Tissue Suspensions Research Articles

Glioma stem cells reconstruct similar immunoinflammatory microenvironment in different transplant sites and induce malignant transformation of tumor microenvironment cells.

This study aimed to examine whether the different tumor-transplanted sites could construct a similar immunoinflammatory microenvironment and to investigate the interactions between tumor microenvironment cells. The red fluorescent protein-SU3 (SU3-RFP) or SU3 glioma stem cells (GSC) were inoculated into the brain, liver, abdominal cavity, and subcutis of green fluorescent protein (GFP)-nude mice. The tumor tissues were taken to observe the tissue cell distribution. The single cell suspension of tumor tissues was prepared and cultured, while the SU3-RFP cells were co-cultured with the cells from GFP-transgenic mice. The RFP+, GFP+, and RFP+/GFP+ cells were traced by fluorescence microscope, and their protein expressions were determined by Western blot analysis. The markers of immunoinflammatory cells, including F4/80, CD11b, CD11c, CD80, CD47, and SIRP-α, were determined by RT-PCR and immunocytochemistry assays, respectively. The xenograft models of all transplant sites were inducible, and the red tumor cells of tumor tissues were encircled by a great quantity of host-derived green cells, including immunoinflammatory cells with CD80, F4/80, CD11b, and CD11c expressions, which might generate the cell colonies and possess the pseudopodia. Additionally, the interactions between red tumor cells and green immunoinflammatory cells, including cell fusion process and yellow fusion cell formation, were observed in cultured cells. The fusion cells-derived B4 cells with expressions of CD47 and SIRP-α proteins had the strong proliferation ability and tumorigenic effect. The similar tumor immunoinflammatory microenvironment was constructed by GSC in different transplant sites, and the cell fusion indicated a malignant transformation of the tumor microenvironment cells.

MOLECULAR CHARACTERIZATION OF EQUINE HERPES VIRUS -1 (EHV-1) IN EGYPT

Equine Herpes Virus -1 is one of the most viruses affecting equide family(Horse, Donkey &Mule) causing severe economic losses to horse industry all over the world, due to respiratory manifestations, abortion of pregnant mares ,and mayeloencephalopathy. Little reports have been made for investigation the prevalence and existence of equine herpes viruses (EHVs) in Egypt. In the present study, seventy clinical samples (6 placentas & 64 nasal swabs) collected from different governorate in Egypt. Virus isolation by inoculating tissue suspensions into MDBK cells revealed characteristic CPE for EHV after the fourth passage within 7 days from inoculation in the form of aggregation of the cells together, rounding, and finally cell detachment. Serological confirmation of the isolated sample by FAT revealed intracytoplasmic greenish yellow fluorescence. DNA extraction and PCR of suspected samples existed Successful amplification of 700 bp of UL45 gene of EHV-1 in 8 out of 70 examined samples.

A novel group A rotavirus associated with acute illness and hepatic necrosis in pigeons (Columba livia), in Australia.

Cases of vomiting and diarrhoea were reported in racing pigeons in Western Australia in May, 2016. Morbidity and mortality rates were high. Similar clinical disease was seen in Victoria in December and by early 2017 had been reported in all states except the Northern Territory, in different classes of domestic pigeon–racing, fancy and meat bird–and in a flock of feral pigeons. Autopsy findings were frequently unremarkable; histological examination demonstrated significant hepatic necrosis as the major and consistent lesion, often with minimal inflammatory infiltration. Negative contrast tissue suspension and thin section transmission electron microscopy of liver demonstrated virus particles consistent with a member of the Reoviridae. Inoculation of trypsin-treated Vero, MDBK and MA-104 cell lines resulted in cytopathic changes at two days after infection. Next generation sequencing was undertaken using fresh liver samples and a previously undescribed group A rotavirus (genotype G18P[17]) of avian origin was identified and the virus was isolated in several cell lines. A q-RT-PCR assay was developed and used to screen a wider range of samples, including recovered birds. Episodes of disease have continued to occur and to reoccur in previously recovered lofts, with variable virulence reported. This is the first report of a rotavirus associated with hepatic necrosis in any avian species.

Enhancing Facelift With Simultaneous Submalar Implant Augmentation.

The midface is particularly prone to the senescent changes of soft tissue ptosis and volume loss, which in individuals with aging or low adiposity can manifest as submalar hollowing. Facelift alone in those with submalar hollowing inadequately addresses the volume loss and may result in a gaunt appearance postoperatively. Submalar implant augmentation is a powerful tool for permanent midface volume restoration for a more youthful and natural contour, as opposed to soft tissue fillers that diminish over time. When performed together, submalar augmentation and facelift synergistically enhance facial rejuvenation results. Determine the long-term safety and efficacy of submalar implant augmentation as an adjunct to facelift. Retrospective review evaluating results and complications in all consecutive patients who had submalar implant augmentation with SMAS-plication facelift in a single surgeon private practice setting from January 1, 1991, to December 31, 2017. Forty-eight patients underwent submalar augmentation with simultaneous facelift with an overall satisfaction rate of 95.7%. Complications included 2.1%transient infraorbital hypoesthesia, 1.1% prolonged swelling, and 1.1% capsular contraction that required a minor adjustment under local anesthesia. No infection, implant migration, or extrusion or facial nerve injury occurred. Submalar implant augmentation is a safe and effective means of enhancing facelift results through midface volume restoration, subperiosteal release, and improved soft tissue suspension in a more favorable vector. Submalar implant augmentation performed simultaneously with facelift may be an attractive alternative to repeated soft tissue filler or fat injections for patients with submalar hollowing who are interested in facial rejuvenation surgery.

The immune functions of sessile hemocytes in three organs of kuruma shrimp Marsupenaeus japonicus differ from those of circulating hemocytes

Shrimp, as invertebrates, have an open vasculature that allows circulating hemocytes to infiltrate the tissues, where they are referred to as sessile hemocytes. Sessile hemocytes are known to express immune-related genes, but it is not known whether their functions differ from those of circulating hemocytes. To answer this question, we enriched them from suspensions of different tissues using discontinuous density gradient centrifugation and analyzed their transcripts by RNA-seq. The results suggest that circulating hemocytes and sessile hemocytes of the gills are in a state that could react quickly to pathogens, immune-related genes expression of sessile hemocytes differ from circulating hemocytes, and the gills, heart and lymphoid organs have cells that express immune-related genes that are different from hemocytes.

Activated innate lymphoid cell populations accumulate in human tumour tissues

BackgroundInnate lymphoid cells (ILC) are part of a heterogeneous family of haematopoietic effector cells which lack re-arranged antigen-specific receptors. They promote host defense and contribute to tissue and metabolic homeostasis, wound healing and immune surveillance. Their role in human cancer immunity is less defined, and therefore we aimed to identify the frequency and phenotype of distinct ILC groups in various types of cancer.MethodsTissue samples and peripheral blood were collected from patients undergoing surgical resection of gastrointestinal and breast tumours. Single cell suspension of tumour tissue was immediately obtained following surgery using tumour dissociation.ResultsWe observed significantly higher frequencies of ILC2 (p value: 0.04) in malignant breast cancer tissue and significantly higher frequencies of group 1 ILC (p value: 0.001) in malignant gastrointestinal tumours. Tumour infiltrating ILC were found to show an activated phenotype with higher expression of MHC-II, KLRG1, early activation marker CD69 and CD44.ConclusionsActivated innate lymphoid cells infiltrate tumours dependent on tumour type and location.

Extended Transconjunctival Lower Eyelid Blepharoplasty with Release of the Tear Trough Ligament and Fat Redistribution

Sir: In the August of 2017 issue of Plastic and Reconstructive Surgery, Wong and Mendelson published an interesting article in which the authors described in detail their methods of subtotally releasing the tear trough ligament and the orbicularis retaining ligament and redistributing the free orbital fat for the correction of the tear trough (lacrimal groove, nasojugal crease) and palpebromalar groove (lid-cheek junction) through the transconjunctival approach.1 Because the authors and we have similar treatment subjects (mainly Asians), and the periorbital rejuvenation is a subject on which we have worked intensively, we would like to share our thoughts about this technique. Wong and Mendelson have been consistently committed to studies of the anatomy of the periorbital region and midcheek and the antiaging treatments of the middle face.1–4 Their research achievements and surgical techniques of course contribute to better understanding the structures of these regions and choosing the optimum surgical procedure. The tear trough deformity is considered to be directly associated with the tear trough ligament, and the formation of the palpebromalar groove is mainly attributable to the orbicularis retaining ligament.2 It is also well known that the degree of infraorbital groove depends on the status of the tissue above (i.e., eye protrusion, prominence of the orbital fat pads, and looseness of the orbicularis oculi muscle and skin) and below (i.e., atrophy and descent of the malar fat pad and retrusion of maxillary) this furrow. Once the groove is seen, does it have to deal with the region? Everyone has the structure of the tear trough ligament (although it may be fragile and thus invisible), and almost all of the patients with eye bags have different degrees of prominence of the nasojugal crease. With these factors in mind, it is important for us to classify the tear trough deformity according to the morphologic features of the lacrimal groove and the situations of the surrounding tissue (because of the limit of article length, this section is not described here in detail). In other words, a judgment of which factor (i.e., the tear trough ligament, the surrounding tissues, or a combination of them) plays the major role in increasing the prominence of the tear trough must be correctly made preoperatively. In the article by Wong and Mendelson,1 the extended transconjunctival lower eyelid blepharoplasty with ligament release was performed on the patient (shown in Fig. 11) who had a mild eye bag and a slight tear trough even when smiling on the frontal view. According to our experience, we usually attribute the formation of this type of crease to the protruded orbital fat compartment. We do believe that transconjunctival blepharoplasty only with orbital fat removal can achieve a similar effect. The tear trough deformity that is visible in the static condition should be treated by ligament release, as shown in Figures 6 through 10. However, the surgical approach should be appropriately selected based on the laxity of the orbicularis oculi muscle and skin. Thus, we think that a transcutaneous approach may be preferential for the patient shown in Figure 10. Skin removal and tightening/suspending the orbicularis oculi can be performed simultaneously for better cosmetic outcome. It has been widely accepted that lifting is an essential step after releasing the tear trough ligament and orbicularis retaining ligament for treatment of the aging middle face, and that tissue suspension or repositioning seems unnecessary following the release of the two ligaments when aiming at correcting the periorbital groove. Although releasing the ligament does not lead to tissue ptosis in young patients or in short-term follow-up (as most of the cases presented in previously published articles), it is uncertain whether such a release procedure without suspension or repositioning will contribute to tissue ptosis over a long observation period such as 10 or 20 years. After all, a ligament, although it may create a depression or a crease/groove, may play a role in balancing the related tissue and preventing ptosis of tissue. DISCLOSURE The authors declare no potential conflicts of interest with the respect to the research, authorship, or publication. Qianwen Wang, M.D., Ph.D.Jiaqi Wang, M.D.Head & Neck Cosmetic Surgery CenterPlastic Surgery HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing, People’s Republic of China

Astrogliosis has Different Dynamics after Cell Transplantation and Mechanical Impact in the Rodent Model of Parkinson's Disease.

Background:Transplantation of fetal mesencephalic tissue is a well-established concept for functional reinnervation of the dopamine-depleted rat striatum. However, there is no extensive description of the glial response of the host brain following this procedure.Aims:The present study aimed to quantitatively and qualitatively analyse astrogliosis surrounding intrastriatal grafts and compare it to the reaction to mechanical injury with the transplantation instrument only.Study Design:Animal experimentation.Methods:The standard 6-hydroxydopamine-induced unilateral model of Parkinson’s disease was used. The experimental animals received transplantation of a single-cell suspension of E14 ventral mesencephalic tissue. Control animals (sham-transplanted) were subjected to injury by the transplantation cannula, without injection of a cell suspension. Histological analyses were carried out 7 and 28 days following the procedure by immunohistochemistry assays for tyrosine hydroxylase and glial fibrillary acidic protein. To evaluate astrogliosis, the cell density and immunopositive area were measured in distinct zones within and surrounding the grafts or the cannula tract.Results:Statistical analysis revealed that astrogliosis in the grafted striatum increased from day 7 to day 28, as shown by a significant change in both cell density and the immunopositive area. The cell density increased from 816.7±370.6 to 1403±272.1 cells/mm2 (p<0.0001) аnd from 523±245.9 to 1164±304.8 cells/mm2 (p<0.0001) in the two zones in the graft core, and from 1151±218.6 to 1485±210.6 cells/mm2 (p<0.05) for the zone in the striatum immediately adjacent to the graft. The glial fibrillary acidic protein-expressing area increased from 0.3109±0.1843 to 0.7949±0.1910 (p<0.0001) and from 0.1449±0.1240 to 0.702±0.2558 (p<0.0001) for the same zones in the graft core, and from 0.5277±0.1502 to 0.6969±0.1223 (p<0.0001) for the same area adjacent to the graft zone. However, astrogliosis caused by mechanical impact only (control) did not display such dynamics. This finding suggests an influence of the grafted cells on the host’s glia, possibly through cross-talk between astrocytes and transplanted neurons.Conclusion:This bidirectional relationship is affected by multiple factors beyond the mechanical trauma. Elucidation of these factors might help achieve better functional outcomes after intracerebral transplantation.

Establishment of a murine breast tumor model by subcutaneous or orthotopic implantation.

A number of murine models are used to mimic the pathology of breast cancer. Tissue inoculation and cell inoculation using orthotopic implantation (OS) and subcutaneous implantation (SQ) are commonly used to generate murine models to investigate cancer. However, limited information is available in regard to the variations of these methods. The present study compared growth, metastasis, survival and histopathology of tumors produced using OS and SQ to characterize features of the tumors produced by the two distinct methods. Additionally, the present study aimed at providing increased options for investigators when designing experiments. 4T1-luc2 cell suspension or 4T1-luc2 tissue suspension was inoculated using either OS or SQ into BALB/c mice. Tumor growth and metastasis were detected using an in vivo imaging system and calipers. Excised tumors and lung were assessed by tissue staining with hematoxylin and eosin, and the vessel marker cluster of differentiation 31. The results of the present study revealed that the cell suspension generated breast tumors of increased size, which was visualized and determined, following inoculation, using calipers at an earlier time point compared with tumors produced by tissue suspension. The increasing bioluminescent trend of OS tumors was more marked compared with that of SQ tumors. The volume of OS tumor was increased with decreased variation, compared with that of SQ tumors. In addition, the OS tumor exhibited increased microvessel density. Bioluminescent signals and histological results in regard to metastasis were consistent: OS implantation produced increased lung metastasis compared with that of SQ implantation, although they exhibited similar survival times. The results of the present study indicated that the inocula from distinct sources (tissue or cell) affected tumor growth. Furthermore, breast tumor progression and histopathological characteristics were distinct between OS and SQ, whereas OS exhibited increased malignant behavior. Understanding the characteristics of murine breast cancer models established by diverse methods may aid investigators to select appropriate animal models, according to the requirements of the study.

Involvement of breast cancer stem cells in tumor angiogenesis.

The aim of the present study was to investigate the role of breast cancer stem cells (BCSCs) in the angiogenesis of breast cancer tumors. The expression levels of mutant p53, cluster of differentiation (CD)31, vascular endothelial factor (VEGF), in addition to human epidermal growth factor (HER)2, were detected in the blood vessels of human breast cancer (BC) tissue samples. CD44+/CD24-/low cells were selected from single-cell suspensions of BC tissues to assess the expression of CD31 and CD105, in addition to the ability of these cells to metabolize acetylated low-density lipoprotein (Ac-LDL). Furthermore, vascular-like structures were observed histologically. Mutant p53, CD31 and VEGF were all expressed in these tissues. CD44+ cells comprised 7.5±2.6 and 94.3±4.7% of the cell population prior to and following sorting, respectively. CD24+ cells comprised 48.2±9.4 and 4.3±4% of the cell population prior to and following sorting, respectively. A low proportion of CD24+ cells corresponded to a high proportion of CD24-/low cells. The percentages of CD105+ and CD31+ glomus cells in the mammary gland were 4.5±0.9 and 6.2±1.3%, respectively, and following passaging for three generations, these increased to 79.6±9.3 and 84.1±10.7%, respectively (P<0.05). Cells were cultured using an endothelial cell culture system, and they internalized DiL-Ac-LDL. Here, vascular endothelial cells formed vascular-like structures, whereas the control group demonstrated no such structures. Overall, the results suggest that BCSCs-derived endothelial cells may contribute to tumor angiogenesis.

Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform.

SummaryEffective control and monitoring of foot‐and‐mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype‐specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD‐endemic countries have motivated the development of simple diagnostic platforms to support local decision‐making. Using a portable thermocycler, the T‐COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan‐serotype‐specific real‐time RT‐PCR (rRT‐PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa‐specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan‐serotype‐specific lyophilized assay were comparable to that of an OIE‐recommended laboratory‐based rRT‐PCR (determined using a panel of 57 FMDV‐positive samples and six non‐FMDV vesicular disease samples for differential diagnosis). The FMDV‐typing assay was able to correctly identify the serotype of 33/36 FMDV‐positive samples (no cross‐reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre‐clinical, clinical and clinically recovered cattle. These data support the use of field‐ready rRT‐PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

Abstract LB-199: Imprime PGG modulates immunosuppressive myeloid components of the tumor microenvironment and drives enhanced antitumor efficacy in combination with checkpoint inhibitor therapies

Abstract Checkpoint inhibitor therapies (CPI) have shown great promise, however in a limited percentage of patients. One of the key mechanisms behind the limited efficacy of CPI therapy is immune resistance mediated by immunosuppressive myeloid cells at the tumor microenvironment (TME), namely M2 macrophages and myeloid-derived suppressor cells (MDSC). Multiple therapeutic interventions are being developed to target these cell types with the intention of reshaping the TME and enhancing the effector functions of the infiltrating cytotoxic T cells.Molecules containing pathogen associated molecular patterns (PAMPS) are one of the unique combination partners that can sensitize tumors to respond to CPI. Imprime PGG (Imprime), an intravenously administered soluble yeast β-1,3/1,6 glucan PAMP, is being clinically developed in combination with tumor-targeting antibodies, anti-angiogenics, and CPI. Imprime has shown promising results in two randomized phase 2 studies in non-small cell lung cancer. Mechanistic studies have shown Imprime to repolarize M2 macrophages and MDSC in in vitro human systems as well as multiple xenograft models. The objective of this study was to evaluate Imprime’s ability to counteract immunosuppression and thereby influence the effector functions of T cells in a syngeneic tumor model. To this end, we first evaluated the anti-tumor efficacy of Imprime in combination with anti-PD-1 or anti-PD-L1 antibody in the MC-38 colon cancer model and found that both combinations repressed tumor growth more effectively than either single agent. Flow cytometric evaluation of single cell suspensions of spleen and tumor tissue after one week of Imprime dosing revealed that the tumor associated macrophages showed a shift to an M1-like phenotype with increased expression of PD-L1, CD86, inducible nitric oxide synthase, MHC class II and downmodulation of Arginase-1. qRT-PCR analyses also demonstrated an increase in transcripts for M1 markers (Il12b p35, Ifng, Tnfa) with a coincident decrease in transcripts for M2 markers (VEGF, Fizz1, CCL17). Adaptive immune resistance, increased PD-L1 expression on the tumor cells as a result of immune activation was observed. In the spleen, the monocytic MDSC also showed increased expression of M1 markers. Furthermore, increased number of CD8 cells in the spleen were of effector memory phenotype with enhanced expression of PD-1, granzyme B, and Ki-67. At the tumor site, the CD8 cells from Imprime treated mice demonstrated increased proliferative and cytokine producing capabilities (IL-2, IFNg, and TNFa) in response to CD3/CD28 stimulation. Collectively, these data show that Imprime’s ability to remold the TME such that the myeloid cells are less suppressive and the cytotoxic T cells are more functionally active can have a tremendous impact on overcoming resistance to CPI therapy. Citation Format: Adria B. Jonas, Anissa SH Chan, Xiaohong Qiu, Kathryn Fraser, Nadine Ottoson, Takashi Kangas, Richard Walsh, Steven M. Leonardo, Ross Fulton, Keith Gorden, Mark Uhlik, Jeremy Graff, Nandita Bose. Imprime PGG modulates immunosuppressive myeloid components of the tumor microenvironment and drives enhanced antitumor efficacy in combination with checkpoint inhibitor therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-199. doi:10.1158/1538-7445.AM2017-LB-199

Stem cells from human hair follicles: first mechanical isolation for immediate autologous clinical use in androgenetic alopecia and hair loss.

Hair follicles are known to contain a well-characterized niche for adult stem cells: the bulge, which contains epithelial and melanocytic stem cells. Stem cells in the hair bulge, a clearly demarcated structure within the lower permanent portion of hair follicles, can generate the interfollicular epidermis, hair follicle structures, and sebaceous glands. The bulge epithelial stem cells can also reconstitute in an artificial in vivo system to a new hair follicle. In this study, we have developed a new method to isolate human adult stem cells by mechanical centrifugation of punch biopsy from human hair follicles without culture condition. Here, we used human follicle stem cells (HFSCs), to improve the hair density in 11 patients (38 to 61 years old) affected by AGA in stage 3-5 as determined by the Norwood-Hamilton classification scale. The primary outcomes were microscopic identification and counting of HFSCs. The secondary outcomes were clinical preliminary results and safety and feasibility in HFSCs-treated scalp. Each scalp tissue suspension contained about 3,728.5±664.5 cells. The percentage of hair follicle-derived mesenchymal stem cells CD44+ [from dermal papilla (DP)] was about 5%+0.7% whereas the percentage of hair follicle epithelial stem cells CD200+ (from the bulge) was about 2.6%+0.3%. In total, 23 weeks after the last treatment with HFSCs mean hair count and hair density increases over baseline values. In particular, a 29%±5% increase in hair density for the treated area and less than a 1% increase in hair density for the placebo area. We have shown that the isolated cells are capable to improve the hair density in patients affected by androgenetic alopecia (AGA). These cells appear to be located in the bulge area of human.

Real-time computed tomography fluoroscopy-guided solitary lung tumor model in a rabbit.

Preclinical studies of lung cancer require suitable large-animal models to allow evaluation and development of surgical and interventional techniques. We assessed the feasibility and safety of a novel rabbit lung cancer model of solitary tumors, in which real-time computed tomography fluoroscopy is used to guide inoculation of VX2 carcinoma single-cell suspensions. Thirty-eight rabbits were divided into four groups according to the volume of the VX2 tissue or cell suspension, the volume of lipiodol, the volume of Matrigel, and the injection needle size. The mixtures were percutaneously injected into rabbit lungs under real-time computed tomography fluoroscopy guidance. Two weeks later, VX2 lung carcinomas were confirmed via positron emission tomography/computed tomography, necropsy, and histology. Real-time computed tomography fluoroscopy allowed the precise inoculation of the tumor cell suspensions containing lipiodol, while the use of Matrigel and a small needle prevented leakage of the suspensions into the lung parenchyma. Solitary lung tumors were successfully established in rabbits (n = 22) inoculated with single-cell suspensions (150 μL), lipiodol (150 μL), and Matrigel (150 μL) using a 26-gauge needle. This combination was determined to be optimal. Pneumothorax was observed in only two of the 38 rabbits (5.3%), both of which survived to the end of the study without any intervention. Real-time computed tomography fluoroscopy-guided inoculation of VX2 single-cell suspensions with lipiodol and Matrigel using a small needle is an easy and safe method to establish solitary lung tumors in rabbits.

Smooth Muscle PPARγ Mutation Causes Salt‐sensitive Hypertension

Systemic loss of peroxisome proliferator‐activated receptor‐γ (PPARγ) function causes hypertension, whereas its activation lowers blood pressure largely via PPARγ activity in the vasculature. We have recently shown that loss of PPARγ in smooth muscle cells increases mesenteric myogenic tone, promotes vascular remodeling, and predisposes to deoxycorticosterone acetate (DOCA)‐salt‐induced hypertension in mice. However, it is unclear whether these effects were attributable to altered salt sensitivity. In this study, we examined the role of smooth muscle PPARγ in regulating renal function and salt‐sensitivity. Transgenic mice expressing dominant negative PPARγ in smooth muscle cells (S‐P467L) and non‐transgenic littermates (NT) were fed normal chow diet (containing 0.3% salt) or a 4% salt diet for a total of 8 weeks and metabolic cage studies were performed at baseline, 3 weeks, 6 weeks, and at the end of the protocol. S‐P467L and NT mice had similar food intake, feces weight, and weight gain on high salt diet throughout the study. While 24‐hour water intake tripled in both strains (6.3 ± 0.21 mL S‐P467L vs. 5.9 ± 0.40 mL NT, n=7) when they were switched to high salt diet from normal chow, S‐P467L mice excreted 32% less urine in the third week (24‐hour urine 1.96 ± 0.13 mL S‐ P467L vs. 2.90 ± 0.17 mL NT, two‐way ANOVA p&lt;0.01). In order to assess renal function, mice were subjected to a bolus i.p. injection of normal saline equal to 10% of their body weight and the urine volume excreted in the subsequent 4 hours were plotted as a percentage of saline injected. We observed a marked decline in their capacity to excrete this volume challenge in S‐ P467L mice (28.2 ± 2.1% S‐P467L vs. 39.9 ± 5.1% NT, p&lt;0.05) in the third week of high salt diet. Because nitric oxide (NO) is known to regulate sodium and water balance, we determined daily renal NO production as indicated by 24‐hour urinary nitrate/nitrite. Three‐week dietary salt induced a 4‐fold increase in urinary NO metabolites in NT mice, but this was blunted in S‐P467L mice (2.82 ± 0.19 μmoL/24 hours S‐P467L vs. 4.42 ± 0.27 μmoL/24 hours NT, p&lt;0.01). In parallel with these renal phenotypes, S‐P467L mice developed marked elevation of blood pressure that peaked in the third week of high salt feeding (telemetry systolic pressure 136.4 ± 3.4 mmHg S‐P467L vs. 124.2 ± 2.4 mmHg NT, p&lt;0.01) and remained elevated throughout the study. At the end of 8 weeks, vascular function was assessed in freshly isolated carotid and basilar arteries, and flow cytometry analysis was performed in single cell suspensions of aorta and kidney tissues. S‐P467L mice, but not NT littermates, exhibited severe impairment of acetylcholine‐ and sodium nitroprusside‐induced vascular relaxation in the carotid and basilar arteries in response to high salt. CD45+ total leukocytes, CD3+ T cells, CD4+/CD8+ T cell subsets, and F4/80+ monocytes/macrophages were not altered in the aortas but were paradoxically reduced in the kidneys of S‐P467L mice in response to high salt. These data indicate that loss of PPARγ in smooth muscle cells predispose to impaired vascular relaxation, renal dysfunction, and salt‐sensitive hypertension. This study highlights the significance of vascular PPARγ in regulating systemic physiological responses.Support or Funding InformationNIH and AHA Grants

Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics

Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

Nrf2基因缺陷增强烟雾浓缩物对小鼠的免疫刺激反应—Nrf2基因缺陷影响着小鼠的免疫应激反应

目的：分析烟雾浓缩物刺激下的Nrf2基因缺陷的小鼠脾脏淋巴细胞亚群百分比和腹腔巨噬细胞炎症因子变化，初步探讨Nrf2基因缺陷对烟雾浓缩物刺激下小鼠免疫反应的影响。方法：36只雌性C57BL/6 WT和Nrf2-/-小鼠随机分别分为两组；其中两组WT和Nrf2-/-小鼠连续饮用含DMSO溶解烟雾浓缩物的纯净水4个月；另两组小鼠饮用含等比DMSO的纯净水4个月。取小鼠脾脏，拍照，坏死评分；制备单细胞悬液，进行计数；采用流式细胞术分析脾脏淋巴细胞各亚群的变化。同时提取小鼠腹腔巨噬细胞，检测分析相关细胞因子的表达。结果：Nrf2基因缺陷促使了烟雾浓缩物刺激下小鼠脾脏肿大，并伴有坏死，降低了其脾脏总细胞数；增高了烟雾浓缩物刺激下小鼠调节免疫相关的脾脏T淋巴细胞和CD8淋巴细胞的百分比，同时能够降低B淋巴细胞；降低了CD4和CD8中央型记忆T细胞(TCM)或效应性记忆T细胞(TEM)的百分比，增加了效应T细胞(TE)；增强了多数炎症性细胞因子、集落刺激因子和趋化因子在小鼠腹腔巨噬细胞中的表达。结论：Nrf2缺陷增强了烟雾浓缩物激活的小鼠天然免疫和适应性免疫的应答。 Objective: To investigate the variation of spleen lymphocyte subsets percentages and abdominal macrophage inflammatory factor of the cigarette smoke extract-induced Nrf2-/- mice, and to dis-cuss the role of Nrf2-/- in cigarette smoke extract-induced immuno-stimulus response of mice. Methods: 36 female C57BL/6 WT and Nrf2-/- mice were randomly divided into 2 groups, respectively. Two groups of WT and Nrf2-/- mice drank pure water containing DMSO dissolved cigarette smoke extract for 4 months continuously; the other two groups’ mice drank pure water containing the same amount of DMSO for four months. Spleens were harvested for taking pictures and necrosis scores; the single cell suspension of spleen tissue was prepared for splenocyte counting and the variation of spleen lymphocyte subsets was analyzed with flow cytometry. Meanwhile, the abdominal macrophages were harvested for the detecting of the related inflammatory factor. Results: Nrf2-/- prompted the spleen swelling with necrosis and lowered the number of splenocytes of cigarette smoke extract-induced mice; Nrf2-/- increased the percents of immune regulation related spleen total T cells and CD8 T cells and reduced the B cells; Nrf2-/- reduced the CD4 and CD8 TCM or TEM cells and increased the TE cells; Nrf2-/- enhanced the majority of inflammatory cytokines, colony stimulating factors and chemokines expression in abdominal macrophage of cigarette smoke extract-induced mice. Conclusion: Nrf2-/- enhances the cigarette smoke extract-induced natural immune and regulating immune response in mice.

Preliminary Characterization of Occipodins, Natural Product Anticoagulants from Hematophagous Marine Parasites, Isopod Elthusa vulgaris and Copepod Phrixocephalus Cincinnatus.

We report identification and preliminary characterization of novel proteinaceous anticoagulants from two marine crustacea, Elthusa vulgaris and Phrixocephalus cincinnatus. We also propose naming them Occipodins in honor of the college where this undergraduate research has been initiated. The organisms were collected at 25-125 meters depth off the coast of Los Angeles. Both organisms are hematophagous arthropods that anchor onto their host species and induce the extravasation of host blood into their digestive organs. Female P. cincinnatus display host specificity infecting the eyes of flatfish by anchoring to the choroid membrane and subsequently developing rami, an elaborate dendritic network, that facilitates the ingestion of extravasated blood from retinal blood vessels (Figure 1). The described species E. vulgaris is an isopod that similarly parasitizes a variety of marine fish by anchoring to the blood-rich gill filaments of their hosts (Figure 2). The accumulation of blood and attenuation of coagulation responses visually observed in the copepod trunk prompted us to initiate the identification of the anticoagulant factors in both hematophagous marine species.A crude cellular extract was prepared by homogenizing 10 specimens with a mortar and pestle frozen in liquid nitrogen resulting in a fine powder. The homogenate was resuspended in 15ml of 50mM Hepes buffer, pH7.4, containing 150mM NaCl and 3mM CaCl2 and mixed on a rotary shaker for 20min at room temperature. The resulting tissue suspension was centrifuged at 15,000xg 15 min and the supernatant was filtered through a 0.2 μm syringe filter. 10μl aliquots of extracts were used for all anticoagulant activity tests using FACT plasma for aPTT and PT assays on a Stago STart 4 hemostasis analyzer (Table 1). The extracts from both parasites were found to have exclusive anticoagulation activity on the intrinsic pathway (APTT assay) but no prolongation of coagulation time was observed in PT assays. Several biochemical tests were employed to further characterize the occipodins. First, size exclusion tests were performed using series of Millipore centrifugal filter units ranging from 10 to 100kD molecular weight cutoff. The size of the occipodin from P. cincinnatus was determined to be larger than 100kD while the anticoagulant isolated from Elthusa was estimated to be ~50kD. In order to identify whether these large macromolecules require a folded structure for activity we employed heat denaturation tests at 96°C for 15min. The P. cincinnatus occipodin showed substantial loss of anticoagulant activity following heat denaturation while E. vulgaris retained some residual anticoagulant activity, indicating the presence of non-proteinaceous, heat-resistant components (Table 1).A preliminary purification procedure of the anticoagulant isolated from Elthusa was established using a DEAE anion exchange chromatography. The crude tissue extract was diluted two fold with 50mM Hepes buffer, pH7.4 and applied on a 22ml column packed with DEAE-Sephacel (GE Healthcare) anion exchange matrix equilibrated with 50mM HEPES buffer, pH 7.4, containing 75mM NaCl. To elute occipodins, a step gradient ranging from 150 to 500mM NaCl (in the same HEPES buffer) was applied, after an extensive washing step with 120ml equilibration buffer. The anticoagulant activity was detected in fractions eluted between 125mM and 500mM NaCl with main pick centered at fractions eluted with buffer containing 150mM NaCl (Table 2)Identification of the site of inhibitory action of occipodins on the intrinsic coagulation pathway is currently in progress. In addition, further experiments will be targeted towards purification of occipodins and identification of their partial amino acid sequences. This sequence information will be used for cloning of cDNAs encoding these proteins. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of DNA extraction protocols to detect Mycobacterium bovis in bovine tissue by PCR

The current scenario of international beef trading has increased the pressure for better and faster diagnosis of bovine tuberculosis. Although traditional culture remains the gold standard method to confirm Mycobacterium bovis infection, it is exceedingly time consuming, and demands viable mycobacteria. Molecular methods overcome the flaws of the bacteriological methods with faster detection and identification. However, mycobacterial features like a complex cell wall and pathogen–host interaction make the molecular detection a challenge. Three protocols for DNA extraction (A, B and C) from bovine tissues were tested to verify the most suitable technique for routine diagnostic assessment of their specificity and sensitivity. Thirty culture-positive and thirty culture-negative granulomatous lesions were included in the trial. From each sample, three tissue suspensions at different dilutions (10-1, 10-2 and 10-3) were prepared and submitted to DNA extraction. PCR procedures targeting IS6110 were performed, employing two volumes of DNA: 5 µL of all three dilutions, and 2.5 µL of the 10-1 dilution. Protocol A was able to detect members of the M. tuberculosis complex in most samples. The sensitivity of the test decreased with increase in tissue-suspension dilution. Although Protocol A presented the highest sensitivity followed by C and B, it showed the lowest specificity, which can be due to a failure in primary isolation caused by the lack of viable organisms or incubation time. Regardless classical bacteriological methods are still recommended by OIE, after evaluating the sensitivity of DNA extraction protocols and PCR procedures, we conclude that the best strategy for M. bovis detection is to follow Protocol A on concentrated tissue suspensions.

Treatment of large, complex, non-healing wounds with cryopreserved amniotic suspension allograft: a case series

Cryopreserved amniotic suspension allograft (CASA) is a regenerative tissue suspension containing amniotic membrane and amniotic fluid components used for soft-tissue repair, replacement, and reconstruction. The aim of this case series is to examine its effects in different wound types. In a retrospective series CASA was applied to chronic non-healing wounds. All patients were treated at a single center between June 2013 and December 2014. The criterion for application of CASA was lack of progress toward wound healing despite standard treatments and adjuvant therapies. We assessed five patients aged between 39 to 86 years old. Three patients had surgical/radiation wounds, one had a sacral pressure ulcer, and one had a diabetic foot ulcer (DFU). All patients experienced progressive improvement in wound area and volume during treatment with CASA. One patient (postmastectomy tissue necrosis) required surgical closure in conjunction with removal of retained sutures. CASA applied topically and subcutaneously was particularly well suited to application in these wounds with large sinus tracts or undermining. All five patients experienced complete wound closure following one to two applications of CASA over 5 to 22 weeks. This series highlights the effective use of CASA in recalcitrant large soft-tissue wounds, DFUs, and deep wounds with tendon exposure. This study was supported by a grant from Derma Sciences (Princeton, NJ, US).