Decellularized Lung Extracellular Matrix Scaffold Promotes Human Embryonic Stem Cell Differentiation towards Alveolar Progenitors.

OBJECTIVE: Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cell replacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environment and mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM (dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonic stem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this study was to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derived lung progenitor cells. MATERIALS AND METHODS: This study was an experimental study. In the first step, a sheep lung was decellularized to achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen and glycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheep lung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were compared in their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm (DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chain reaction (PCR) assessments. RESULTS: We found that the dECM-derived scaffold preserved its composition and native porous structures while lacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by the RNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderived hydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium. DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expression of SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker) genes. CONCLUSION: Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towards lung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.

Introducing a noninvasive, non-surgical, and reversible animal model for fecal incontinence using abobotulinumtoxinA in dogs.

The aim of this study was to introduce a new animal model of fecal incontinence (FI) by injecting abobotulinumtoxinA in the external anal sphincter (EAS) muscle of dogs which replaces models based on anal sphincter destructions that are invasive, mostly require surgical procedures, expensive, permanent, and painful to the animals. 4 healthy mongrel dogs were used in this study. First, they were received NaCl 0.09% (as control) injections in EAS muscle and effects were assessed by means of Electromyography (EMG) and clinically evaluated by sphincter pinch test and presence of leakage of feces for 2 weeks. Then, they received abobotulinumtoxinA in EAS muscle and reevaluated for 6 weeks to see short-term and medium-term effects of abobotulinumtoxinA injection. Saline had no significant changes in results obtained from EMG, however, there were significant decreases in amplitudes of action potentials after receiving abobotulinumtoxinA in comparison with no injection or saline injection in EAS muscle. Pinch tests were normal after saline injection assessment period, however, then started to be negative, ranging from two days after abobotulinumtoxinA injection to seven days after receiving abobotulinumtoxinA. Animals also had significant presentations of fecal incontinence (leakage of feces and cage contamination with feces) from the 1st week after receiving abobotulinumtoxinA until the 6th week after receiving abobotulinumtoxinA. AbobotulinumtoxinA caused paralysis in the EAS and producd FI conditions in dogs. This animal model was an appropriate substitute to the various invasive, expensive and also complicated procedures with an easy, feasible, noninvasive and non-painful single-stage abobotulinumtoxinA injection.

Hydrocortisone Promotes Differentiation of Mouse Embryonic Stem Cell-Derived Definitive Endoderm toward Lung Alveolar Epithelial Cells.

In this article which was published in Cell J, Vol 20, No 4, winter 2019, on pages 469-479, the authors regret to acknowledge that we failed to mention in our article that a patent based on this study had been filed by Royan Institute and Tehran University with S.S.C., M.R.M.D., H.B., and Y.T. as inventors. The authors would like to apologies for any inconvenience caused.

Prevention of LPS-induced acute respiratory distress syndrome in sheep by bone marrow-derived mesenchymal stem/stromal cells.

MATERIALS AND METHODS: In this study, 10 male Shall sheep were used in two groups and bone marrow samples were collected and BM-MSCs isolated. Then experimental model of ARDS was induced by intrapulmonary injection of LPS to dose of 400 µg/kg. Twenty-four hours after LPS injection, 5 × 107 cells of BM-MSCs were autologous transferred in the group of treatment and 1 ml PBS was infused in the group of control as intrapulmonary. Then, the symptoms of clinical, complete blood count, analysis of arterial blood gases and the concentrations of IL6,IL10,TNF-α,total protein, Ig M and albumin BAL were determined before and at times of 3,6,12,24,48,72, and 168 after transplantation/infusion. KEY FINDINGS: The results of the investigations 24 h post-LPS injection(time 0) indicated the occurrence of acute inflammation which confirmed ARDS model. These changes included increase in RR, HR and RT, decrease in PO2 and SatO2 and increase in PCO2, WBC, neutrophils, macrophages, total protein,IL6,IL10, TNF-α,Ig M and albumin. But the stem/stromal cells transplantation reduced the severity of clinical signs induced by LPS, caused significant increase in PO2, SatO2 and IL-10 and significant decrease in PCO2, the total protein, TNF-α,IL-6, Ig M, albumin, WBCs, neutrophils and macrophages at different times of sampling both in compared with before transplantation(time 0) and in compared with the group of control. While in the control group, inflammation continued until the end of the study. SIGNIFICANCE: These results showed that BM-MSCs are able to reduce inflammation and have an important role in reconstruction of the damaged lung.

Mesenchymal stem/stromal cells: the therapeutic effects in animal models of acute pulmonary diseases.

The pulmonary diseases are one of the most important causes of death in the world. The successful therapies in the field of lung diseases are very limited and the medical treatments available are ineffective in many of the lung diseases. Many studies have evaluated the new therapies in the acute pulmonary diseases, and the transplantation of mesenchymal stem/stromal cells (MSCs), which is a branch of cell therapy, has a special place among the new medical techniques. The MSCs are present throughout the body and are thought to play a role in tissue regeneration and inflammation control. In the event of injury, the local MSCs traverse the shortest possible distance from the tissue or blood vessels to reach the affected site. But, there are few undifferentiated cells in the tissues. The exogenous MSCs are used to immunity modify or regenerative treatments in preclinical models of acute pulmonary diseases. Several studies have shown the positive effects of MSCs replacement in the acute lung disorders. The effection mechanism of the MSCs include the differentiation ability and the secretion of paracrine agents such as the anti-inflammatory mediators. Many studies suggest that this treatment method is safe and is probably to be widely used in future clinical trials. This review will describe the therapeutic effects of the MSCs in the experimental models of the acute pulmonary diseases for use as a method of treatment in clinical trials in future.

A report on Coenuruses cerebralis infection in a wild goat (Capra aegagrus).

Coenurus cerebralis is the larval stage of Taenia multiceps inhabiting the small intestine of dogs and wild carnivores as the definitive hosts. A two-year-old wild female goat (Capra aegagrus) was referred with signs of lateral recumbency and seizure for four days and loss of appetite. In clinical examination, paddling, convulsion, and unconsciousness were observed indicating central nervous system disorder. Biochemical analyses showed increased levels of hematocrit, creatinine phosphorous, total bilirubin, direct bilirubin, blood urea nitrogen and calcium. No bacteria has been grown on culture medium taken from cerebrospinal fluid (CSF). The amount of total protein of the CSF was 1.10 g dL-1 (normal range = 20 - 40 mg dL-1). Hematological changes represented a left shift degenerative leukocytosis. At necropsy, two cysts sized over the 3 × 3 cm were detected, one on occipital lobe of the right hemisphere and the other on superior colliculi. The cysts contained a translucent fluid with a number of clusters of scolice growing from the inner layer of the cysts. To the best of our knowledge, this is the first report of coenurosis occurrence in Capra aegagrus.

Hydrocortisone Promotes Differentiation of Mouse Embryonic Stem Cell-Derived Definitive Endoderm toward Lung Alveolar Epithelial Cells.

OBJECTIVE: The ability to generate lung alveolar epithelial type II (ATII) cells from pluripotent stem cells (PSCs) enables the study of lung development, regenerative medicine, and modeling of lung diseases. The establishment of defined, scalable differentiation methods is a step toward this goal. This study intends to investigate the competency of small molecule induced mouse embryonic stem cell-derived definitive endoderm (mESC-DE) cells towards ATII cells. MATERIALS AND METHODS: In this experimental study, we designed a two-step differentiation protocol. mESC line Royan B20 (RB20) was induced to differentiate into DE (6 days) and then into ATII cells (9 days) by using an adherent culture method. To induce differentiation, we treated the mESCs for 6 days in serum-free differentiation (SFD) media and induced them with 200 nM small molecule inducer of definitive endoderm 2 (IDE2). For days 7-15 (9 days) of induction, we treated the resultant DE cells with new differentiation media comprised of 100 ng/ml fibroblast growth factor (FGF2) (group F), 0.5 µg/ml hydrocortisone (group H), and A549 conditioned medium (A549 CM) (group CM) in SFD media. Seven different combinations of factors were tested to assess the efficiencies of these factors to promote differentiation. The expressions of DE- and ATII-specific markers were investigated during each differentiation step. RESULTS: Although both F and H (alone and in combination) promoted differentiation through ATII-like cells, the highest percentage of surfactant protein C (SP-C) expressing cells (~37%) were produced in DE-like cells treated by F+H+CM. Ultrastructural analyses also confirmed the presence of lamellar bodies (LB) in the ATII-like cells. CONCLUSION: These results suggest that hydrocortisone can be a promoting factor in alveolar fate differentiation of IDE2-induced mESC-DE cells. These cells have potential for drug screening and cell-replacement therapies.

Intrapulmonary autologous transplant of bone marrow-derived mesenchymal stromal cells improves lipopolysaccharide-induced acute respiratory distress syndrome in rabbit.

BACKGROUND: Lung diseases such as acute respiratory distress syndrome (ARDS) have a high incidence worldwide. The current drug therapies for ARDS have supportive effects, making them inefficient. New methods such as stromal cell therapy are needed for this problem. METHODS: This research was performed with ten New Zealand rabbits in two groups. Bone marrow aspiration was performed on the treated group, and mesenchymal stem cells were isolated and cultured. The experimental model of ARDS was induced using LPS from Escherichia coli strain O55:B5. Then, 1010 bone marrow mesenchymal stem cells (BM-MSCs) were autologously transplanted intrapulmonary in the treatment group, and 1-2 ml of PBS in the control group. The clinical signs, computed tomographic (CT) scans, echocardiography, blood gas analysis, complete blood count, and cytokine levels were measured before and at 3, 6, 12, 24, 48, 72, and 168 h after BM-MSC transplant. Finally, the rabbits were killed, and histopathological examination was performed. RESULTS: The results showed that BM-MSCs decreased the severity of clinical symptoms, the number of white blood cells and heterophils in the blood, the total cell count, and number of heterophils and macrophages in bronchoalveolar lavage, and balanced the values of arterial blood gases (increase in partial pressure of oxygen and O2 saturation and decrease in the partial pressure of carbon dioxide). They also downregulated the tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentrations and increased the IL-10 concentrations at different times compared with time 0 and in the control group, significantly. In the CT scan, a significant decrease in the Hounsfield units and total lung volume was found by echocardiography, and in comparing the two groups, a significant difference in the parameters was noticed. The histopathology demonstrated that the BM-MSCs were able to reduce the infiltration of inflammatory cells and pulmonary hemorrhage and edema. CONCLUSIONS: This study indicated that BM-MSCs play a significant role in the repair of lung injury.

The Therapeutic Potential of Differentiated Lung Cells from Embryonic Stem Cells in Lung Diseases.

Lung diseases cause great morbidity and mortality. The choice of effective medical treatment is limited and the number of lung diseases are difficult to treat with current treatments. The embryonic stem cells (ESCs) have the potential to differentiate into cell types of all three germinal layers, including lung epithelial cells. So they can be a potential source for new cell therapies for hereditary or acquired diseases of the airways and lungs. One method for treatment of lung diseases is cell therapy and the use of ESCs that can replace the damaged epithelial and endothelial cells. Progress using ESCs has developed slowly for lung regeneration because differentiation of lung cells from ESCs is more difficult as compared to differentiation of other cells. The review studies the therapeutic effects of differentiated lung cells from embryonic stem cells in lung diseases. There are few studies of differentiation of ESCs into a lineage of respiratory and then investigation of this cell in experimental model of lung diseases.

The first report of peste des petits ruminants (PPR) in camels (Camelus dromedarius) in Iran.

In mid-July 2013, an outbreak of peste des petits ruminants (PPR) was observed in a herd of camels after they were imported from Kuwait to the Khuzestan province in southwest of Iran. The clinical signs of the affected animals included sudden death, fever, oral erosion, and ecthyma like lesions, yellowish diarrhea, pneumonia and respiratory distress, enlargement of lymph node, severe dehydration, dermatitis, ulcerative keratitis, and conjunctivitis. Necropsy findings included keratoconjunctivitis, congestion and consolidation of the lung, paleness of the liver, and enlargement and edema of lymph nodes. Histopathological exam revealed degeneration and acute hyperemia of the lungs, fatty change and necrotic foci in the liver, tubular necrosis in the kidneys, and necrotic dermatitis. We used immunocapture enzyme linked immunosorbent assay (ELISA) to confirm peste des petits ruminants virus (PPRV) and differentiate it from rinderpest virus. Then virus genome was studied by molecular analysis for detecting of strain and substrain of the virus. Immunocapture ELISA of all specimens reacted positively against PPRV antigens. Also, reverse transcription polymerase chain reaction (RT-PCR) results in the lung and lymph nodes of the dead camels consolidated the cause of disease to be PPRV. The present study is the first report of the PPRV outbreak in camels in Iran.

Blood electrolytes changes in peritonitis of cattle.

Peritonitis is an inflammation of the peritoneal cavity and is one of the main causes of animal deaths. It has been reported that many diseases such as peritonitis cause electrolyte imbalance in the body. The present study has been conducted to evaluate the serum electrolyte concentration in cattle with peritonitis. In order to perform this study, 45 cattle with peritonitis were selected in the Karaj area, and 20 healthy cattle were used as the control group. After diagnosis of peritonitis in the infected cattle, 10-ml blood samples were taken from the jugular vein, the concentrations of calcium, phosphorus, magnesium, and chloride were estimated using the spectrophotometric method, and sodium and potassium concentrations were assessed by a flame photometer device. The results showed that the concentrations of calcium, magnesium, sodium, potassium, and chloride in cattle affected with peritonitis were reduced compared with the control group, but the differences were not statistically significant. The concentration of phosphorus in the peritonitis-infected cattle was significantly higher than in the healthy cattle. On the basis of the results of the present study, it can be concluded that inflammation of the peritoneal cavity in cattle causes blood electrolyte deterioration, and more attention needs to be focused on this factor in the treatment of infected animals.

Effect of lactation on functional and morphological echocardiographic variables in adult dairy cows.

OBJECTIVES: To study the effect of pregnancy and lactation on echocardiographic parameters in Holstein dairy cows. ANIMALS: Nine multiparous high milk producing (HMP) dairy cows (producing more than 40 kg of milk per day in peak production) and 9 low milk producing (LMP) cows (producing less than 30 kg or milk per day in peak production). METHODS: Echocardiography was performed twice; one month before calving and two months after calving. RESULTS: The heart rate of HMP cows in the early lactation period was significantly higher than in the dry period. In LMP cows, there was a significant increase in left ventricular dimension in the early lactation period as compared to the dry period, and the interventricular septum in systole (IVSs) in the dry period was significantly thicker than early lactation period. In HMP cows, there was an increase in the right ventricular diameter in systole in the early lactation period as compared to the dry period. Left ventricular and aortic dimensions in the dry period of HMP were significantly higher than those of LMP cows. When the data were corrected for body weight, comparison of values of the dry period of HMP and LMP cows showed that left ventricular volume in systole in HMP was significantly higher and that IVSs, left ventricular fractional shortening and ejection fraction were significantly lower than in LMP cows. CONCLUSIONS: This study demonstrated that lactation influences the intracardiac dimensions. The amount of milk production can influence echocardiographic parameters in dairy cows.

The therapeutic effect of Tarentula cubensis extract (Theranekron®) in foot-and-mouth disease in cattle: a randomised trial in an endemic setting.

BACKGROUND: Foot-and-mouth disease (FMD) is a contagious viral disease of ruminant animals. Eradication of disease in western countries is by slaughter of infected and in contact animals but this is not possible in endemic countries. There is no standard treatment for FMD in endemic countries, but anti-inflammatory drugs and mild disinfectant and protective dressing to inflamed areas to prevent secondary infection is recommended. METHOD: A randomised controlled clinical trial of a homeopathic preparation of Tarentula cubensis (Theranekron®) was conducted during an outbreak of FMD in cattle in Iran. A single subcutaneous injection of Theranekron® was used as sole treatment in 50 infected animals (treatment group). The control group comprised 15 infected animals treated with standard medication including: daily injection of flunixin meglumine and oxytetracycline and daily dressing of lesions with 4% sodium carbonate. Systemic and local signs were recorded over 14 days. RESULTS: Rectal temperature in treatment group subsided to normal range within 1 day of homeopathic treatment, and was significantly lower in test group than in control group on several successive days (P < 0.05). Healing of inflamed mucosal areas and appetite score of the treatment was significantly better than control during first 3 days of treatment (P < 0.05). CONCLUSION: It appears that Theranekron® is effective for treatment of systemic and local signs of FMD-infected cattle. Further research is justified.

The effects of oral consumption of selenium nanoparticles on chemotactic and respiratory burst activities of neutrophils in comparison with sodium selenite in sheep.

The present study was designed to compare the effects of nano-selenium and of sodium selenite on the chemotactic and respiratory burst activities of neutrophils in sheep. Fifteen sheep were randomly divided into three groups. Groups 1 and 2 received selenium nanoparticles (1 mg/kg) or sodium selenite (1 mg/kg) orally, respectively, for ten consecutive days, and the third group was considered as the control. To determine the chemotactic and respiratory burst activities of the neutrophils, the leading front assay and the NBT test were used on heparinized blood samples that were collected at different intervals (days 0, 10th, 20th, and 30th). The results obtained showed that the chemotactic activities in groups 1 and 2 increased significantly on the 10th, 20th, and 30th day, compared to day 0, and on the 20th day in comparison with the 10th day, while in group 2, there was a significant decrease on the 30th day compared to the 20th day. The chemotactic activities in group 1 were significantly higher than in group 2 on the 10th day and in the control group on the 10th, 20th, and 30th day, but the chemotactic activities in group 2 were significantly higher than those in the control group only on the 20th day. On the 30th day into the experiment, the respiratory bursts in groups 1 and 2 were significantly stronger in comparison with those at day 0. Overall, nano-selenium increased the chemotactic and respiratory burst activities more significantly than sodium selenite, which is suggestive of a stronger stimulatory effect of the Se nanoparticles on intracellular activities.

The role of route of vitamin E administration on the plasma antioxidant activity and lipid peroxidation in newborn calves.

The aim of our study was to evaluate plasma values of alpha-tocopherol, malondialdehyde (MDA) and antioxidant activity after a single-dose administration of vitamin E as intramuscular injection, oral supplementation and intramuscular injection plus oral supplementation at 4 hr after birth. Thirty calves were bled at birth and assigned to treatments as follows: control (n = 8), intramuscular injection (40 IU/kg, n = 7), oral supplementation (25 IU/kg, n = 7) and intramuscular injection (20 IU/kg) plus oral supplementation (12.5 IU/kg, n = 8). Blood was collected at 12 and 24 hr after birth and plasma alpha-tocopherol, MDA and antioxidant activity values were determined. Results showed that no changes in MDA values were observed after oral administration (P > 0.05). However, antioxidant activity values showed an increase at both 12 (9.57 +/- 0.65 mmol/l) and 24 hr (10.42 +/- 0.54 mmol/l) after birth when compared to control (3.73 +/- 0.75 mmol/l). Injection with or without oral supplementation increased serum antioxidant activity values at 12 (about 102%, 46%) and 24 hr (94%, 115%) after birth, when compared to control. In addition, MDA values were found to be lower in those animals receiving an injection of vitamin E or injection plus oral supplementation of vitamin E as compared to control at both time-points (P < 0.001). Injection of vitamin E provided beneficial effects to plasma antioxidant activity and MDA values. Therefore, injection may be the best method of vitamin E administration in newborn calves for protecting them in the stressful postnatal condition.