/GT-rich sequences within target genes to influence cellular homeostasis in mammals. Previously, we demonstrated that increased expression of BTEB1 in a human endometrial epithelial cell line Hec-1-A resulted in serum dependent-enhanced proliferation, which was accompanied by heightened expression of cell cycle- and growth-associated genes. In the present study, we examined the mechanism underlying the altered proliferative potential associated with BTEB1 by the identification of additional BTEB1 downstream gene targets and by the demonstration of BTEB1 transactivation of promoters for a number of growth-associated genes. Using mRNA differential display in the analysis of RNA populations from Hec-1-A sublines with high (4S, 9S) and low (2As, 3As) BTEB1 cellular content, we identified 10 distinct differentially expressed transcripts, nine of which had higher levels in S than in As sublines. The expression levels of two of these cDNAs, Axl receptor tyrosine kinase and mitosin, whose encoded products are implicated in cellular proliferation, were modestly induced by serum, albeit in a BTEB1-independent manner. Moreover, insulin-like growth factor-I, a mitogen present in serum, had no significant effect on their expression in either subline. In transient reporter assays, the basal activities of the Axl gene promoter and those for two other growth-regulatory genes, namely p21(WAF1) and IGFBP-2, were increased by serum and were significantly higher in 4S than in 2As lines. However, while BTEB1 and its ubiquitous family member Sp1 increased basal p21(WAF1) and IGFBP-2 transcription when added as expression constructs in the parental Hec-1-A cell line, only Sp1 activated Axl transcription, despite the presence in all three gene promoters of GC-enriched regions that presumably can bind BTEB1 and Sp1 with similar affinities. To elucidate intracellular signaling pathways that might involve BTEB1, inhibitors of specific kinase-dependent transducers were used in transient transfection assays involving the IGFBP-2 gene promoter in 4S and 2As sublines. While inhibitors of the MAPK, PI-3K, and PKA pathways elicited similar effects on the IGFBP-2 gene promoter activity, irrespective of cellular BTEB1 content, that for JNK had a more pronounced effect on Hec-1-A sublines exhibiting higher BTEB1 expression levels. Taken together, the results suggest that BTEB1 mediates the expression of growth-associated genes through direct and indirect transactivation mechanisms, one of which may involve the participation of a JNK family member.