Influenza is one of the most common causes of virus diseases worldwide. Virus detection requires determination of Influenza RNA in the upper respiratory tract. Efficient screening is not possible in this way. Analysis of volatile organic compounds (VOCs) in breath holds promise for non-invasive and fast monitoring of disease progression. Breath VOC profiles of 14 (3 controls and 11 infected animals) swine were repeatedly analyzed during a complete infection cycle of Influenza A under high safety conditions. Breath VOCs were pre-concentrated by means of needle trap micro-extraction and analysed by gas chromatography mass spectrometry before infection, during virus presence in the nasal cavity, and after recovery. Six VOCs could be related to disease progression: acetaldehyde, propanal, n-propyl acetate, methyl methacrylate, styrene and 1,1-dipropoxypropane. As early as on day four after inoculation, when animals were tested positive for Influenza A, differentiation between control and infected animals was possible. VOC based information on virus infection could enable early detection of Influenza A. As VOC analysis is completely non-invasive it has potential for large scale screening purposes. In a perspective, breath analysis may offer a novel tool for Influenza monitoring in human medicine, animal health control or border protection.

Influenza A is a serious pathogen itself, but often leads to dangerous co-infections in combination with bacterial species such asStreptococcus pyogenes. In comparison to classical biochemical methods, analysis of volatile organic compounds (VOCs) in headspace above cultures can enable destruction free monitoring of metabolic processesin vitro. Thus, volatile biomarkers emitted from biological cell cultures and pathogens could serve for monitoring of infection processesin vitro. In this study we analysed VOCs from headspace above (co)-infected human cells by using a customized sampling system. For investigating the influenza A mono-infection and the viral-bacterial co-infectionin vitro, we analysed VOCs from Detroit cells inoculated with influenza A virus andS. pyogenesby means of needle-trap micro-extraction (NTME) and gas chromatography mass spectrometry (GC-MS). Besides the determination of microbiological data such as cell count, cytokines, virus load and bacterial load, emissions from cell medium, uninfected cells and bacteria mono-infected cells were analysed. Significant differences in emitted VOC concentrations were identified between non-infected and infected cells. After inoculation withS. pyogenes, bacterial infection was mirrored by increased emissions of acetaldehyde and propanal. N-propyl acetate was linked to viral infection. Non-destructive monitoring of infections by means of VOC analysis may open a new window for infection research and clinical applications. VOC analysis could enable early recognition of pathogen presence and in-depth understanding of their etiopathology.

Background: The healing of a bone injury is a highly complex process involving a multitude of different tissue and cell types, including immune cells, which play a major role in the initiation and progression of bone regeneration. Methods: We histologically analyzed the spatio-temporal occurrence of cells of the innate immune system (macrophages), the adaptive immune system (B and T lymphocytes), and bone cells (osteoblasts and osteoclasts) in the fracture area of a femoral osteotomy over the healing time. This study was performed in a bone osteotomy gap mouse model. We also investigated two key challenges of successful bone regeneration: hypoxia and revascularization. Results: Macrophages were present in and around the fracture gap throughout the entire healing period. The switch from initially pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype coincided with the revascularization as well as the appearance of osteoblasts in the fracture area. This indicates that M2 macrophages are necessary for the restoration of vessels and that they also play an orchestrating role in osteoblastogenesis during bone healing. The presence of adaptive immune cells throughout the healing process emphasizes their essential role for regenerative processes that exceeds a mere pathogen defense. B and T cells co-localize consistently with bone cells throughout the healing process, consolidating their crucial role in guiding bone formation. These histological data provide, for the first time, comprehensive information about the complex interrelationships of the cellular network during the entire bone healing process in one standardized set up. With this, an overall picture of the spatio-temporal interplay of cellular key players in a bone healing scenario has been created. Conclusions: A spatio-temporal distribution of immune cells, bone cells, and factors driving bone healing at time points that are decisive for this process—especially during the initial steps of inflammation and revascularization, as well as the soft and hard callus phases—has been visualized. The results show that the bone healing cascade does not consist of five distinct, consecutive phases but is a rather complex interrelated and continuous process of events, especially at the onset of healing.

The interaction of hematopoietic cells and the bone microenvironment to maintain bone homeostasis is increasingly appreciated. We hypothesized that the transfer of allogeneic T lymphocytes has extensive effects on bone biology and investigated trabecular and cortical bone structures, the osteoblast reconstitution, and the bone vasculature in experimental hematopoietic stem cell transplantations (HSCT). Allogeneic or syngeneic hematopoietic stem cells (HSC) and allogeneic T lymphocytes were isolated and transferred in a murine model. After 20, 40, and 60 days, bone structures were visualized using microCT and histology. Immune cells were monitored using flow cytometry and bone vessels, bone cells and immune cells were fluorescently stained and visualized. Remodeling of the bone substance, the bone vasculature and bone cell subsets were found to occur as early as day +20 after allogeneic HSCT (including allogeneic T lymphocytes) but not after syngeneic HSCT. We discovered that allogeneic HSCT (including allogeneic T lymphocytes) results in a transient increase of trabecular bone number and bone vessel density. This was paralleled by a cortical thinning as well as disruptive osteoblast lining and loss of B lymphocytes. In summary, our data demonstrate that the adoptive transfer of allogeneic HSCs and allogeneic T lymphocytes can induce profound structural and spatial changes of bone tissue homeostasis as well as bone marrow cell composition, underlining the importance of the adaptive immune system for maintaining a balanced bone biology.

Therapeutic interventions for diseases such as leukemia and autoimmune disorders are increasingly designed to selectively target and deplete specific immune cell subsets over prolonged periods. This can disrupt the homeostasis of the adaptive immune system, with consequences not only for peripheral blood but also for the “cradle of the immune system”, the bone marrow. This study hypothesized that such immune imbalance due to immune cell depletion therapies impairs the differentiation capacity of mesenchymal stromal cells (MSCs), which are key progenitors of bone cells and are thus essential for maintaining bone health. To validate this hypothesis, a series of cell culture experiments were conducted in which MSCs were stimulated with immune-conditioned media derived from various immune cell subsets. A comprehensive analytical approach was employed to evaluate the differentiating MSCs, including their associated supernatants, deposited collagen, mineralized matrix, and lipid deposition at defined time points during their maturation. Therefore, fluorescence and nonfluorescent histological staining, enzyme-linked immunosorbent assays (ELISAs), and expression analyses with Ribonucleic acid (RNA) were performed. This study demonstrated that, compared with immune-conditioned media from T cells or peripheral blood mononuclear cells (PBMCs), B cell-immune-conditioned media enriched with interleukin 4, bone morphogenetic protein 2 and Dickkopf 1 enhanced the osteogenic differentiation of MSCs in vitro. This positive osteogenic effect is driven primarily by elevated cytokine secretion by stimulated B cells, which in vivo in turn potentially stimulate bone turnover and modulate immune system function. In summary, these data demonstrate an pro-osteogenic and pro-adipogenic influence of B cells on differentiating MSCs in vitro, suggesting that an imbalance in immune cells in the bone marrow perturbs bone homeostasis.