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There is a growing recognition that sexual problems can adversely impact health, well-being, and quality of life. Sex therapy provides individuals with an effective means of understanding, improving, and resolving their sexual difficulties. Yet, few individuals access a sex therapist when experiencing sexual difficulties and research on perceptions toward this service remains limited. The current study aimed to explore attitudes and beliefs held by a sample with a current or previous history of sexual problems toward sex therapy and therapists. A total of 27 individuals aged 19-53 participated in unmoderated structured interviews that were analyzed using reflexive thematic analysis. The results were organized into five categories, (1) overall perceptions of sex therapy, (2) when and for whom is sex therapy, (3) expectations, (4) beliefs about sex therapists, and (5) sources of beliefs. While the results indicated that participants held positive attitudes toward the service and about others seeking sex therapy, none had accessed sex therapy for their previous or current sexual problems. Numerous barriers relating to stigma, cost, and accessibility appeared to hinder the utilization of the service. The inaccurate and unrealistic beliefs about sex therapy and sex therapists highlight the need for increased education regarding the profession, which could reduce barriers and increase accessibility., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: The Surviving Sepsis Campaign recommends systematic screening for sepsis. Although many sepsis screening tools include parent or healthcare professional concern, there remains a lack of evidence to support this practice. We aimed to test the diagnostic accuracy of parent and healthcare professional concern in relation to illness severity, to diagnose sepsis in children., Design: This prospective multicenter study measured the level of concern for illness severity as perceived by the parent, treating nurse and doctor using a cross-sectional survey. The primary outcome was sepsis, defined as a pSOFA score >0. The unadjusted area under receiver-operating characteristic curves (AUC) and adjusted Odds Ratios (aOR) were calculated., Setting: Two specialised pediatric Emergency Departments in Queensland., Patients: Children aged 30 days to 18 years old that were evaluated for sepsis., Intervention: None., Main Results: 492 children were included in the study, of which 118 (23.9%) had sepsis. Parent concern was not associated with sepsis (AUC 0.53, 95% CI: 0.46-0.61, aOR: 1.18; 0.89-1.58) but was for PICU admission (OR: 1.88, 95% CI: 1.17-3.19) and bacterial infection (aOR: 1.47, 95% CI: 1.14-1.92). Healthcare professional concern was associated with sepsis in both unadjusted and adjusted models (nurses: AUC 0.57, 95% CI-0.50, 0.63, aOR: 1.29, 95% CI: 1.02-1.63; doctors: AUC 0.63, 95% CI: 0.55, 0.70, aOR: 1.61, 95% CI: 1.14-2.19)., Conclusions: While our study does not support the broad use of parent or healthcare professional concern in isolation as a pediatric sepsis screening tool, measures of concern may be valuable as an adjunct in combination with other clinical data to support sepsis recognition., Clinical Trial Registration: ACTRN12620001340921., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Sever, Schlapbach, Gilholm, Jessup, Phillips, George, Gibbons and Harley.)

This paper present seasonal variation in the equivalent concentration (BaP eq ) of PAHs in order to assess the potential cancer risk for two different groups of residents via ingestion, dermal contact and inhalation pathways. The possible ecological risk caused by PAH atmospheric deposition based on risk quotient was also estimated. A bulk (total, wet and dry) deposition and PM 10 particle fraction (particles with an equivalent aerodynamic diameter < 10 µm) were collected from June 2020 to May 2021 at an urban residential location in the northern part of Zagreb, Croatia. The monthly average of total equivalent BaP eq mass concentrations of PM 10 varied from 0.057 ng m -3 in July to 3.656 ng m -3 in December; the annul ∑BaP eq average was 1.348 ng m -3 . In bulk deposition, ∑BaP eq mass concentrations varied from 1.94 to 57.60 ng L -1 . In both investigated media, BaP had the highest contribution in carcinogenic activity. For PM 10 media, dermal absorption implied the greatest potential cancer risk, followed by ingestion and inhalation. For bulk media, a moderate ecological risk for BaA, BbF and BaP was observed according to the risk quotient approach.

Digital health interventions can address governments' aims of providing better care, better outcomes, and lower costs. No previous research has examined attitudes toward digital sex therapy to understand what might facilitate, or hinder, the uptake of these interventions. This sequential mixed-methods study with qualitative structured interviews ( n  = 27) followed by a quantitative survey ( n  = 334) aimed to understand participants' expectations and attitudes toward digital sex therapy interventions. Participants reported a mixture of positive and negative attitudes to digital sex therapy. More positive attitudes, higher education level, and previous engagement in therapy predicted greater openness to using these interventions.

First data on polycyclic aromatic hydrocarbons (PAHs) and nitro-aromatic compounds (NACs) in aerosols as well as of PAHs, polychlorinated biphenyls (PCBs) and NACs in bulk and wet atmospheric deposition samples were simultaneously obtained during 6-month-long field campaign at the costal central Adriatic area. Special attention was given to open-fire biomass burning episodes as extreme events common for the overall Mediterranean coastal area in order to gain a better understanding of the atmospheric variabilities and potential sources of trace organic pollutants in coastal environments. Diesel and gasoline combustion related to land and maritime traffic as well as occasional open-fire episodes (forest fires) were found to be the dominant pollution sources of PAHs in PM 10 particles. NACs were determined almost exclusively in samples affected by biomass burning episodes. Open-fire episodes had a strong contribution to the overall NACs atmospheric deposition fluxes. Several chlorinated congeners of PCBs were predominantly contributed in deposition samples., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: Paediatric sepsis is a major contributor to morbidity and mortality worldwide. Assessing concern from parents and healthcare professionals to determine disease severity in a child evaluated for sepsis remains a field requiring further investigation. This study aims to determine the diagnostic accuracy of parental and healthcare professional concern in the diagnosis of children evaluated for sepsis., Methods and Analysis: This prospective multicentre observational study will be conducted over a 24-month period in the paediatric emergency department (ED) at two tertiary Australian hospitals. A cross-sectional survey design will be used to assess the level of concern in parents, nurses and doctors for children presenting to ED and undergoing assessment for sepsis. The primary outcome is a diagnosis of sepsis, defined as suspected infection plus organ dysfunction at time of survey completion. Secondary outcomes include suspected or proven infection and development of organ dysfunction, defined as a Paediatric Sequential Organ Failure Assessment Score >0, within 48 hours of presentation, paediatric intensive care unit admission, confirmed or probable bacterial infection independent of organ dysfunction, and hospital length of stay., Ethics and Dissemination: Ethics approval was obtained from Children's Health Queensland's Human Research Ethics Committee (HREC/17/QRCH/85). Findings will be shared with relevant stakeholders and disseminated via conferences and peer-reviewed journals TRIAL REGISTRATION NUMBER: WHO Universal Trial Number, U1111-1256-4537; ANZCTR number, ACTRN1262000134092., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Airborne particles are composed of inorganic species and organic compounds. PM 1 particles, with an aerodynamic diameter smaller than 1 μm, are considered to be important in the context of adverse health effects. Many compounds bound to particulate matter, such as polycyclic aromatic hydrocarbons (PAH), are suspected to be genotoxic, mutagenic, and carcinogenic. In this study, PAHs in the PM 1 particle fraction were measured for one year (1/1/2018-31/12/2018). The measuring station was located in the northern residential part of Zagreb, the Croatian capital, close to a street with modest traffic. Significant differences were found between PAH concentrations during cold (January-March, October-December) and warm (April-September) periods of the year. In general, the mass concentrations of PAHs characteristic for car exhausts (benzo(ghi)perylene (BghiP), indeno(1,2,3-cd)pyrene (IP), and benzo(b)fluoranthene (BbF)) were higher during the whole year than concentrations of fluoranthene (Flu) and pyrene (Pyr), which originated mostly from domestic heating and biomass burning. Combustion of diesel and gasoline from vehicles was found to be one of the main PAH sources. The incremental lifetime cancer risk (ILCR) was estimated for three age groups of populations and the results were much lower than the acceptable risk level (1 × 10 -6 ). However, more than ten times higher PAH concentrations in the cold part of the year, as well as associated health risk, emphasize the need for monitoring of PAHs in PM 1 . These data represent a valuable tool in future plans and actions to control PAH sources and to improve the quality of life of urban populations., Competing Interests: The authors declare that they have no conflict of interest.

Polycyclic aromatic hydrocarbons (PAHs) are compounds known for their adverse effects on human health. Many of them are proven carcinogens, especially those with 5 and 6 aromatic rings, which under normal tropospheric conditions are found in the particle-phase. Benzo(a)pyrene (BaP) is often measured as their general representative. Sarajevo, the capital of Bosnia and Herzegovina, is among the European cities with the poorest air quality. However, in Sarajevo PAHs are neither routinely measured within the air quality monitoring network nor have been a subject of extended, continuous field studies during the most polluted cold periods of the year. The capital of Croatia, Zagreb, is located approximately 300 km air distance north-west from Sarajevo. PAH mass concentrations in Zagreb have been measured continuously since 1994 within air quality monitoring networks. During winter 2017/2018, the SAFICA project (Sarajevo Canton Winter Field Campaign 2018) was carried out in order to characterize the chemical composition of organic and inorganic aerosol in the Sarajevo Canton. This paper presents the results of PAH measurements in the cities of Sarajevo and Zagreb at one urban location per city. Daily (24 h), continuous samples of PM 10 (particulate matter with aerodynamic diameters ≤10 μm) were collected during heating season, from December 27, 2017 to February 27, 2018. Mass concentrations of eleven particle-phase PAHs in Sarajevo and Zagreb from filter samples collected during the same period were compared. The average BaP ambient mass concentrations in Sarajevo and Zagreb were 6.93 ng m -3 and 3.11 ng m -3 , respectively. The contribution of BaP to the total PAH mass concentration was similar at both locations (11%). However, much higher contributions of particle-phase fluoranthene and pyrene were found in Sarajevo. Contributions of individual PAH, diagnostic ratios and factor analysis indicate that combustion of gasoline and diesel from vehicle traffic are a potential source of PAHs at both locations, as well as combustion of other liquid fossil fuels (petroleum and fuel oil). Wood burning was occasionally indicated as a PAH emission source in Zagreb, while in Sarajevo the contribution of PAHs from wood and coal combustion was more evident. Calculated value for total carcinogenic potency (TCP) of PAHs, which was estimated using toxic equivalence factors from the literature, in PM 10 samples collected in Sarajevo was more than twice higher than in Zagreb (10.6 ng m -3 and 4.7 ng m -3 , respectively). BaP had the highest contribution to the TCP at both locations (69 and 67%)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives and Background: Familial Mediterranean Fever (FMF) is characterized by recurrent fever episodes as a result of inflammation of serous membranes. Changes in the number of different mtDNA copy number variations, detected in FMF patients, who developed amyloidosis, might be an important parameter in the understanding of the pathophysiology of the disease., Methods: Changes in the mtDNA copy number between 50 patients with FMF, who had M694V homozygote mutation and amyloidosis, and 50 healthy controls, who had not any MEFV mutation or FMF clinical finding, were examined. The 22 MEFV mutations were analyzed by Pyromark Q24 system. Quantitative analysis was performed on RT-PCR. The level of mtDNA was calculated using the delta Ct (ΔCt) of average Ct of mtDNA and nDNA (ΔCt = Ct mtDNA-Ct nDNA) in the same well as an exponent of 2 (2ΔCt)., Results: A significant decrease in the amount of mtDNA was detected in FMF patients with M694V homozygous mutation carriers, who developed amyloidosis compared to the control group (p < 0.001)., Conclusion: In this study, mitochondrial dysfunction, which has been identified through changes in the mitochondrial genome in many diseases, was identified by showing that the copy number variations of mtDNA in leukocytes also decreased for FMF disease (Tab. 3, Fig. 1, Ref. 21).

Several species of the genus Fusarium can cause apple fruit to rot while stored. Since Fusarium taxonomy is very complex and has constantly been revised and updated over the last years, the aim of this study was to identify Fusarium species from rotten apples, based on combined morphological characteristics and molecular data. We identified 32 Fusarium isolates from rotten apple fruit of cultivars Golden Delicious, Jonagold, Idared, and Pink Lady, stored in Ultra Low Oxygen (ULO) conditions. Fusarium rot was detected in 9.4 % to 33.2 % of naturally infected apples, depending on the cultivar. The symptoms were similar in all four cultivars: a soft circular brown necrosis of different extent, with or without visible sporulation. Fusarium species were identified by the morphology of cultures grown on potato-dextrose agar (PDA) and carnation leaf agar (CLA). Twenty one isolates were identified as Fusarium avenaceum and confirmed as such with polymerase chain reaction (PCR) using specific primer pair FA-ITSF and FA-ITSR. F. pseudograminearum,F. semitectum, F. crookwellense, and F. compactum were identified by morphological characteristics. F.avenaceum can produce several mycotoxins and its dominance in Fusarium rot points to the risk of mycotoxin contamination of apple fruit juices and other products for human consumption. Pathogenicity tests showed typical symptoms of Fusarium rot in most of the inoculated wounded apple fruits. In this respect Fusarium avenaceum, as the dominant cause of Fusarium rot in stored apple fruits is a typical wound parasite.

The present study tested the hypothesis that the scavenger receptor SR-A modulates granuloma formation in response to pulmonary infection with Mycobacterium tuberculosis (MTB). To test this hypothesis, we monitored survival and histopathology in WT and SR-A-deficient mice following aerosol infection with MTB Rv. SR-A-deficient (SR-A-/-) mice infected with MTB survived significantly longer than WT mice; the mean survival of SR-A-/- mice exceeded 430 days compared to 230 days for WT mice. Early granuloma formation was not impaired in SR-A-/- mice. The extended survival of SR-A-/- mice was associated with 13- and 3-fold higher number of CD4+ lymphocytes and antigen presenting cells in SR-A-/- lungs compared to WT mice 280 after infection. The histopathology of chronically infected SR-A-/- lungs, however, was marked by abundant cholesterol clefts in parenchymal lesions containing infection in multinucleated giant cells. The present study indicates SR-A as a candidate gene of the innate immune system influencing the chronic phase of M. tuberculosis infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Alveolar type II epithelial or other pulmonary cells secrete GM-CSF that regulates surfactant catabolism and mucosal host defense through its capacity to modulate the maturation and activation of alveolar macrophages. GM-CSF enhances expression of scavenger receptors MARCO and SR-A. The alveolar macrophage SP-R210 receptor binds the surfactant collectin SP-A mediating clearance of respiratory pathogens. The current study determined the effects of epithelial-derived GM-CSF in host resistance to influenza A pneumonia. The results demonstrate that GM-CSF enhanced resistance to infection with 1.9×10(4) ffc of the mouse-adapted influenza A/Puerto Rico/8/34 (PR8) H1N1 strain, as indicated by significant differences in mortality and mean survival of GM-CSF-deficient (GM(-/-)) mice compared to GM(-/-) mice in which GM-CSF is expressed at increased levels. Protective effects of GM-CSF were observed both in mice with constitutive and inducible GM-CSF expression under the control of the pulmonary-specific SFTPC or SCGB1A1 promoters, respectively. Mice that continuously secrete high levels of GM-CSF developed desquamative interstitial pneumonia that impaired long-term recovery from influenza. Conditional expression of optimal GM-CSF levels at the time of infection, however, resulted in alveolar macrophage proliferation and focal lymphocytic inflammation of distal airways. GM-CSF enhanced alveolar macrophage activity as indicated by increased expression of SP-R210 and CD11c. Infection of mice lacking the GM-CSF-regulated SR-A and MARCO receptors revealed that MARCO decreases resistance to influenza in association with increased levels of SP-R210 in MARCO(-/-) alveolar macrophages. In conclusion, GM-CSF enhances early host resistance to influenza. Targeting of MARCO may reinforce GM-CSF-mediated host defense against pathogenic influenza., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.

The assessment of tumor vascularization by color flow Doppler sonography (CFDS) has been suggested for the distinction between benign and malignant thyroid nodules. Our objective was to investigate if the CFDS results reflect the percentage of histologically determined microvessels in adenomas (As), adenomatous nodules (ANs), and papillary carcinomas (PCs). Tissue sections from 10 adenomas, 8 ANs and 13 PC and surrounding tissue of 10 PCs and 2 benign nodules were immunostained for CD34. A computerized image analysis was used to determine the microvessel density in four hot spots and ten systematically selected fields. Preoperatively CFDS was performed and classified according to Frates et al. We found a consistent percentage increase of CD34 stained microvessels in PCs (83 and 96%) as compared to adenomas and ANs (38 and 49%) determined by the hot spot analysis and systematic field analysis. A ROC analysis on the basis of the histologically determined number of microvessels demonstrated 70% microvessels as an optimal cut point for the diagnosis of PC with the highest sensitivity of 92% and highest specificity of 89%. The analysis of the CFDS-classification IV for the distinction between PCs and adenomas and ANs showed a sensitivity of 62% with a specificity of 100%. The lower sensitivity of the CFDS classification as compared with the immunohistologic determination of the microvessel density indicates that the CFDS classification detects the pathognomonic intranodular microvessels only incompletely. The higher CFDS specificity is most likely due to the detection of other vascular aspects of malignancy in addition to intranodular microvessels., (Copyright Georg Thieme Verlag KG Stuttgart New York.)

Pulmonary surfactant has two crucial roles in respiratory function; first, as a biophysical entity it reduces surface tension at the air water interface, facilitating gas exchange and alveolar stability during breathing, and, second, as an innate component of the lung's immune system it helps maintain sterility and balance immune reactions in the distal airways. Pulmonary surfactant consists of 90% lipids and 10% protein. There are four surfactant proteins named SP-A, SP-B, SP-C, and SP-D; their distinct interactions with surfactant phospholipids are necessary for the ultra-structural organization, stability, metabolism, and lowering of surface tension. In addition, SP-A and SP-D bind pathogens, inflict damage to microbial membranes, and regulate microbial phagocytosis and activation or deactivation of inflammatory responses by alveolar macrophages. SP-A and SP-D, also known as pulmonary collectins, mediate microbial phagocytosis via SP-A and SP-D receptors and the coordinated induction of other innate receptors. Several receptors (SP-R210, CD91/calreticulin, SIRPalpha, and toll-like receptors) mediate the immunological functions of SP-A and SP-D. However, accumulating evidence indicate that SP-B and SP-C and one or more lipid constituents of surfactant share similar immuno-regulatory properties as SP-A and SP-D. The present review discusses current knowledge on the interaction of surfactant with lung innate host defense., (2010 S. Karger AG, Basel)

Mycobacterium tuberculosis comes in contact with pulmonary surfactant, alveolar macrophages and type II epithelial cells. Alveolar type II epithelial cells secrete pulmonary surfactant, a complex mixture of phospholipids and proteins lining the alveolar surface, while alveolar macrophages are involved in surfactant catabolism. Surfactant proteins SP-A and SP-D modulate phagocytosis of M. tuberculosis by alveolar macrophages. We have reported that mice with decreased surfactant catabolism resulting from GM-CSF deficiency are highly susceptible to acute aerosol infection with 100 cfu of M. tuberculosis. Here, we evaluated the lungs of WT, GM-CSF-deficient, and GM-CSF-corrected mice surviving six months after sub-acute aerosol infection of 5-10 cfu M. tuberculosis. We show that GM-CSF-deficient mice develop intra-bronchial and intra-alveolar tuberculosis lesions with numerous mycobacteria, inflammatory cells, and extracellular proteinaceous material containing surfactant protein B (SP-B). In contrast, WT and GM-CSF-corrected mice develop typical epithelioid granulomas containing lymphocytes, SP-B positive cells, and M. tuberculosis bacilli inside macrophages. Our findings support the concept that whole pulmonary surfactant is an important component of host mycobacterial infection in the distal lung.

Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

The mechanisms by which GM-CSF mediates bacterial clearance and inflammation during mycobacterial infection are poorly understood. The objective of this work was to determine how GM-CSF alters pulmonary mycobacterial infection in vivo. Differences in GM-CSF levels in the lungs of normal mice (GM(+/+)), transgenic GM-CSF-deficient (GM-CSF(-/-)), and transgenic mice with high GM-CSF expression only in lung epithelial cells (SP-C-GM-CSF(+/+)/GM(-/-)) did not affect pulmonary infection rates caused by either the attenuated Mycobacterium bovis BCG or the virulent Mycobacterium tuberculosis H37Rv. However, in contrast to findings with BCG, all GM-CSF(-/-) and SP-C-GM-CSF(+/+)/GM(-/-) mice succumbed prematurely to virulent H37Rv. Granuloma formation was impaired in both GM-CSF(-/-) and SP-C-GM-CSF(+/+)/GM(-/-) mice regardless of mycobacterial virulence. However, H37Rv-infected GM-CSF(-/-) mice suffered broncho-alveolar destruction, edema, and necrosis while only short-lived granulomas were observed in SP-C-GM-CSF(+/+)/GM(-/-) mice. Bone marrow-derived macrophages, but not dendritic cells of SP-C-GM-CSF(+/+)/GM(-/-) mice, were hypo-responsive to mycobacterial infection. Surfactant protein levels were differentially influenced by BCG and H37Rv. We conclude that GM-CSF has an essential protective role first in preserving alveolar structure and second in regulating macrophages and dendritic cells to facilitate containment of virulent mycobacteria in pulmonary granulomas. However, precise regulation of lung GM-CSF is vital to effective control of M. tuberculosis.

Mass spectrometric characterization of the surfactant protein A (SP-A) receptor 210 (SP-R210) led to the identification of myosin (Myo) XVIIIA and nonmuscle myosin IIA. Antibodies generated against the unique C-terminal tail of MyoXVIIIA revealed that MyoXVIIIA, MyoIIA, and SP-R210 have overlapping tissue distribution, all being highly expressed in myeloid cells, bone marrow, spleen, lymph nodes, and lung. Western blot analysis of COS-1 cells stably transfected with either MyoXVIIIA or MyoIIA indicated that SP-R210 antibodies recognize MyoXVIIIA. Furthermore, MyoXVIIIA but not MyoIIA localized to the surface of COS-1 cells, and most importantly, expression of MyoXVIIIA in COS-1 cells conferred SP-A binding. Western analysis of recombinant MyoXVIIIA domains expressed in bacteria mapped the epitopes of previously derived SP-R210 antibodies to the neck region of MyoXVIIIA. Antibodies raised against the neck domain of MyoXVIIIA blocked the binding of SP-A to macrophages. Together, these findings indicate that MyoXVIIIA constitutes a novel receptor for SP-A.

DNA-damage evokes cell cycle checkpoints, which function to maintain genomic integrity. The retinoblastoma tumor suppressor (RB) and mismatch repair complexes are known to contribute to the appropriate cellular response to specific types of DNA damage. However, the signaling pathways through which these proteins impact the cell cycle machinery have not been explicitly determined. RB-deficient murine embryo fibroblasts continued a high degree of DNA replication following the induction of cisplatin damage, but were inhibited for G(2)/M progression. This damage led to RB dephosphorylation/activation and subsequent RB-dependent attenuation of cyclin A and CDK2 activity. In both Rb+/+ and Rb -/- cells, cyclin D1 expression was attenuated following DNA damage. As cyclin D1 is a critical determinant of RB phosphorylation and cell cycle progression, we probed the pathway through which cyclin D1 degradation occurs in response to DNA damage. We found that attenuation of endogenous cyclin D1 is dependent on multiple mismatch repair proteins. We demonstrate that the mismatch repair-dependent attenuation of endogenous cyclin D1 is critical for attenuation of CDK2 activity and induction of cell cycle checkpoints. Together, these studies couple the activity of the retinoblastoma and mismatch repair tumor suppressor pathways through the degradation of cyclin D1 and dual attenuation of CDK2 activity.

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G(1) and S-phase progression. While RB-mediated G(1) inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition.

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.

The regulatory role of activator protein-1 (AP-1) family members in mouse surfactant protein (SP) B (mSP-B) promoter function was assessed in the mouse lung epithelial cell line MLE-15. Expression of recombinant Jun B and c-Jun inhibited mSP-B promoter activity by 50-75%. Although c-Fos expression did not alter mSP-B transcription, Jun D enhanced mSP-B promoter activity and reversed inhibition of mSP-B by c-Jun or Jun B. A proximal AP-1 binding site (-18 to -10 bp) was identified that overlaps a thyroid transcription factor-1 binding site. Mutation of this proximal AP-1 site blocked both Jun B inhibition and Jun D enhancement and partially blocked c-Jun inhibition of promoter activity. Promoter deletion mutants were used to identify additional sequences mediating the inhibitory effects of c-Jun in the distal region from -397 to -253 bp. The AP-1 element in this distal site (-370 to -364 bp) is part of a composite binding site wherein AP-1, cAMP response element binding protein, thyroid transcription factor-1, and nuclear factor I interact. Point mutation of the distal AP-1 binding site partially blocked c-Jun-mediated inhibition of the SP-B promoter. Both stimulatory (Jun D) and inhibitory (c-Jun/Jun B) effects of AP-1 family members on mSP-B promoter activity are mediated by distinct cis-acting elements in the mSP-B 5'-flanking region.

Retinoids are known to play important roles in organ development of the lung. Retinoids exert their activity by modulating the expression of numerous genes, generally influencing gene transcription, in target cells. In the present work, the mechanism by which retinoic acid (RA) regulates surfactant protein (SP) B expression was assessed in vitro. RA (9-cis-RA) enhanced SP-B mRNA in pulmonary adenocarcinoma cells (H441 cells) and increased transcriptional activity of the SP-B promoter in both H441 and mouse lung epithelial cells (MLE-15). Cotransfection of H441 cells with retinoid nuclear receptor (RAR)-alpha, -beta, and -gamma and retinoid X receptor (RXR)-gamma further increased the response of the SP-B promoter to RA. Treatment of H441 cells with RA increased immunostaining for the SP-B proprotein and increased the number of cells in which the SP-B proprotein was detected. An RA responsive element mediating RA stimulation of the human SP-B promoter was identified. RAR-alpha and -gamma and RXR-alpha but not RAR-beta or RXR-beta and -gamma were detected by immunohistochemical analysis of H441 cells. RA, by activating RAR activity, stimulated the transcription and synthesis of SP-B in pulmonary adenocarcinoma cells.

Surfactant protein B (SP-B) is selectively expressed in bronchiolar and alveolar epithelial cells of the lung. We identified an upstream enhancer located in the 5'-flanking region of the human SP-B gene (-439 to -331 base pair, hSP-B(-439/-331)) by deletion analysis of SP-B-luciferase constructs assessed in transfection assays in vitro. The element cis-activated the expression of an SV40 promoter-luciferase reporter gene in a human pulmonary adenocarcinoma cell line (H441-4). Three distinct binding sites for the nuclear transcription protein, thyroid transcription factor 1 (TTF-1), were identified, and the purified TTF-1 homeodomain was bound to bhe region of hSP-B(-439/-331). Co-transfection of H441-4 cells with the expression vector pCMV-TTF-1 trans-activated the native human SP-B promoter and the SV40 promoter fused with the SP-B enhancer. Mutations of the TTF-1 binding sites in the upstream enhancer blocked TTF-1 binding and transactivation activity. In summary, TTF-1 interacts with distinct proximal (-80 to -110) and distal (-439 to -331) cis-acting elements than regulate lung epithelial cell-specific transcription of the human SP-B gene.

We analyzed the expression of P-glycoprotein (Pgp) by immunohistochemistry using JSB-1 monoclonal antibody (MAb) on paraffin-embedded sections of the multi-drug resistant (MDR) (CHrC5 and CEM-VLB), and sensitive (AuxB1 and CEM) cell lines, and also in normal kidney, colon, adrenal and in kidney and colon carcinomas. After comparing the sensitivity of three different immunohistochemical techniques the peroxidase-antiperoxidase method was found to be the best. We then tested six different fixation methods. The MDR cell lines and human tissues demonstrated the strongest staining with B-5 fixative. Both MDR cell lines, but not the tissues fixed in 1% paraformaldehyde and Zamboni's fixative demonstrated weak staining. No immuno- reactivity could be detected in MDR cell lines and tissues fixed in 10% buffered or nonbuffered formalin or by the AMeX method of tissue processing. The present study clearly shows that the type of fixative is critical for the preservation of Pgp epitope recognized by JSB-1 MAb, and that B-5 fixative is expected to be equally applicable for the detection of Pgp in normal and neoplastic tissues.