Expression of immune regulatory genes correlate with the abundance of specific Clostridiales and Verrucomicrobia species in the equine ileum and cecum.

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFß, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

Mesenchymal stromal cells from human umbilical cords display poor chondrogenic potential in scaffold-free three dimensional cultures.

Many researchers world over are currently investigating the suitability of stromal cells harvested from foetal tissues for allogeneic cell transplantation therapies or for tissue engineering purposes. In this study, we have investigated the chondrogenic potential of mesenchymal stromal cells (MSCs) isolated from whole sections of human umbilical cord or mixed cord (UCSCs-MC), and compared them with cells isolated from synovial membrane (SMSCs), Hoffa's fat pad (HFPSCs) and cartilage. All MSCs were positive for surface markers including CD73, CD90, CD105, CD44, CD146 and CD166, but negative for CD11b, CD19, CD34, CD45 and HLA-DR in addition to CD106 and CD271. Chondrogenic potential of all cell sources was studied using 3D pellet cultures incubated in the presence of different combinations of anabolic substances such as dexamethasone, IGF-1, TGF-ß1, TGF-ß3, BMP-2 and BMP-7. BMP-2 and dexamethasone in combination with TGF-ß1 or TGF-ß3 excelled at inducing chondrogenesis on SMSCs, HFPSCs and chondrocytes, as measured by glycosaminoglycans and collagen type II staining of pellets, quantitative glycosaminoglycan expression, quantitative PCR of cartilage signature genes and electron microscopy. In contrast, none of the tested growth factor combinations was sufficient to induce chondrogenesis on UCSCs-MC. Moreover, incubation of UCSCs-MC spheroids in the presence of cartilage pieces or synovial cells in co-cultures did not aid chondrogenic induction. In summary, we show that in comparison with MSCs harvested from adult joint tissues, UCSCs-MC display poor chondrogenic abilities. This observation should alert researchers at the time of considering UCSCs-MC as cartilage forming cells in tissue engineering or repair strategies.

TL1A regulates TCRγδ+ intraepithelial lymphocytes and gut microbial composition.

TL1A is a proinflammatory cytokine, which is prevalent in the gut. High TL1A concentrations are present in patients with inflammatory bowel disease (IBD) and in IBD mouse models. However, the role of TL1A during steady-state conditions is relatively unknown. Here, we used TL1A knockout (KO) mice to analyse the impact of TL1A on the intestinal immune system and gut microbiota. The TL1A KO mice showed reduced amounts of small intestinal intraepithelial TCRγδ(+) and CD8(+) T cells, and reduced expression of the activating receptor NKG2D. Moreover, the TL1A KO mice had significantly reduced body weight and visceral adipose tissue deposits, as well as lower levels of leptin and CXCL1, compared with wild-type mice. Analysis of the gut microbial composition of TL1A KO mice revealed a reduction of caecal Clostridial cluster IV, a change in the Firmicutes/Bacteroidetes ratio in caecum and less Lactobacillus spp. in the mucosal ileum. Our results show that TL1A deficiency impacts on the gut microbial composition and the mucosal immune system, especially the intraepithelial TCRγδ(+) T-cell subset, and that TL1A is involved in the establishment of adipose tissue. This research contributes to a broader understanding of TL1A inhibition, which is increasingly considered for treatment of IBD.

Efficient rotational cooling of Coulomb-crystallized molecular ions by a helium buffer gas.

The preparation of cold molecules is of great importance in many contexts, such as fundamental physics investigations, high-resolution spectroscopy of complex molecules, cold chemistry and astrochemistry. One versatile and widely applied method to cool molecules is helium buffer-gas cooling in either a supersonic beam expansion or a cryogenic trap environment. Another more recent method applicable to trapped molecular ions relies on sympathetic translational cooling, through collisional interactions with co-trapped, laser-cooled atomic ions, into spatially ordered structures called Coulomb crystals, combined with laser-controlled internal-state preparation. Here we present experimental results on helium buffer-gas cooling of the rotational degrees of freedom of MgH(+) molecular ions, which have been trapped and sympathetically cooled in a cryogenic linear radio-frequency quadrupole trap. With helium collision rates of only about ten per second--that is, four to five orders of magnitude lower than in typical buffer-gas cooling settings--we have cooled a single molecular ion to a rotational temperature of 7.5(+0.9)(-0.7) kelvin, the lowest such temperature so far measured. In addition, by varying the shape of, or the number of atomic and molecular ions in, larger Coulomb crystals, or both, we have tuned the effective rotational temperature from about 7 kelvin to about 60 kelvin by changing the translational micromotion energy of the ions. The extremely low helium collision rate may allow for sympathetic sideband cooling of single molecular ions, and eventually make quantum-logic spectroscopy of buffer-gas-cooled molecular ions feasible. Furthermore, application of the present cooling scheme to complex molecular ions should enable single- or few-state manipulations of individual molecules of biological interest.

Ampicillin-improved glucose tolerance in diet-induced obese C57BL/6NTac mice is age dependent.

Ampicillin has been shown to improve glucose tolerance in mice. We hypothesized that this effect is present only if treatment is initiated prior to weaning and that it disappears when treatment is terminated. High-fat fed C57BL/6NTac mice were divided into groups that received Ampicillin at different ages or not at all. We found that both diet and Ampicillin significantly changed the gut microbiota composition in the animals. Furthermore, there was a significant improvement in glucose tolerance in Ampicillin-treated, five-week-old mice compared to nontreated mice in the control group. At study termination, expressions of mRNA coding for tumor necrosis factor, serum amyloid A, and lactase were upregulated, while the expression of tumor necrosis factor (ligand) superfamily member 15 was downregulated in the ileum of Ampicillin-treated mice. Higher dendritic cell percentages were found systemically in high-fat diet mice, and a lower tolerogenic dendritic cell percentage was found both in relation to high-fat diet and late Ampicillin treatment. The results support our hypothesis that a "window" exists early in life in which an alteration of the gut microbiota affects glucose tolerance as well as development of gut immunity and that this window may disappear after weaning.

Breast size increment during pregnancy and breastfeeding in mothers with polycystic ovary syndrome: a follow-up study of a randomised controlled trial on metformin versus placebo.

OBJECTIVE: To study the significance of breast size increment in pregnancy, and the impact of metformin during pregnancy on breastfeeding in women with polycystic ovary syndrome (PCOS). DESIGN: A follow-up study of a randomised controlled trial (the PregMet study). SETTING: Eleven secondary care centres. POPULATION: Women with PCOS during pregnancy and postpartum. METHODS: Women with PCOS were randomised to treatment with metformin or placebo from the first trimester to delivery. Questionnaires were sent to 240 participants 1 year postpartum: 186 responded. MAIN OUTCOME MEASURES: Pre-pregnancy and late-pregnancy brassiere size and breastfeeding patterns were registered, and androgen levels were measured in the mothers. RESULTS: No difference in breast size increment and breastfeeding were found between the placebo and metformin groups. Breast size increment correlated positively with the duration of both exclusive and partial breastfeeding, whereas body mass index (BMI) correlated negatively with the duration of partial breastfeeding. Dehydroepiandrostenedione-sulphate (DHEAS), testosterone and free testosterone index (FTI) in pregnancy did not correlate with breast size increment or duration of breastfeeding. Women with no change in breast size were more obese, had higher blood pressure, serum triglycerides and fasting insulin levels, and had a shorter duration of breastfeeding compared with those with breast size increment. CONCLUSIONS: Metformin and androgens had no impact on breastfeeding. Women with PCOS who had no breast size increment in pregnancy seem to be more metabolically disturbed and less able to breastfeed.

A novel method for transuterine identification of piglets.

Implantable microchips provide a secure, permanent and unique identification of individual animals. When performing fetal intervention studies in experimental animal models easy and secure identification of fetuses is desirable, as having test and control groups within the same uterus reduces the total number of animals used in a study. The aims of this study were: (1) to establish a protocol to identify porcine fetuses in utero by microchip implantation and (2) to assess postnatally whether clinical or pathological reactions to the implant occurred. Two Danish Landrace/Danish Large White crossbred sows at day 100 of gestation were used. The sows were sedated with azaperone and induced with propofol intravenously. Anaesthesia was maintained with isoflurane and oxygen. Antibiotics were administered intramuscularly (i.m.) at induction and analgesia was given pre-, intra- and postoperatively. A laparotomy was performed and the uterus exteriorized. The rump of the first fetus was recognized through the uterine wall and the thigh muscle of the fetus was fixed between the thumb and the forefinger. The microchip was then implanted into the fetus at an angle of 45 degrees i.m. in the lateral hindleg using an insertion device with a 12G needle. The same procedure was done in every fetus. The uterus was returned to the abdomen and the abdominal wall closed. The sows gave birth to 24 liveborn piglets and one stillborn. None of the liveborn piglets were limping at the time of birth and no visible cutaneous or palpable reactions on the hindlegs were observed. Following euthanasia, the microchip was easily localized and no macroscopic reactions at the implantation site were seen. None of the piglets had more than one microchip implanted. Histology showed a chronic mild foreign body granulomatous inflammatory response with peripheral eosinophils surrounding the microchip. No inflammation was evident in the adjacent muscles. It is concluded that transuterine identification of piglets two weeks before delivery is feasible using a microchip implant as an effective, easy and reliable method for identification of individuals after birth.

Accuracy of clinical data in a population based vascular registry.

INTRODUCTION: Clinical databases are increasingly being employed to evaluate the quality of treatments, including patients with peripheral vascular disease. Valid data is vital to the value of these analyses. OBJECTIVE: To assess the validity of clinical data in a population-based national vascular registry. DESIGN: Traditional reproducibility study was supplemented by refilling of data by an independent observer, thereby creating three data sets for comparison. MATERIALS AND METHODS: Twenty prospectively recorded electronic forms from each department were selected randomly from the Danish National Vascular Registry. Data forms were refilled by the surgeons of the department concerned, and by an independent member of the board of the Danish National Vascular Registry. Refilling was performed blinded to the original forms. CONCLUSIONS: A high degree of accuracy of clinical data can be achieved. An independent observer makes it possible to evaluate the classification of observer dependent parameters and explain differences in the reproducibility of data.

Effect of pre-analytical handling on haematological variables in minipigs.

Pre-analytical handling may be an important determinant of haematological variables, if analysis is delayed. We investigated the effect of anticoagulants, i.e. tripotassium ethylenediamine-tetraacetic acid (EDTA) and citric acid, theophylline, adenosine, dipyridamole (CTAD), storage time (0.5, 1.5, 3.5, 5.5, 7.5, 25.5 and 27.5 h after blood sampling), and storage temperature (5 degrees C and 20 degrees C) on the variation in haemoglobin (HGB), red blood cell count (RBC), haematocrit (HCT), white blood cell count (WBC), and platelet count (PLT) in minipigs. Medians of HGB, RBC, HCT, WBC and PLT were significantly higher in EDTA tubes than in CTAD tubes due to the dilution effect of the anticoagulant. We found a minor significant increase in HCT after 25.5 h in blood stored at 20 degrees C, and at the same time a minor significant increase in WBC in EDTA tubes stored at 20 degrees C. We found a significant decrease in PLT in blood stored at 5 degrees C, especially in EDTA tubes. Minor variations were also observed in HGB and RBC. Our results indicate that PLT should only be measured in tubes placed at room temperature. If HCT or WBC analyses are to be performed on the day after blood sampling, the samples must be stored in a refrigerator until analysis. Our studies underline that time delay before analysis of haematological variables can cause increased variation, and should therefore be limited as far as possible in order to reduce the number of animals needed to make reliable conclusions.

A serological indication of the existence of a guineapig poliovirus.

Attempts were made to clarify whether laboratory guineapigs may harbour a poliovirus which, in 1911, was described as the cause of a disease called guineapig lameness. By the use of ELISA for antibodies against the poliovirus, Theiler's murine encephalomyelitis virus (TMEV), it was shown that two pet shop guineapigs suffering from lameness had extremely high titres against poliovirus, while healthy guineapigs from the same pet shop were negative. Clearly positive results were also found in 35 out of 152 laboratory guineapig sera. Positive results were found in only two out of six breeding centres, but in three out of three experimental units, all of which purchased guineapigs from one of the seropositive breeding colonies. The diseased guineapigs recovered fully after treatment with vitamins in the drinking water, a treatment used for guineapig lameness by small animal practitioners. A theory that vitamin C deficient guineapigs are, due to an impaired steroid secretion, predisposed to succumbing to infection and develop demyelinating disease similar to that in TMEV infected mice is discussed briefly. Guineapig sera were also tested serologically for other infections. Antibodies against lymphocytic choriomeningitis virus, Clostridium piliforme and Toxoplasma gondii were not found, but one breeding colony was infected with adenovirus, pneumonia virus of mice, reovirus type 3, Sendai virus, parainfluenza (simian) virus type 5 and Encephalitozoon cuniculi. Two other breeding colonies were infected with both reovirus type 3 and E. cuniculi. In all three experimental units infection with adenovirus was observed, and in two of these Sendai virus and E. cuniculi antibodies were also found. The pet shop guineapigs were infected with adenovirus, reovirus type 3 and E. cuniculi.

Postoperative hypophosphataemia and muscle function.

Muscle function tests of the triceps brachii muscle were performed before operation and on the third postoperative day in ten patients undergoing elective cholecystectomy. Electromyograms (EMGs) were recorded by surface electrodes during sustained isotonic and isometric muscle contraction with a constant force of 20 per cent of the preoperative maximal voluntary contraction. Root-mean-square of the EMG was calculated together with the neuromuscular efficiency and measures of the fatiguability. These parameters were compared with changes in the simultaneously measured serum phosphate concentrations. Mean (s.e.m.) neuromuscular efficiency measured after 32-40 s of muscle contraction decreased 14(5) per cent after operation (P less than 0.01), whereas the mean fatiguability of the muscle was unchanged. Mean serum phosphate concentration was 0.87(0.06) mmol/l before operation and 0.79(0.06) mmol/l 3 days after the operation (P greater than 0.05). Two patients developed severe postoperative hypophosphataemia (serum phosphate concentration less than 0.50 mmol/l). Postoperative muscle function deterioration was not associated with changes in the serum phosphate level (P greater than 0.10; r = 0.03). We conclude that patients undergoing cholecystectomy develop postoperative deterioration in skeletal muscle function, which is not associated with serum phosphate concentration.

Profundaplasty as the only reconstructive procedure in patients with severe ischemia of the lower extremity.

Twenty-one profundaplasties were performed as the only reconstructive procedure. All of the patients had severe ischemia of the lower extremity with serious symptoms, such as rest pain or gangrene, or both. There was no postoperative mortality. Two patients had a crural amputation, and one patient, a femoral amputation performed during the first three months postoperatively. All of the other patients improved, and there was a significant rise in distal blood pressures after one week, further increasing after three months. At follow-up study, 15 of the patients had considerably less symptoms than preoperatively or were free of symptoms. There was a limb salvage rate of 83 per cent after 60 months. The operation is recommended whenever possible in patients with severe ischemia of the lower extremity when the possibilities of other reconstructive procedures are considered to be poor.

Tibial hypo-/aplasia with preaxial syn- and polydactyly.

Tibial hypo-/aplasia with preaxial syn- and polydactyly is a rare autosomal dominant condition. Fewer than 20 cases have so far been described. One is presented here.

Serum and urinary phosphate during and after prolonged muscular ischaemia in non-exercising men and women.

Serum inorganic phosphate (Pi) concentrations and urinary Pi excretions were measured in nine patients undergoing abdominal aortic bypass grafting (group I) and in nine patients undergoing lower limb arterial embolectomy (group II). In group I, serum Pi concentrations were normal until 24 h after reperfusion, when they decreased, reaching their nadir at 48 h (median Pi 0.45 mmol 1(-1). The urinary phosphate clearance relative to the creatinine clearance (Cp/Cc) increased 5 min after reperfusion reaching a maximal median value (0.454) in the 2-24-h collection period. In group II, eight patients had a normal and one had a subnormal serum Pi before reperfusion. None developed hypo- or hyperphosphataemia and the Cp/Cc remained unchanged. Daily urinary Pi excretion was higher in group I than in group II (P less than 0.03). The authors concluded that the regional muscular ischaemia in non-exercising men and women was not associated with hyperphosphataemia. The possible influence of major surgery and anaesthesia on phosphate homeostasis should be considered.

[Social aspects of reconstructive vascular surgery in patients under 60 years of age]. / Sociale aspekter ved rekonstruktiv karkirurgi hos patienter yngre end 60 år.

The effect of initial elective arterial reconstructive surgery was evaluated in relation to employment and social status. Ninety patients with intermittent claudication were followed for a period of 3.5 years. The male to female ratio was 1:12. At the time of operation, all the patients were under the age of 60 years and potentially professionally active. The indication for operation in fifty-nine patients was intermittent claudication. For thirty-one patients, the indication for operation was ischaemic rest pain, gangrene or ulcer. Fifty-six patients (74%) returned to active employment following vascular reconstructive surgery. It is concluded that reconstructive vascular surgery is of great importance for rehabilitation and in the success of resumption of active employment.

Operative pleurodesis in spontaneous pneumothorax. Indications and results.

In 61 cases of spontaneous pneumothorax treated with thoracotomy and pleurodesis, sometimes with resection of bullous lung tissue, the records were reviewed. There were few postoperative complications and no treatment-requiring recurrences. The postoperative drainage time was significantly longer in patients with, than in those without, chronic obstructive pulmonary disease preoperatively, but the long-term results were the same in both groups. Age did not influence the results. The treatment policy in this selected group of patients seemed to be highly satisfactory.

Popliteal arterial aneurysm.

Popliteal arterial aneurysms are rare in young individuals. A case without a penetrating trauma presenting in a young woman is described. Complications may lead to vascular emergencies. Early diagnosis and surgical treatment is important.