Maintenance Of Microenvironment Research Articles

Glycosaminoglycans' Ability to Promote Wound Healing: From Native Living Macromolecules to Artificial Biomaterials.

Glycosaminoglycans (GAGs) are important for the occurrence of signaling molecules and maintenance of microenvironment within the extracellular matrix (ECM) in living tissues. GAGs and GAG-based biomaterial approaches have been widely explored to promote in situ tissue regeneration and repair by regulating the wound microenvironment, accelerating re-epithelialization, and controlling ECM remodeling. However, most approaches remain unacceptable for clinical applications. To improve insights into material design and clinical translational applications, this review highlights the innate roles and bioactive mechanisms of native GAGs during in situ wound healing and presents common GAG-based biomaterials and the adaptability of application scenarios in facilitating wound healing. Furthermore, challenges before the widespread commercialization of GAG-based biomaterials are shared, to ensure that future designed and constructed GAG-based artificial biomaterials are more likely to recapitulate the unique and tissue-specific profile of native GAG expression in human tissues. This review provides a more explicit and clear selection guide for researchers designing biomimetic materials, which will resemble or exceed their natural counterparts in certain functions, thereby suiting for specific environments or therapeutic goals.

Mesenchymal stem cells, as glioma exosomal immunosuppressive signal multipliers, enhance MDSCs immunosuppressive activity through the miR-21/SP1/DNMT1 positive feedback loop

BackgroundThe immunosuppressive microenvironment in glioma induces immunotherapy resistance and is associated with poor prognosis. Glioma-associated mesenchymal stem cells (GA-MSCs) play an important role in the formation of the immunosuppressive microenvironment, but the mechanism is still not clear.ResultsWe found that GA-MSCs promoted the expression of CD73, an ectonucleotidase that drives immunosuppressive microenvironment maintenance by generating adenosine, on myeloid-derived suppressor cells (MDSCs) through immunosuppressive exosomal miR-21 signaling. This process was similar to the immunosuppressive signaling mediated by glioma exosomal miR-21 but more intense. Further study showed that the miR-21/SP1/DNMT1 positive feedback loop in MSCs triggered by glioma exosomal CD44 upregulated MSC exosomal miR-21 expression, amplifying the glioma exosomal immunosuppressive signal. Modified dendritic cell-derived exosomes (Dex) carrying miR-21 inhibitors could target GA-MSCs and reduce CD73 expression on MDSCs, synergizing with anti-PD-1 monoclonal antibody (mAb).ConclusionsOverall, this work reveals the critical role of MSCs in the glioma microenvironment as signal multipliers to enhance immunosuppressive signaling of glioma exosomes, and disrupting the positive feedback loop in MSCs with modified Dex could improve PD-1 blockade therapy.Graphical abstract

Roles of the calcified cartilage layer and its tissue engineering reconstruction in osteoarthritis treatment.

Sandwiched between articular cartilage and subchondral bone, the calcified cartilage layer (CCL) takes on both biomechanical and biochemical functions in joint development and ordinary activities. The formation of CCL is not only unique in articular cartilage but can also be found in the chondro-osseous junction adjacent to the growth plate during adolescence. The formation of CCL is an active process under both cellular regulation and intercellular communication. Abnormal alterations of CCL can be indications of degenerative diseases including osteoarthritis. Owing to the limited self-repair capability of articular cartilage and core status of CCL in microenvironment maintenance, tissue engineering reconstruction of CCL in damaged cartilage can be of great significance. This review focuses on possible tissue engineering reconstruction methods targeting CCL for further OA treatment.

Prussian Blue Nanozyme Promotes the Survival Rate of Skin Flaps by Maintaining a Normal Microenvironment.

Ischemia-reperfusion (I/R) injury leads to a low success rate of skin flap transplantation in reconstruction surgery, thus requiring development of new treatments. Necroptosis and apoptosis pathways, along with overexpression of reactive oxygen species and pro-inflammatory factors in skin flap transplantation, are deemed as potential therapeutic targets. This study provides a paradigm for nanozyme-mediated microenvironment maintenance to improve the survival rate of the transplanted skin flap. Prussian blue nanozyme (PBzyme) with multiple intrinsic biological activities was constructed and selected for this proof-of-concept study. The prepared PBzyme shows anti-inflammatory, antiapoptotic, antinecroptotic, and antioxidant activities in both in vitro and in vivo models of I/R injured skin flaps. The multiple inhibitory effects of PBzyme maintained a normal microenvironment and thus significantly promoted the survival rate of the I/R injured skin flap (from 37.21 ± 8.205% to 79.61 ± 7.5%). Of note, PBzyme regulated the expression of the characteristic signal molecules of necroptosis, including Rip 1, Rip 3, and pMLKL, indicating that PBzyme may be a therapeutic agent for necroptosis-related diseases. This study shows great prospects for clinical application of PBzyme in the treatment of skin flaps via local administration.

HnRNPU in Sertoli cells cooperates with WT1 and is essential for testicular development by modulating transcriptional factors Sox8/9.

Background: Sertoli cells are essential regulators of testicular fate in the differentiating gonad; however, its role and underlying molecular mechanism of regulating testicular development in prepubertal testes are poorly understood. Although several critical regulatory factors of Sertoli cell development and function have been identified, identifying extrinsic factors that regulate gonocyte proliferation and migration processes during neonatal testis development remains largely unknown.Methods: We used the Sertoli cell-specific conditional knockout strategy (Cre/Loxp) in mice and molecular biological analyses (Luciferase assay, ChIP-qPCR, RNA-Seq, etc.) in vitro and in vivo to study the physiological roles of hnRNPU in Sertoli cells on regulating testicular development in prepubertal testes.Results: We identified a co-transcription factor, hnRNPU, which is highly expressed in mouse and human Sertoli cells and required for neonatal Sertoli cell and pre-pubertal testicular development. Conditional knockout of hnRNPU in murine Sertoli cells leads to severe testicular atrophy and male sterility, characterized by rapid depletion of both Sertoli cells and germ cells and failure of spermatogonia proliferation and migration during pre-pubertal testicular development. At molecular levels, we found that hnRNPU interacts with two Sertoli cell markers WT1 and SOX9, and enhances the expression of two transcriptional factors, Sox8 and Sox9, in Sertoli cells by directly binding to their promoter regions. Further RNA-Seq and bioinformatics analyses revealed the transcriptome-wide of key genes essential for Sertoli cell and germ cell fate control, such as biological adhesion, proliferation and migration, were deregulated in Sertoli cell-specific hnRNPU mutant testes.Conclusion: Our findings demonstrate an essential role of hnRNPU in Sertoli cells for prepubertal testicular development and testis microenvironment maintenance and define a new insight for our understanding of male infertility therapy.

Observation of Solute Transport between Articular Cartilage and Subchondral Bone in Live Mice.

To establish a method for investigating the permeability of calcified cartilage zone (CCZ) and to observe solute transport between articular cartilage (AC) and subchondral bone (SB) through intact CCZ in vivo. We developed a novel fixing device combined with un-decalcified fluorescence observation method to address the permeability of CCZ in live mice. Twenty-four Balb/c female mice aged 1 to 8 months were used to observe the development of CCZ. Eighty-four Balb/c female mice (aged 1 or 6 months) with mature or immature CCZ of distal femur were used to investigate the permeability of intact CCZ in vivo. Diffusivity of rhodamine B (476 Da) and tetramethyl-rhodamine isothicyanate-dextran (TRITC-Dextran, 20 kDa) was tested from AC to SB in 0 minutes, 1 minute, 15 minutes, 30 minutes, 1 hour, and 2 hours. None diffused knee joints (0 minutes) served as blank control, while in vitro immersion of distal femurs in rhodamine B or TRITC-Dextran for 72 hours served as positive control. CCZ was well developed in 6-month mice. Both tracers penetrated immature CCZ down to SB in less than 1 hour in live mice, while the diffusion of both tracers decreased rapidly at tidemark in all testing time points. Current study provided direct evidence of blocking effect of CCZ in solute transportation during short diffusion period in live animal, indicating the important role of CCZ in joint development and microenvironment maintenance.

Microbiome Conundrum in Colon Cancer: Development, Progression, and Therapeutics.

Colon cancer (CC) is a major global health challenge. Diet, microbiome, obesity, and family history are some of the etiological factors that contribute to the occurrence of CC. Recent investigations have established a strong correlation between colonic microbiota composition and CC. The microbiota protects or damages colonic cells, depending on the type of metabolites and their mechanism of action. Microbiota dysbiosis enhances CC-cell proliferation and promotes metastasis. The microbiota modulates CC progression by epigenetic modifications mediated through either microbial metabolites or structural component interactions with host colon epithelial cells. In addition, gut homeostasis correlates with modulation of host inflammatory and immune responses. This review highlights the microbiome conundrum in tumor growth and metastasis, epigenetic regulation, tumor microenvironment maintenance, microbiome-derived reactive oxygen species in tumor progression, and microbiome-derived metabolites in CC prevention. Understanding the microbiome conundrum in CC development and progression will aid in developing a diet-based therapeutic strategy for colon tumorigenesis management.

Role of angiocrine signals in bone development, homeostasis and disease.

Skeletal vasculature plays a central role in the maintenance of microenvironments for osteogenesis and haematopoiesis. In addition to supplying oxygen and nutrients, vasculature provides a number of inductive factors termed as angiocrine signals. Blood vessels drive recruitment of osteoblast precursors and bone formation during development. Angiogenesis is indispensable for bone repair and regeneration. Dysregulation of the angiocrine crosstalk is a hallmark of ageing and pathobiological conditions in the skeletal system. The skeletal vascular bed is complex, heterogeneous and characterized by distinct capillary subtypes (type H and type L), which exhibit differential expression of angiocrine factors. Furthermore, distinct blood vessel subtypes with differential angiocrine profiles differentially regulate osteogenesis and haematopoiesis, and drive disease states in the skeletal system. This review provides an overview of the role of angiocrine signals in bone during homeostasis and disease.

Abstract P3-04-05: Outliers—extreme long-term survivors with metastatic breast cancer

Abstract Breast cancer has an extremely heterogeneous clinical course, but the specific causes for this diversity are largely unknown. Understanding the basis for extreme long-term survival with incurable cancer can be of value for prognosis or possible interventions if they arise from modifiable factors. Methods: We identified patients at our institution with persistent and distant metastases who are alive greater than 5 years from initial diagnosis, and evaluated the clinical characteristics of this population. Additionally we evaluated the published METABRIC cohort of patients who died of cancer to compare patients with greater than median survival (late) to those who died with less than median survival (early). In doing so, we evaluated the non-exclusive alternative hypotheses that long-survival is dictated by cancer genomes versus by host immunity. To do this, we grouped somatic mutations into 11 common pathways and compared the likelihood of being present in the tumors causing early versus late death, when stratified for hormone receptor [HR] status. To evaluate the effect of immune infiltration, we used gene expression profiles using the CIBERSORT algorithm (Nat Methods 12:453, 2015). Results: We identified 53 individuals who are alive with metastatic breast cancer greater than 5 years from the original diagnosis. The four longest-term survivors were diagnosed with breast cancer in 1978, 1978, 1979, and 1980 and developed metastasis in 1982, 2007, 1996, and 2000 respectively. The median time to metastasis was 4.5 years. Of these, 80% had ER and/or PR-positive breast cancer, 9% triple negative and 18% had HER2+ disease. The longest-term survivors tend to have well-differentiated histology. Analysis of the METABRIC data, demonstrated, as expected, that long survival correlates with hormone-receptor-positive disease. Among 647 subjects who died of cancer, 192 were HR-negative (median survival 36 mo.) and 455 were HR-positive (median survival 76 mo.). Among HR-positive there were no statistical differences in genome profiles between long-versus-short survivors. By contrast, among HR-negative tumors, mutations PI3K-pathway genes and microenvironment maintenance were associated with longer survival (p=0.041 and p=0.047, respectively). CIBERSORT was used to provide quantitative estimates of infiltrating tumor cells, allowing patient groups to be divided by quartiles. However, no significant differences were found in survival among quartiles in T-cell infiltration, in T-cell subsets (activated CD4 memory, CD8, and Tregs) or in monocyte/macrophage subsets. Conclusions: Some patients live for extended periods of time with metastatic breast cancer. Long-term survival of breast cancer can be explained, in part, by the cell-autonomous features of the tumor, including HR status and genes involved in PI3K signaling and microenvironment maintenance, but no major differences were found in immune infiltrates. Although these data are representative of longer-than-median survivors, extreme long-term survivors may have additional unique features in genomic and immune characteristics. Citation Format: Ejebe IH, Denu RA, Longhurst C, Bauman JD, MacGregor S, Lee K, Burkard ME. Outliers—extreme long-term survivors with metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-05.

Sertoli Cells Are Susceptible to ZIKV Infection in Mouse Testis.

Flaviviruses including Dengue virus (DENV), Yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) are global health problems that caused several serious diseases such as fever, hemorrhagic fever, and encephalitis in the past century. Recently, Zika virus (ZIKV) which spreads from Asia to American and causes millions of infections emerges as a new dangerous member of the genus of Flavivirus. Unlike other well-known flaviviruses, ZIKV can be transmitted sexually and infect testes in murine models. Its impacts on sperm functions, and the exact susceptible cells, however, are not entirely clear. To investigate these issues, we infected interferon α/β and γ receptors deficient AG6 mice with ZIKV and examined the outcomes of infection using an assortment of physiological, histopathological, immunological, and virological techniques. We found that infected mice displayed signs of reproductive system disorder, altered androgen levels in serum, and high viral load in semen and testes. Additionally, histopathological examinations revealed marked atrophy of seminiferous tubules and significant reduction in lumen size. Notably, these were accompanied by positive staining of ZIKV antigens on sertoli cells, detection of viral particles and vacuole changes within cytoplasm of sertoli cells. The susceptibility of sertoli cells to ZIKV was further validated in vitro study using cell lines. Importantly, the disruption of tight junctions within testis and altered sperm morphology were also observed in ZIKV infected mice. It is well-known that tight junctions formed by adjacent sertoli cells are major component of blood testis barrier, which plays important roles in maintenance of microenvironment for spermagenesis in testis. Taken together, these results demonstrate that sertoli cells are susceptible to ZIKV infection, which results in the disruption of tight junctions in testis and causes abnormal spermatogenesis in mice. These results also imply that long-term impact of ZIKV infection on human male reproductive system requires close monitoring.

Predominant overexpression of CD25/FOXP3, IFN-γ, and suppressive cytokines in high-grade lesion samples infected with human papillomavirus

Introduction: Human papillomavirus (HPV) persistent infection is the leading cause of cervical cancer and its precursor lesions, and the inappropriate immune response is among the factors that contribute to viral persistence. This may be influenced by regulatory T (Treg) cells and the production of immunosuppressive cytokines, such as transforming growth factor beta (TGF-β) and interleukin-10 (IL-10). Objective: We established the profile of the predominant response, Th1 or immunosuppressive response, in the tissue microenvironment, by detecting interferon-gamma (IFN-γ), TGF-β, and IL-10, as well as the co-expression of IL-2 receptor alpha (CD25) and forkhead box P3 (FOXP3). Methods: Seventy-four samples from uterine cervix biopsies that underwent HPV deoxyribonucleic acid (DNA) detection and histopathology analysis were immunostained to detect CD25/FOXP3, IFN-γ and suppressive cytokines in lymphocytes. Results: The microenvironment of high-grade squamous intraepithelial lesion (HSIL) samples with high numbers of viral particles (≥ 10,000 copies/ml) contained high numbers of CD25/FOXP3+, TGF-β+, IL-10+, and IFN-γ+ cells. Conclusion: The co-expression of CD25/FOXP3 and the expression of TGF-β, and IL-10 in HSIL samples suggest the existence of Treg cells in these locations, although IFN-γ expression was observed in several cells in these samples. Our data suggest that this cytokine could be related to immunosuppressed microenvironment maintenance, favoring the persistent HPV infection and the progression to carcinoma.

Evaluating the methylation status of CXCR4 promoter: A cost-effective and sensitive two-step PCR method

The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region.

Research on control measures for the stability of micro-environmental indicators in clean operating department

Objective To explore the effective measures for the stability of micro-environmental indicators in clean operating department. Methods Professionals of clean technology were invited to participate in unit operation and maintenance of management. Besides, operating room managers conducted regular sampling analysis of five micro-environmental indicators, including temperature, humidity, differential pressure, illumination and decibel, in 100-level, 1 000-level, 10 000-level, 100 000-level clean operating rooms, and compared the test results with parameters of target environmental indicators regulated in national standards. Then the test results of annual bacteria subsidence from different operating rooms in clean operating department were compared. Results Under the management of professionals, the values of temperature, humidity, differential pressure, illumination and decibel from micro-environment in 100-level, 1 000-level, 10 000-level, 100 000-level clean operating rooms were in accordance with rated values in the industry with (P>0.05); the annual bacteria subsidence in 100-level, 1 000-level, 10 000-level, 100 000-level clean operating rooms in 2013 was in accordance with the standards specified in GB50333 and clearly less than rated values. Conclusions Professional participating in the management and maintenance of micro-environment in clean operating department and irregular supervision and sampling test by operating room managers, the micro-environmental indicators in operation department can be well controlled. Key words: Operation room; Clean technology; Micro-environment; Stability of indicators; Control measures

Hiding in Plain Sight: Interplay between Staphylococcal Biofilms and Host Immunity

Staphylococcus aureus and Staphylococcus epidermidis are notable for their propensity to form biofilms on implanted medical devices. Staphylococcal biofilm infections are typified by their recalcitrance to antibiotics and ability to circumvent host immune-mediated clearance, resulting in the establishment of chronic infections that are often recurrent in nature. Indeed, the immunomodulatory lifestyle of biofilms seemingly shapes the host immune response to ensure biofilm engraftment and persistence in an immune competent host. Here, we provide a brief review of the mechanisms whereby S. aureus and S. epidermidis biofilms manipulate host–pathogen interactions and discuss the concept of microenvironment maintenance in infectious outcomes, as well as speculate how these findings pertain to the challenges of staphylococcal vaccine development.

Expression of carbonic anhydrase in the fetal eye and extra-ocular tissues

Carbonic anhydrases (CAs) plays a critical functional role in the ciliary body and retina for maintenance of microenvironment. With immunohistochemistry using orbital contents from 8 human fetuses (12-16 weeks of gestation), we examined expressions of CAs isozymes-1, 2, 3, 6, 7 9 and 12 and found strong reactivity of CA9 in extra-ocular fibrous tissues in the anterior and posterior eyes. CA9 is known to express in the fetal joint cartilage to maintain pH against hypoxia: actually, in the present specimens, the SO pulley and its tendon was strongly positive for CA9. The CA9-positive anterior fibrous tissues were positive for smooth muscle actin and connected the orbital aspect of the 4 rectus muscle with the palpebral conjunctiva, whereas the posterior tissue was negative for smooth muscle actin and corresponded to the lateral insertion tendon of the orbitalis muscle. The anterior CA9-positve tissues seemed to correspond to the primitive form of the sleeve and pulley system. Any of matrix substances (collagen types I and II, aggrecan, versican, fibronectin, tenascin and hyaluronan) displayed a distribution pattern specific for the CA9-positive fibrous tissues. Therefore, whether or not CA9 was positive in the fibrous tissue seemed not to depend on the tissue components such as the extracellular matrix and intermediate filaments but to suggest a stressful condition such as hypoxia, unsuitable base balance and/or under mechanical stress.

Ultrastructure of Anterior Uterus of the Oviduct and the Stored Sperm in Female Soft‐Shelled Turtle,Trionyx sinensis

Ultrastructure of sperm storage in female soft-shelled turtle, Trionyx sinensis was examined under light and electron microscopes. Sperm storage tubules are restricted to the anterior of the uterus. These tubules developed either by folding or fusion of the oviductal mucosal folds and are lined by both ciliated and secretory cells. Ciliated cells are characterized by a few microvilli and prominent cilia in the apical membranes. A prominent feature of the secretory cell is the presence of secretory granules in the supranuclear region. The size, shape, and electron density of these granules vary markedly. The secretory product is released mainly by exocytosis into the oviductal lumen, where it appears as flocculent material. The unique structure in the base of the epithelium, the basal border of the cell -- the basal lamina -- and a blood vessel layer, is presumed to be a important barrier, by which the nourishment exchange and the microenvironment maintenance are ensured. The gland cell is presented with numerous, round, membrane-bound secretory granules of moderate to high electron densities. We divide these granules into three types according to their appearance: (1) membrane bounded granules with high-homogeneous electron density, (2) membrane bounded granules with moderate-homogeneous electron density, (3) membrane bounded, electron dense granules with concentric structures. These granules are presented as different stages of the secretions in the gland cell. The junction complexes are markedly distributed between cells, which are important in keeping stability and the microenvironment maintenance of the sperm storage tubules. Sperm stored in the tubules are heterogeneous in cytology. In addition to the mature sperm in the lumen, sperm with large chromatic granules are found, which are presumed to be immature sperm and are being in the process of nuclear condensation. Several spermatozoa in the tubules are exhibited with definitive indications of degeneration of the nuclei. The nuclear volume increases. The electron density of the central cores in mitochondria declines, combined with the deterioration of concentric membrane structure. Those changes are possibly due to the long time storage of the sperm in sperm storage tubules, and the leakage of reactive oxygen species is suggested to be a major cause. We conclude that the ultrastructure character of sperm storage in the oviduct of Trionyx sinensis is unique, in addition to having a basal function in secretion and the cilia swing, the tubules also provide an available microenvironment for the sperm to long time stored. The degenerative sperm in the tubules might be related to paternity-specific reproductive adaptations, and the sperm competition might occur during long time storage.

Effect of micro-environment maintenance on embryo culture after in-vitro fertilization: comparison of top-load mini incubator and conventional front-load incubator

To investigate the effect of microenvironment maintenance on embryo culture and clinical results using two types of incubators. Temperature and oxygen concentration in a mini-incubator and a conventional incubator were compared following a 5-s door opening/closing procedure. Embryos of 30 in-vitro fertilization embryo transfer (IVF-ET) cases were randomly allocated to either one of the incubator, cultured, and the early-stage good embryo formation rate and the good blastocyst formation rate were compared, as indicators for micro-environment maintenance ability. Temperature recovery after a 5-s door opening/closing procedure was approximately 5 min for the mini-incubator and 30 min for the conventional incubator. The oxygen concentration return was significantly improved in the mini-incubator (3.0 +/- 0 min) compared with the conventional incubator (7.8 +/- 0.9 min). Both the early-stage good embryo formation rate and the good blastocyst formation rate were significantly higher in the mini-incubator (39.5% and 15.1%) than the conventional incubator (28.4% and 7.8%). The microenvironment maintenance ability of incubators appears to significantly influence the formation of good embryos.

Whole-cell recording from non-dissociated taste cells in mouse taste bud

A method for the whole-cell recording from non-dissociated taste cells within mouse taste bud is described. The lingual epithelial sheet containing the taste buds was peeled free from the tongue by injecting a proteolytic enzyme, elastase, under the lingual epithelium and by incubating it in normal Tyrode solution at 30°C. The preparation consisting of a taste bud and a small piece of the lingual epithelium was obtained by further the incubation in divalent cation-free Tyrode solution. After holding the small piece of the epithelium by a holding pipette loaded with continuous negative pressure for keeping the orientation of the taste bud, whole-cell configuration was established in a non-dissociated taste cell within the taste bud with a patch pipette containing Lucifer Yellow. Taste stimuli or blockers were applied from the third pipette placed near the taste pore under the continuous flow of bathing solution. Under this condition, we could simultaneously accomplish patch-clamping, visualization of taste cell morphology, localized taste stimulation and maintenance of microenvironment around the taste organ. Rapid responses to a relatively high concentration of salt stimuli were also obtained.