Search

Tuberculosis pleurisy was induced by inoculation of virulent (H37Rv strain or Erdman strain) or attenuated (H37Ra strain) green-fluorescent protein-expressing Mycobacterium tuberculosis into guinea pigs that had or had not been vaccinated with Bacille-Calmette Guérin (BCG). Pleural fluid and cells were analyzed for phagosome-lysosome (P-L) fusion, on the basis of confocal microscopy, intracellular and extracellular bacterial survival, and production of cytokine mRNA. BCG vaccination increased fluid volume and cellular accumulation, significantly enhanced P-L fusion, and significantly decreased intracellular bacterial survival in pleural-effusion macrophages of the guinea pigs infected with the 2 virulent strains. Furthermore, significant increases in interferon-gamma, transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-12p40 cytokine mRNA were seen in the pleural cells of the BCG-vaccinated guinea pigs. Copyright © 2008 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Data collected by animal shelters can provide an overview of population numbers and recommendations for shelter management and community programming. While studies utilize data from shelter software, questions remain on whether such data are reliable. The objective of the online experiment was to determine the agreement in data input for surrender reason, breed, and color across shelter staff ( n  = 81) when presented with four complex narratives of fictional owners surrendering dogs. Additionally, we aimed to understand how staff select surrender reasons for data input through qualitative analysis. Out of 40 possible surrender reasons, the number of unique reasons selected for each scenario ranged from 12-16, suggesting a variety of possible data entries for the same surrender narrative. Agreement was also low for breed and color. Shelter staff described a variety of different methods of determining the surrender reason for input into shelter software, such as asking the owner for their most influential reason or inferring the underlying reason. Further research is required to understand how animal shelter data can be collected consistently in a way that can meaningfully inform shelter management decisions.

As animals experience distress in animal shelters, leaders call for increased efforts to divert intake of companion animals away from shelters. One novel intake diversion strategy is supported self-rehoming, where owners find new homes for their animals without surrendering to a physical shelter. This study aimed to identify predictors of successful diversion of animals through the AdoptaPet.com 'Rehome' online platform. Data for dogs (n = 100,342) and cats (n = 48,484) were analysed through logistic regression to assess the association of animal- and owner-related factors and outcome. Overall, 87.1% of dogs and 85.7% of cats were successfully diverted from animal shelters, out of which, 37.8% of dogs and 35.3% of cats were kept by their original owner. Multiple animal-related factors predicted increased odds of diversion (e.g. younger, smaller). Dog and cat owners who set a longer rehoming deadline (i.e. > 8 weeks) were over twice as likely to keep or adopt out their animal. Dog owners who surrendered for owner-related reasons had increased odds of diversion in comparison to animal behaviour issues. We conclude that online-supported, self-rehoming platforms provide pet owners with an alternative to relinquishment that may reduce the intake of animals to shelters; however, owners with animals that are not preferred by adopters may have to decide whether to keep their animal or relinquish their animal to a shelter or rescue. These results provide guidance for animal shelter professionals on the likelihood of successful diversion programmes given certain animal and owner characteristics., Competing Interests: None., (© The Author(s) 2023.)

Despite the advances in single-cell transcriptomics, the reconstruction of gene regulatory networks remains challenging. Both the large amount of zero counts in experimental data and the lack of a consensus preprocessing pipeline for single-cell RNA sequencing (scRNA-seq) data make it hard to infer networks. Imputation can be applied in order to enhance gene-gene correlations and facilitate downstream analysis. However, it is unclear what consequences imputation methods have on the reconstruction of gene regulatory networks. To study this, we evaluate the differences on the performance and structure of reconstructed networks before and after imputation in single-cell data. We observe an inflation of gene-gene correlations that affects the predicted network structures and may decrease the performance of network reconstruction in general. However, within the modest limits of achievable results, we also make a recommendation as to an advisable combination of algorithms while warning against the indiscriminate use of imputation before network reconstruction in general., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

There is increasing awareness among animal shelter professionals regarding the role of shelters in perpetuating inequities in pet ownership, although the relationship between owner vulnerabilities and animal shelter services is largely understudied. Currently, there is no literature comparing the sociodemographic conditions of communities where surrendered animals originate and communities where they are adopted. The present study compared the "flow" of surrendered animals between originating communities (incoming) and communities where they were adopted (outgoing; n = 21,270). To analyze community-level vulnerability, we used the Canadian Index of Multiple Deprivation (CIMD), which has four dimensions of social vulnerability. We found that three of the four CIMD dimensions were significantly different between surrendering and adopting communities (Ethnocultural Composition, Situational Vulnerability (SV), Economic Dependency, but not Residential Instability). For further investigation, we also grouped our analysis by intake groups (small animal n = 2,682; puppy n = 973; dog n = 3,446; kitten n = 6,436; cat n = 7,733) and found multiple relationships for which the incoming and outgoing CIMD quintiles were different. For example, for both puppies and kittens, the median outgoing SV quintile ranks were statistically significantly lower (less vulnerable) than incoming quintile ranks, with the effect size being moderate (puppy r = 0.31, kitten r = 0.30; p ≤ 0.0025), supporting the concern of the flow of certain animals from more vulnerable to less vulnerable communities. The results of this research provide a basis for understanding potential inequities in the use of shelter services to surrender or adopt an animal. Furthermore, these methods allow animal shelters to assess community needs and create interventions to reduce intake and increase adoption of animals. Finally, these data provide further support that animal sheltering is best considered from a One Welfare perspective., Competing Interests: Data were obtained from the BC SPCA, who is the employer of one of the authors EG. The remaining 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., (Copyright © 2021 Ly, Gordon and Protopopova.)

Recent natural disasters and weather extremes are a stark reminder that we live in a climate crisis. Climate scientists and policymakers have asked each discipline to anticipate and create mitigation and adaptation plans in preparation for a worsening future. Companion animals both impact and are impacted by the changing climate through their intrinsically linked relationships to human society. In this theoretical paper, we argue that companion animal scientists are well-suited to address climate change issues. We identify several anticipated climate change outcomes, such as an increase in extreme weather events, human migration, disasters, and an increase in human inequity, and connect these outcomes to identified or hypothesized impacts on companion animals and the human-animal bond. We suggest opportunities to reduce climate change impacts on companion animals that include alterations to owner caretaking behaviors and breeding practices, and education of owners and governments on zoonosis and disaster preparedness. Furthermore, building climate resilience through decreasing inequity in companion animal fields is paramount; and we propose that a starting place can be in animal sheltering and other support services. We also summarize how companion animals and owners' caretaking behaviors are impacting climate change through the use of finite natural resources as well as pollution and carbon emissions. We propose that replacement, reduction, and refinement, that guide laboratory animal research, can also be useful to mitigate the effects of companion animals on the environment. We suggest criteria for successful mitigation and adaptation plans to include equitability, sustainability, respect for animals, and measurability. Finally, we end on a call to all companion animal professionals to actively consider their role in mitigating the impact of companion animals on the climate and preparing for the fallout of climate change in their communities., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Dairy farm systems have intensified to meet growing demands for animal products, but public opposition to this intensification has also grown due, in part, to concerns about animal welfare. One approach to addressing challenges in agricultural systems has been through the addition of new technologies, including genetic modification. Previous studies have reported some public resistance towards the use of these technologies in agriculture, but this research has assessed public attitudes toward individual practices and technologies and few studies have examined a range of practices on dairy farms. In the present study, we presented participants with four scenarios describing dairy practices (cow-calf separation, the fate of excess dairy calves, pasture access and disbudding). Citizens from Canada and the United States (n = 650) indicated their support (on a 7-point scale) toward five approaches (maintaining standard farm practice, using a naturalistic approach, using a technological approach, or switching to plant-based or yeast-based milk production) aimed at addressing the welfare issues associated with the four dairy practices. Respondents also provided a text-based rationale for their responses and answered a series of demographic questions including age, gender, and diet. Participant diet affected attitudes toward milk alternatives, with vegetarians and vegans showing more support for the plant-based and yeast-based milk production. Regardless of diet, most participants opposed genetic modification technologies and supported more naturalistic practices. Qualitative responses provided insight into participants' values and concerns, and illustrated a variety of perceived benefits and concerns related to the options presented. Common themes included animal welfare, ethics of animal use, and opposition toward technology. We conclude that Canadian and US citizens consider multiple aspects of farm systems when contemplating animal welfare concerns, and tend to favor naturalistic approaches over technological solutions, especially when the latter are based on genetic modification., Competing Interests: The authors have declared that no competing interests exist.

Previous studies identify owner-related issues, such as cost and housing, as common reasons for relinquishment of companion animals to animal shelters. It is likely that the burden of surrendering for owner-related reasons falls on those who are socially vulnerable (e.g., low income, unemployed); however, very few studies have assessed social determinants as a predictor of animal relinquishment. The present study used the Canadian Index of Multiple Deprivation (CIMD), which uses four factors of social vulnerability (Ethnocultural Composition, Economic Dependency, Residential Instability, and Situational Vulnerability) to predict risk of surrender for various reasons, of various species and breeds, and of various health statuses across British Columbia, Canada ( n = 29,236). We found that CIMD factors predicted increased risk of surrender across many shelter variables. For further understanding of differences between areas in the province, the present study also analyzed the relationship between CIMD factors and animal surrender variables in two areas of interest: Metro Vancouver ( n = 3,445) and Kamloops ( n = 2,665), and plotted these relationships on a geospatial scale. We found that there were some similarities across areas, such as Situational Vulnerability predicting increased odds of surrendering pit bull-labeled dogs vs. all other dog breeds. There were also differences in predictors of animal surrender variables, suggesting that provision of animal services, such as veterinary care, for vulnerable groups may be specific to location. For example, whereas Ethnocultural Composition predicted increased risk of owner surrender for multiple owner-related reasons in Metro Vancouver, these same reasons for surrender were predicted by Residential Instability in Kamloops, indicating demographic differences that affect animal shelter service use. The results of this research validate the use of geospatial analysis to understand relationships between human vulnerability and animal welfare, but also highlight the need for further interventions in marginalized populations to increase retention of animals., Competing Interests: Data was obtained from the BC SPCA, who is the employer of one of the authors EG. The remaining 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., (Copyright © 2021 Ly, Gordon and Protopopova.)

People often express concern for the welfare of farm animals, but research on this topic has relied upon self-report. Facial expressions provide a quantifiable measure of emotional response that may be less susceptible to social desirability bias and other issues associated with self-report. Viewing other humans in pain elicits facial expressions indicative of empathy. Here we provide the first evidence that this measure can also be used to assess human empathetic responses towards farm animals, showing that facial expressions respond reliably when participants view videos of farm animals undergoing painful procedures. Participants (n = 30) were asked to watch publicly sourced video clips of cows and pigs undergoing common management procedures (e.g. disbudding, castration, tail docking) and control videos (e.g. being lightly restrained, standing). Participants provided their subjective rating of the intensity of 5 negative emotions (pain, sadness, anger, fear, disgust) on an 11-point Likert scale. Videos of the participants (watching the animals) were scored for intensity of unpleasantness of the participants' facial expression (also on an 11-point Likert scale) by a trained observer who was blind to treatment. Participants showed more intense facial expressions while viewing painful procedures versus control procedures (mean ± SE Likert; 2.4 ± 0.08 versus 0.6 ± 0.17). Participants who reported more intense negative responses also showed stronger facial expressions (slope ± SE = 0.4 ± 0.04). Both the self-reported and facial measures varied with species and procedure witnessed. These results indicate that facial expressions can be used to assess human-animal empathy., Competing Interests: The authors have declared that no competing interests exist.

Interleukin-4 is a signature cytokine of T-helper type 2 (Th2) cells that play a major role in shaping immune responses. Its role in highly relevant animal model of tuberculosis (TB) like guinea pig has not been studied till date. In the current study, the guinea pig IL-4 gene was cloned and expressed using a prokaryotic expression vector (pET30 a(+)). This approach yielded a recombinant protein of 19 kDa as confirmed by mass spectrometry analysis and named as recombinant guinea pig (rgp)IL-4 protein. The authenticity of the expression of rgpIL-4 protein was further verified through polyclonal anti-IL4 antiserum raised in rabbits that showed specific and strong binding with the recombinant protein. The biological activity of the rgpIL-4 was ascertained in RAW264.7 cells where LPS-treated nitric oxide (NO) production was found to be suppressed in the presence of this protein. The three-dimensional structure of guinea pig IL-4 was predicted by utilizing the template structure of human interleukin-4, which shared a sequence homology of 58%. The homology modeling result showed clear resemblance of guinea pig IL-4 structure with the human IL-4. Taken together, our study indicates that the newly expressed, biologically active rgpIL-4 protein could provide deeper understanding of the immune responses in guinea pig to different infectious diseases like TB and non-infectious ones.

Virus replication displays a large cell-to-cell heterogeneity; yet, not all sources of this variability are known. Here, we study the effect of defective interfering (DI) particle (DIP) co-infection on cell-to-cell variability in influenza A virus (IAV) replication. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNA) and are, thus, defective in virus replication. Moreover, they interfere with virus replication. Using single-cell isolation and reverse transcription polymerase chain reaction, we uncovered a large between-cell heterogeneity in the DI vRNA content of infected cells, which was confirmed for DI mRNAs by single-cell RNA sequencing. A high load of intracellular DI vRNAs and DI mRNAs was found in low-productive cells, indicating their contribution to the large cell-to-cell variability in virus release. Furthermore, we show that the magnitude of host cell mRNA expression (some factors may inhibit virus replication), but not the ribosome content, may further affect the strength of single-cell virus replication. Finally, we show that the load of viral mRNAs (facilitating viral protein production) and the DI mRNA content are, independently from one another, connected with single-cell virus production. Together, these insights advance single-cell virology research toward the elucidation of the complex multi-parametric origin of the large cell-to-cell heterogeneity in virus infections., Competing Interests: The authors declare no conflicts of interest.

In some settings, rotavirus vaccines have been associated with a low-level risk of intussusception, the most common cause of bowel obstruction in infants. As Vietnam prepares to introduce rotavirus vaccine into the national immunization program, we sought to better characterize the epidemiology of recurrent intussusception. We enrolled children <2 years of age who were hospitalized for intussusception retrospectively from January 2013 through December 2014 and prospectively from January 2015 through December 2016 at 2 hospitals in Vietnam. We enrolled 2477 children. Nearly all children were successfully treated by enema with low surgery rate (1%). We found 10% of children (n = 254) experienced at least once recurrence (range: 1-6) and 57% of first recurrences happened within the first 12 weeks after treatment of the first episode. The median age at first intussusception was 13 months for children without a recurrent episode and 10 months for children with a recurrence. The symptoms of the recurrent cases were milder with less vomiting (67%), bloody stool (7%) and fever (10%) compared to the initial cases (p < 0.01). We found the rate of recurrences following enema reduction of intussusception to be similar to that reported from other countries. Due to the high rate of intussusception and recurrent episodes in Vietnam, a better understanding of the cause of recurrent intussusception will be critical in assessing intussusception cases after rotavirus introduction., (Published by Elsevier Ltd.)

Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers (https://github.com/vals/umis/). We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.

Differentiation of naïve CD4 + T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo . By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database www.PlasmoTH.org, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates., Competing Interests: Competing interests The authors declare no competing interests.

Rapid, large-scale manufacture of medical countermeasures can be uniquely met by the plant-made-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plant-based manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

About half of the human genome consists of highly repetitive elements, most of which are considered dispensable for human life. Here, we report that repetitive elements originating from endogenous retroviruses (ERVs) are systematically transcribed during human early embryogenesis in a stage-specific manner. Our analysis highlights that the long terminal repeats (LTRs) of ERVs provide the template for stage-specific transcription initiation, thereby generating hundreds of co-expressed, ERV-derived RNAs. Conversion of human embryonic stem cells (hESCs) to an epiblast-like state activates blastocyst-specific ERV elements, indicating that their activity dynamically reacts to changes in regulatory networks. In addition to initiating stage-specific transcription, many ERV families contain preserved splice sites that join the ERV segment with non-ERV exons in their genomic vicinity. In summary, we find that ERV expression is a hallmark of cellular identity and cell potency that characterizes the cell populations in early human embryos., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tumor necrosis factor alpha (TNF-α) is a cytokine which plays opposing roles in the context of infectious disease pathogenesis. TNF-α is essential for the development of a protective immune response to some pathogens, for example, Mycobacterium tuberculosis, by synergizing with other cytokines. However, exorbitant or uncontrolled TNF-α activity may also drive pathology and disease symptoms in many infectious diseases. In order to elucidate the beneficial and detrimental roles of TNF-α in tuberculosis (TB) and other diseases for which the guinea pig is the small animal model of choice, recombinant guinea pig (rgp)TNF-α has been produced using prokaryotic expression systems. However, it is unknown whether posttranslational modifications which cannot be made in the prokaryotic expression systems may be important for rgpTNF-α structure and function. Therefore, we carried out a comparative study by expressing rgpTNF-α in prokaryotic and eukaryotic expression systems and analyzed the eukaryotic-expressed rgpTNF-α for the presence of posttranslational modifications by subjecting it to NanoLC-MS/MS. We conclude that the eukaryotic-expressed rgpTNF-α lacks posttranslational modifications, and we found no significant difference in terms of the biological activity between prokaryotic- and eukaryotic-expressed rgpTNF-α. Taken together, results from our study show that a prokaryotic expression system can be used for generating large amounts of rgpTNF-α without concern for the biological integrity.

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.

Interleukin-4 (IL-4), a pleiotropic cytokine produced by T-helper type 2 (Th2) cells, is involved in promoting humoral immune responses, allergic reactions and asthma. Previous studies suggested an important role for IL-4 in susceptibility to pulmonary tuberculosis; however, the role of IL-4 has not been studied in the guinea pig, a highly relevant model for this disease. In the present study, we cloned a cDNA for guinea pig IL-4 and examined, for the first time, mRNA expression by real-time RT-PCR in cultured guinea pig cells. High levels of IL-4 mRNA expression were detected in spleen T cells of naïve animals after in vitro stimulation with PMA plus ionomycin for 4-24 h. The expression of IL-4 mRNA was low in spleen and lymph node cells immunized with ovalbumin (OVA) plus Complete Freund's Adjuvant (CFA) in response to OVA (Th1), but significantly higher in the guinea pigs immunized with OVA plus alum (Th2). BCG vaccination reduced the expression of IL-4 mRNA in both spleen and lung digest cells compared to naïve guinea pigs, while levels of IFN-γ were similar in both groups. Furthermore, lung cells from Mycobacterium tuberculosis-infected guinea pigs stimulated in vitro with PPD or MPT64 showed low levels of IL-4 mRNA expression. Thus, BCG vaccination or M. tuberculosis infection modulates IL-4 mRNA expression in the guinea pig. Cloning of guinea pig IL-4 will allow us to address the role of IL-4 in vaccine-induced resistance to pulmonary TB in a highly relevant animal model., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We examined the ability of recombinant guinea pig IL-8 (CXCL8) to activate neutrophils upon infection with virulent Mycobacterium tuberculosis. Using a Transwell insert culture system, contact-independent cell cultures were studied in which rgpIL-8-treated neutrophils were infected with virulent M. tuberculosis in the upper well, and AM were cultured in the lower well. IL-1β and TNF-α mRNA expression was significantly upregulated by AM. Neutralizing anti-rgpTNF-α polyclonal antibody abrogated the response of AM to supernatants from the rgpIL-8-treated, infected neutrophils, while an anti-rgpIL-8 polyclonal antibody had no effect. This suggests that TNF-α produced by rgpIL-8 treated, infected neutrophils may play an important role in the activation of AM in the early response of the host against M. tuberculosis infection. Significant induction of apoptosis in M. tuberculosis-infected neutrophils was observed as compared to the uninfected neutrophils. Feeding of infected, apoptotic neutrophils to AM induced a significant up-regulation of TNF-α and IL-1β mRNA compared to AM exposed to staurosporine-treated apoptotic neutrophils. Suppressed intracellular mycobacterial growth was also seen in AM fed with infected, apoptotic neutrophils as compared to the AM infected with M. tuberculosis H37Rv alone. Taken together, these data suggest that neutrophil-macrophage interactions may contribute to host defense against M. tuberculosis infection., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Background: Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance to Mycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. We hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNgamma-mediated signaling pathway., Methodology/principal Findings: Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNgamma, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFalpha, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNgamma receptor with lipid rafts, without affecting surface expression of IFNgamma receptor., Conclusions/significance: We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNgamma, by modulation of IFNgamma receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.

Background: It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models., Methodology/principal Findings: By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates., Conclusions/significance: These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.

Tumour necrosis factor alpha (TNF-alpha) is a prototypic pro-inflammatory cytokine that has a central role in the initial host response to Mycobacterium tuberculosis infection. It is a key player in granuloma formation, macrophage activation, bacterial killing, and pathology in vivo. However, the exact mechanism has not been completely understood. This review summarizes the TNFalpha data acquired from the 'gold standard' guinea pig animal model of tuberculosis. While production of TNFalpha is widely accepted as beneficial to the host response, we have found that this hypothesis is just one side of the story. TNFalpha can up-regulate and down-regulate some key pro-inflammatory cytokines (IFNgamma, IL-12p40) and differentially modulate macrophage activation and intracellular bacterial growth. Neutralization of TNFalpha in vivo allows an antiinflammatory TGFbeta-mediated response to develop. Furthermore, BCG vaccination modulates TNFalpha responses directly in the pulmonary granulomas to reduce tissue damage. The bipolar nature of TNFalpha should be considered as knowledge of this critical molecule continues to grow.

Previously, treatment with anti-gpTNFalpha antibody enhanced TNFalpha mRNA expression in pulmonary granulomas microdissected from non-vaccinated guinea pigs, and modified splenic granuloma architecture. In this study, pleural fluid, cells, and granulomatous tissues were collected 3, 5, and 8 days post-pleurisy induction in guinea pigs treated with anti-gpTNFalpha or normal serum control. Neutralizing TNFalpha reduced the percentage of macrophages in the pleural exudate while increasing the proportions of neutrophils and lymphocytes. Cell-associated mycobacterial loads were increased in guinea pigs treated with anti-gpTNFalpha antibody. Cells from the pleural exudate in both treatment groups at day 3 expressed predominantly TNFalpha and IFNgamma mRNA. By day 5, treatment with anti-gpTNFalpha antibody significantly reduced TNFalpha mRNA and increased TGFbeta and iNOS mRNA expression, a transition which did not occur in the control group until day 8. TNFalpha mRNA overwhelmed the cytokine milieu of microdissected pleural granulomas in the control group at day 3 whereas TNFalpha, IFNgamma, and TGFbeta mRNA dominated the anti-gpTNFalpha-treated group. At day 8, granulomas from the control group began shifting towards an anti-inflammatory profile with increased levels of TGFbeta mRNA. Neutralization of TNFalpha hastened the transition to an anti-inflammatory cytokine response in guinea pig pleural granulomas and exudate cells.

Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a DeltahsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogen's dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; k(cat)/K(m) = 14.4+/-0.5 microM(-1) s(-1)), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the DeltahsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 A revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design.

The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (DeltadosR-dosS [DeltadosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the DeltadosR-S mutant exhibited a growth defect. In the rabbit, the DeltadosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.

Background: Mouse virulence assessments of certain Mycobacterium tuberculosis mutants have revealed an immunopathology defect in which high tissue CFU counts are observed but the tissue pathology and lethality are reduced. M. tuberculosis mutants which grow and persist in the mouse lungs, but have attenuated disease progression, have the immunopathology (imp) phenotype. The antigenic properties of these strains may alter the progression of disease due to a reduction in host immune cell recruitment to the lungs resulting in disease attenuation and prolonged host survival., Results: In this study we focused on the mouse immune response to one such mutant; the M. tuberculosis Delta sigC mutant. Aerosol infection of DBA/2 and SCID mice with the M. tuberculosis Delta sigC mutant, complemented mutant and wild type strain showed proliferation of mutant bacilli in mouse lungs, but with decreased inflammation and mortality in DBA/2 mice. SCID mice shared the same phenotype as the DBA/2 mice in response to the Delta sigC mutant, however, they succumbed to the infection faster. Bronchoalveolar lavage (BAL) fluid analysis revealed elevated numbers of infiltrating neutrophils in the lungs of mice infected with wild type and complemented Delta sigC mutant strains but not in mice infected with the Delta sigC mutant. In addition, DBA/2 mice infected with the Delta sigC mutant had reduced levels of TNF-alpha, IL-1beta, IL-6 and IFN-gamma in the lungs. Similarly, there was a reduction in proinflammatory cytokines in the lungs of SCID mice. In contrast to the mouse model, the Delta sigC mutant had reduced initial growth in guinea pig lungs. A possible mechanism of attenuation in the Delta sigC mutant may be a reduction in neutrophilic-influx in the alveolar spaces of the lungs, and decreased proinflammatory cytokine secretion. In contrast to mouse data, the M. tuberculosis Delta sigC mutant proliferates slowly in guinea pig lungs, a setting characterized by caseating necrosis., Conclusion: Our observations suggest that the immunopathology phenotype is associated with the inability to trigger a strong early immune response, resulting in disease attenuation. While macrophages and T cells have been shown to be important in containing M. tuberculosis disease our study has shown that neutrophils may also play an important role in the containment of this organism.

TB is presenting new challenges as a global health problem, especially with new threats of HIV coinfection and multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis. The current TB vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG), is the most widely used vaccine worldwide but its efficacy against pulmonary TB in adults in many high-burden countries is limited. Different vaccine strategies will probably be required for the various needs that exist within a population in which some individuals have been previously immunized with BCG, coinfected with HIV and/or latently infected with M. tuberculosis. In the last 15 years, new strategies to improve or replace BCG in the laboratory have led to several promising vaccine candidates that are actively being evaluated in human clinical trials. Some of these new vaccines may eventually be recommended for travelers to TB high-burden countries. This paper summarizes the progress of vaccine candidates in animal models to improve, replace or augment BCG vaccination.

The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-beta, with some IFN-gamma and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF-alpha mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-beta mRNA with some IL-12p40 and IFN-gamma mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally "vaccinated" by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF-alpha mRNA in unimmunized animals, and mostly TGF-beta mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.

Background: Mouse and guinea pig models have been used to identify Mycobacterium tuberculosis mutants attenuated for survival. However, unlike mice, M. tuberculosis-infected guinea pigs form caseating granulomas, which may simulate human disease more closely., Methods: We used designer arrays for defined mutant analysis, a high-throughput subtractive competition assay, for genotypically defined M. tuberculosis mutants and compared the survival of the same mutant pools in guinea pig and mouse aerosol models. Selected mutants found to be attenuated in either aerosol model were also analyzed in the mouse hollow-fiber model., Results: M. tuberculosis mutants representing 74 genes were tested. Eighteen M. tuberculosis mutants were attenuated for survival in either aerosol model, with 70% of selected mutants also attenuated in the mouse hollow-fiber model. The majority of attenuated mutants in the mouse aerosol model were detected only after 90 days of infection. There was a high degree of concordance between the genes identified by the 2 aerosol models, with detection being significantly earlier in the guinea pig (P<.0003)., Conclusions: We identified M. tuberculosis genes required for survival in mammalian lungs. The majority of mouse late-stage survival mutants were detected significantly earlier in the guinea pig, which suggests that differences in tuberculosis-induced lung pathologic changes may account for this accelerated detection.

Levels of IL-12p40, TNFalpha, TGFbeta, IFNgamma and IL-10 mRNA were assessed by laser capture microdissection followed by quantitative real-time PCR in the pulmonary granulomas of unimmunized and BCG-vaccinated guinea pigs infected by aerosol with virulent Mycobacterium tuberculosis. Lesions microdissected from unimmunized guinea pigs were overwhelmed by the pro-inflammatory TNFalpha mRNA at both 3 and 6 weeks post infection, indicating the struggle to control the mounting infection. The cytokine profile of granulomas from vaccinated guinea pigs shifted from type 1 cytokine mRNA (IFNgamma and IL-12p40) at 3 weeks to a predominantly anti-inflammatory environment (TGFbeta mRNA) at 6 weeks. The relative proportions of cytokine mRNA transcripts in the periphery of the granuloma were different from the centre, reflecting differences in cell composition and architecture. Moreover, analysis of the individual lung lobes at 6 weeks post infection suggests that heterogeneity exists in the cytokine profile between the lobes of the lung.

To determine the mechanisms by which dietary fish oil (FO) affects antigen-stimulated Th1 cell development, DO11.10 Rag 2(-/-) T cell receptor transgenic mice were fed a control diet (5% corn oil (CO) or a FO diet (1% CO + 4% FO, (n-3) PUFA) for 2 wk. CD4(+) T cells were cultured under neutral or Th1 polarizing conditions. FO feeding suppressed (P < 0.05) ovalbumin peptide-induced proliferation of nonpolarized CD4(+) T cells. Differentiation in vitro to Th1 cells was not affected by dietary FO, as evidenced by similar percentages of KJ1-26(+), IFN-gamma(+), IL-4(-) Th1 cells in cultures from CO-fed (99%) and FO-fed (97%) mice. However, the absolute number of viable Th1 cells in polarized cultures from FO-fed mice was less than half that observed in CO-fed mice (P < 0.05), indicating that FO inhibits in vitro Th1 clonal expansion. The reduced number of Th1 cells in FO cultures was not a result of increased apoptosis, because similar percentages of apoptotic Th1 cells were observed in cultures from FO- and CO-fed mice. IL-2-induced cell proliferation was significantly decreased in polarized Th1 cells from the FO group; however, the suppressed proliferation was not linked to reduced CD25 surface expression on antigen-stimulated CD4(+) T cells. Adoptively transferred CFSE-labeled DO11.10 CD4(+) cells into immunized mice (Th1 polarizing agents) showed that dietary FO reduced (P < 0.05) the number of cell divisions in vivo. These studies suggest that the attenuated inflammatory response which accompanies FO feeding may be explained, at least in part, by suppression of Th1 clonal expansion.

We have demonstrated that downregulation of proliferation by CD4(+) T-cells in mice fed n-3 PUFA diets is dependent on the involvement of CD28. Therefore, we hypothesized that the balance of co-stimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. Mice were fed a control corn oil (CO)-enriched diet devoid of n-3 PUFA, or diets enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 14d. The proliferation of splenic CD4(+) T-cells was suppressed by DHA and EPA following stimulation with anti-CD3 and anti-CD28. Surprisingly, the number of surface CD28 molecules was not reduced in activated CD4(+) T-cells from either group of n-3 PUFA-fed mice. However, in mice fed EPA, CTLA-4 protein levels were enhanced significantly 72 h post-activation (P<0.01). Therefore, we conclude that dietary EPA may suppress CD4(+) T-cell activation by enhancing the downregulatory co-receptor CTLA-4, while not altering the levels of CD28.

Our laboratory has demonstrated that down-regulation of proliferation and cytokine synthesis by CD4(+) T cells in mice fed diets rich in n-3 polyunsaturated fatty acids (PUFA) is highly dependent on the involvement of the co-stimulatory molecule, CD28. It has been reported that the inhibitory cytokine interleukin (IL)-10 acts directly on T cells which up-regulate IL-10 receptor (IL-10R) expression following stimulation via CD28 by efficiently blocking proliferation and cytokine production. Thus, it was hypothesized that dietary n-3 PUFA would suppress T cell function through the effects of IL-10. The proliferation of purified splenic CD4(+) T cells activated in vitro with anti-CD3 and anti-CD28 (alphaCD3/CD28) from conventional mice (C57BL/6) fed either a control corn oil (CO)-enriched diet devoid of n-3 PUFA, docosahexaenoic acid (DHA; 22 : 6) or eicosapentaenoic acid (EPA; 20 : 5) for 14 days was suppressed by dietary DHA and EPA. Surprisingly, a similar trend was seen in IL-10 gene knock-out (IL-10(-/-)) mice fed dietary n-3 PUFA. IL-10R cell surface expression was also significantly down-regulated on CD4(+) T cells from both the C57BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with alphaCD3/CD28. Enzyme-linked immunosorbent assay (ELISA) measurements revealed that C57BL/6 mice fed DHA had significantly reduced interferon (IFN)-gamma and IL-10 levels 48 h post-activation. However, CD4(+) T cells from IL-10(-/-) mice fed dietary n-3 PUFA produced significantly greater levels of IFN-gamma than the CO-fed group. Our data suggest that in the absence of IL-10, CD4(+) T cells from n-3 PUFA-fed mice may up-regulate IFN-gamma. Suppressed CD4(+) T cells from n-3 PUFA-fed C57BL/6 mice may use mechanisms other than IL-10 to down-regulate T cell function.

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.

In vitro evidence indicates that (n-3) polyunsaturated fatty acids (PUFA) suppress T-cell activation in part by displacing proteins from lipid rafts, specialized regions within the plasma membrane that play an important role in T-cell signal transduction. However, the ability of (n-3) PUFA to influence membrane microdomains in vivo has not been examined to date. Therefore, we compared the effect of dietary (n-3) PUFA on raft (liquid ordered) vs. soluble (liquid disordered) microdomain phospholipid composition in mouse T cells. Mice were fed diets containing either 5 g/100 g corn oil (control) or 4 g/100 g fish oil [contains (n-3) PUFA] + 1 g/100 g corn oil for 14 d. Splenic T-cell lipid rafts were isolated by density gradient centrifugation. Raft sphingomyelin content (mol/100 mol) was decreased (P < 0.05) in T cells isolated from (n-3) PUFA-fed mice. Dietary (n-3) PUFA were selectively incorporated into T-cell raft and soluble membrane phospholipids. Phosphatidylserine and glycerophosphoethanolamine, which are highly localized to the inner cytoplasmic leaflet, were enriched to a greater extent with unsaturated fatty acids compared with sphingomyelin, phosphatidylinositol and glycerophosphocholine. These data indicate for the first time that dietary (n-3) PUFA differentially modulate T-cell raft and soluble membrane phospholipid and fatty acyl composition.

1Alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the proliferation of many cancer cells in culture, but not the aggressive human prostate cancer cell line DU 145. We postulated that the 1,25-(OH)2D3-resistant phenotype in DU 145 cells might result from the high levels of expression of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) induced by treatment with 1,25-(OH)2D3. As this P450 enzyme initiates 1,25-(OH)2D3 catabolism, we presumed that a high level of enzyme induction could limit the effectiveness of the 1,25-(OH)2D3 antiproliferative action. To examine this hypothesis we explored combination therapy with liarozole fumarate (R85,246), an imidazole derivative currently in trials for prostate cancer therapy. As imidizole derivatives are known to inhibit P450 enzymes, we postulated that this drug would inhibit 24-hydroxylase activity, increasing the 1,25-(OH)2D3 half-life, thereby enhancing 1,25-(OH)2D3 antiproliferative effects on DU 145 cells. Cell growth was assessed by measurement of viable cells using the MTS assay. When used alone, neither 1,25-(OH)2D3 (1-10 nM) nor liarozole (1-10 microM) inhibited DU 145 cell growth. However, when added together, 1,25-(OH)2D3 (10 nM)/liarozole (1 microM) inhibited growth 65% after 4 days of culture. We used a TLC method to assess 24-hydroxylase activity and demonstrated that liarozole (1-100 microM) inhibited this P450 enzyme in a dose-dependent manner. Moreover, liarozole treatment caused a significant increase in 1,25-(OH)2D3 half-life from 11 to 31 h. In addition, 1,25-(OH)2D3 can cause homologous up-regulation of the vitamin D receptor (VDR), and in the presence of liarozole, this effect was amplified, thus enhancing 1,25-(OH)2D3 activity. Western blot analyses demonstrated that DU 145 cells treated with 1,25-(OH)2D3/liarozole showed greater VDR up-regulation than cells treated with either drug alone. In summary, our data demonstrate that liarozole augments the ability of 1,25-(OH)2D3 to inhibit DU 145 cell growth. The mechanism appears to be due to inhibition of 24-hydroxylase activity, leading to increased 1,25-(OH)2D3 half-life and augmentation of homologous up-regulation of VDR. We raise the possibility that combination therapy using 1,25-(OH)2D3 and liarozole or other inhibitors of 24-hydroxylase, both in nontoxic doses, might serve as an effective treatment for prostate cancer.

We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.