Ahmed Reda

The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models

COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby determining the outcome of the studies.

Particulate Matter from Both Heavy Fuel Oil and Diesel Fuel Shipping Emissions Show Strong Biological Effects on Human Lung Cells at Realistic and Comparable In Vitro Exposure Conditions

Background Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. Objectives To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Methods Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. Results The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon (“soot”). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Conclusions Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture

STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and students t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

The Gut Microbiota and Developmental Programming of the Testis in Mice

Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.

In vitro spermatogenesis - optimal culture conditions for testicular cell survival, germ cell differentiation, and steroidogenesis in rats

Although three-dimensional testicular cell cultures have been demonstrated to mimic the organization of the testis in vivo and support spermatogenesis, the optimal culture conditions and requirements remain unknown. Therefore, utilizing an established three-dimensional cell culture system that promotes differentiation of pre-meiotic murine male germ cells as far as elongated spermatids, the present study was designed to test the influence of different culture media on germ cell differentiation, Leydig cell functionality, and overall cell survival. Single-cell suspensions prepared from 7-day-old rat testes and containing all the different types of testicular cells were cultured for as long as 31u2009days, with or without stimulation by gonadotropins. Leydig cell functionality was assessed on the basis of testosterone production and the expression of steroidogenic genes. Gonadotropins promoted overall cell survival regardless of the culture medium employed. Of the various media examined, the most pronounced expression of Star and Tspo, genes related to steroidogenesis, as well as the greatest production of testosterone was attained with Dulbecco’s modified eagle mediumu2009+u2009glutamine. Although direct promotion of germ cell maturation by the cell culture medium could not be observed, morphological evaluation in combination with immunohistochemical staining revealed unfavorable organization of tubules formed de novo in the three-dimensional culture, allowing differentiation to the stage of pachytene spermatocytes. Further differentiation could not be observed, probably due to migration of germ cells out of the cell colonies and the consequent lack of support from Sertoli cells. In conclusion, the observations reported here show that in three-dimensional cultures, containing all types of rat testicular cells, the nature of the medium per se exerts a direct influence on the functionality of the rat Leydig cells, but not on germ cell differentiation, due to the lack of proper organization of the Sertoli cells.

Male germ cell development in humans.

Background: Germ cells are unique cells that possess the ability to transmit genetic information between generations. Detailed knowledge about the molecular and cellular mechanisms determining the fate of human male germ cells still remains sparse. This is partially due to ethical issues limiting the access to research material. Therefore, the mechanisms of proliferation, differentiation and apoptosis of human male germ cells still remain challenging study objectives. Methods: This review focuses on using English articles accessible in PubMed as well as personal files on the current knowledge of the molecular and cellular mechanisms connected with human testicular germ cell development, maturation failure and the possibility of fertility preservation in patients in whom there is a risk of gonadal failure. However, since rodents, particularly mice, offer the possibility of studying germ cell development by use of genetic modification techniques, some studies using animal models are also discussed. Conclusion: This mini review focuses on the current knowledge about male germ cells. However, the reader is referred to two previous mini reviews focusing on testicular somatic cells, i.e. on Sertoli cells and Leydig cells.

Differentiation of stem cells upon deprivation of exogenous FGF2: a general approach to study spontaneous differentiation of hESCs in vitro

Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added to the media for 12 weeks. None of these genes are related to the germ lineage, whereas genes for neuronal cells (PAX6 and NR6A1) and endothelial cells (FLT-1 and PTF1A) were up-regulated. To induce and support the differentiation towards the germ lineage we stimulated hESCs with different concentrations of ATRA for 7 and 14 days. We observed no significant difference in gene expression on RNA level when combining all cell lines. Whereas, the overall outcome was negative, one of these cell lines demonstrated an up-regulation of DDX4 on RNA and protein level after 7 days of ATRA stimulation. In summary, our data showed that the lack of exogenous FGF2 results in up-regulation of genes crucial for neuronal and endothelial cell differentiation of hESCs, but not in the up-regulation of genes related to germ cell differentiation when cultured on hFFs. Additionally, we demonstrated that ATRA supplementation did not result in a general specific direction of hESCs towards the germ lineage.

A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures

Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have tried to establish the culture conditions in two laboratories, and to improve the reliability of the culture system to generate mature germ cells. Male mice at three days of age were sacrificed. Testes were cut into small pieces which were cultured atop agarose stands, using Minimum Essential Medium alpha supplemented with different supplements; melatonin, Glutamax, and different concentrations of KSR. The results showed that the duration of culture beyond 18xa0days had an impact on the number of differentiated germ cells. Supplementation with melatonin and Glutamax revealed a positive influence on the efficiency of male germ cell differentiation in vitro. Furthermore, the results confirmed that KSR had a positive effect on germ cell maturation and testosterone production, with a concentration of at least 10%. In conclusion, this study emphasizes the beneficial role of at least 10% KSR in the IVM of germ cells.

Untargeted Identification of Wood Type-Specific Markers in Particulate Matter from Wood Combustion

Residential wood combustion emissions are one of the major global sources of particulate and gaseous organic pollutants. However, the detailed chemical compositions of these emissions are poorly characterized due to their highly complex molecular compositions, nonideal combustion conditions, and sample preparation steps. In this study, the particulate organic emissions from a masonry heater using three types of wood logs, namely, beech, birch, and spruce, were chemically characterized using thermal desorption in situ derivatization coupled to a GCxGC-ToF/MS system. Untargeted data analyses were performed using the comprehensive measurements. Univariate and multivariate chemometric tools, such as analysis of variance (ANOVA), principal component analysis (PCA), and ANOVA simultaneous component analysis (ASCA), were used to reduce the data to highly significant and wood type-specific features. This study reveals substances not previously considered in the literature as meaningful markers for differentiation among wood types.