Adipose tissue collection from a living kidney donor; an experimental model for the research of atherosclerosis.

Thanks to an increased number of living-donor kidney transplants the IKEM transplant program offers the possibility of obtaining adipose tissue for scientific purposes from patients with varying degrees of atherosclerosis. Surgery mainly addresses vascular complications of this disease. On the other hand, surgery may also be the reason for the development and acceleration of atherosclerosis - for instance, acceleration of atherosclerosis in the living kidney donor, particularly if, although meeting internationally recognized donation criteria, the donor actually suffers from metabolic syndrome. The effort to refine the examinations of living kidney donors in terms of eliminating the risk of developing atherosclerosis is a long-term project. The aims are to determine the risk factors for living kidney donors and to prevent long-term complications after donation. The paper gives a detailed description of the technique of adipose tissue collection from a living kidney donor and of the experimental model for the research of atherosclerosis.The project has the potential to increase the safety of living kidney donation and to enhance our present knowledge of atherosclerosis development mechanisms.

Mycobacterium avium subsp. paratuberculosis detected in the reproductive tract of cows from an infected herd.

Mycobacterium avium subspecies paratuberculosis (MAP), the causal agent of paratuberculosis, was detected by quantitative real-time IS900 PCR in the follicular fluid from the reproductive tracts of cows originating from one infected herd. As well as being detected in follicular fluid of cows shedding bacteria in their faeces, MAP was also detected in the follicular fluid of one apparently healthy, non-shedding individual cow. The finding of MAP in follicular fluid is unexpected and could contribute to the lower viability of embryos and resultant lower pregnancy rate. In addition to finding contaminated follicular fluid, vaginal and uterine flush fluids were determined to be positive for the presence of MAP in 75% and 56.3% of the time of the cattle currently shedding MAP in their faeces, respectively. The presence of MAP in different parts of the reproductive tract was seen in clinically as well as subclinically infected cows. These findings extend our currently scant and contradictory knowledge about the dissemination of MAP in the reproductive tract of female cattle.

Culture- and quantitative IS900 real-time PCR-based analysis of the persistence of Mycobacterium avium subsp. paratuberculosis in a controlled dairy cow farm environment.

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.

Short communication: Examination of milk filters by real-time PCR as a herd-level indicator of the presence of Mycobacterium avium subspecies paratuberculosis in dairy herds.

The aim of this study was to assess the suitability of real-time quantitative PCR (qPCR) for the detection of Mycobacterium avium ssp. paratuberculosis (MAP) in milk filters as a herd level indicator of paratuberculosis infection. Seventy-nine samples from textile or metal milk filters from 15 herds with defined MAP prevalence (infection status = noninfected, 0-5%, 5-10%, or >10% of animals with clinically confirmed paratuberculosis) were analyzed. The MAP DNA was isolated by a modified commercially available protocol for feces, and detection and quantification of the pathogen was performed by the IS900 qPCR. Mycobacterium avium ssp. paratuberculosis DNA was detected in 63 (79.7%) samples. Determination of MAP infection established by fecal and tissue culture was correctly confirmed by the analysis of milk filters on 11 of 12 infected farms; MAP was not detected in filters from 3 farms where paratuberculosis was never diagnosed. Statistical analysis of the data supports the evidence that milk filters can be used as a template for the direct detection of MAP on the herd level. The probability of successful MAP detection in milk filters in a herd with MAP-infected cows is at least 94.3%. Absolute numbers of MAP detected on the milk filter can be used for a rough estimation of paratuberculosis prevalence >10% in the herd. Analysis of milk filters for the presence of MAP can be a useful tool for the detection of paratuberculosis on the herd level before any individual control strategies.

Cholesterol efflux and macrophage polarization in human adipose tissue.

The pro-inflammatory status of adipose tissue (AT) has been found to be related to reverse cholesterol transport (RCT) from peritoneal macrophages. However, this finding was made in experimental models using induced peritonitis and isolated peritoneal macrophages of animals. This experimental relationship is in agreement with RCT changes in man in two extreme situations, sepsis or cardiovascular complications. Given the above, we sought to test RTC in relationship to macrophage polarization in the visceral AT (VAT) of living kidney donors (LKDs) and the effect of conditioned media obtained from their AT. The influence of ATCM on CE capacity was first assessed in an experiment where standard plasma was used as cholesterol acceptor from [14C] cholesterol labeled THP-1. Conditioned media as a product of LKDs' incubated AT showed no effect on CE. Likewise, we did not find any effect of individual plasma of LKDs on CE when individual plasma of LKDs were used as acceptors. On the other hand, we documented an effect of LKDs' adipose cell size on CE. Our results indicate that the pro-inflammatory status of human AT is not likely induced by disrupted RCT but might be influenced by the metabolic status of LKDs' adipose tissue.

Persistence of Mycobacterium avium subsp. paratuberculosis at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle.

In this study, products from all steps of anaerobic digestion at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle were examined and quantified for the presence of the causal agent of paratuberculosis, Mycobacterium avium subsp. paratuberculosis, using culture and quantitative real-time PCR (qPCR). Viable M. avium subsp. paratuberculosis cells were detected using culture in fermentors for up to 2 months; the presence of M. avium subsp. paratuberculosis DNA (10(1) cells/g) was demonstrated in all anaerobic fermentors and digestate 16 months after initiation of work at a biogas plant, using IS900 qPCR. F57 qPCR was able to detect M. avium subsp. paratuberculosis DNA (10(2) cells/g) at up to 12 months. According to these results, a fermentation process that extended beyond 2 months removed all viable M. avium subsp. paratuberculosis cells and therefore rendered its product M. avium subsp. paratuberculosis free. However, M. avium subsp. paratuberculosis DNA was found during all the examined periods (more than 1 year), which could be explained by either residual DNA being released from dead cells or by the presence of viable cells whose amount was under the limit of cultivability. As the latter hypothesis cannot be excluded, the safety of the final products of digestion used for fertilization or animal bedding cannot be defined, and further investigation is necessary to confirm or refute this risk.

The optimization of the protocol for immunofluorescence on fish spermatozoa.

In comparison with mammals, the fertilization of fish occurs predominantly outside the organism in a water environment, where fish spermatozoa require specific conditions to interact with oocytes. It is evident that optimal conditions for fish and mammalian spermatozoa are quite different. This paper describes a special approach to handling fish (common carp and Siberian sturgeon) spermatozoa in comparison with the samples originating from mammals (boar). This approach concerns not only the differences in the composition of the media applied but also primarily emphasizes the concrete parts of the immunofluorescence protocol determining accurate results. Individual parts of the protocol for indirect immunofluorescence of mammalian sperm were changed step by step and modified protocols were applied to immunofluorescence experiments with carp and sturgeon spermatozoa. By evaluating the changes in the integrity of the fish sperm head and flagellum, we selected the steps and corresponding conditions that are crucial for handling the fish spermatozoa. Based on our results, it may be concluded that when working with fish spermatozoa, the cells attached to the microscopic slides must not desiccate prior to the fixation, which is a usual step when working with mammalian sperm. The second crucial step is the necessity to fix the fish spermatozoa, especially when the research is focused on the structure of the flagellum. The impact of the temperature conditions is rather low, but working at low temperatures, except for the period of incubation with antibodies, leads to a higher number of unaffected cells.

Pro-inflammatory gene expression in adipose tissue of patients with atherosclerosis.

Inflammatory changes, both in the arterial wall and adipose tissue, play a crucial role in the development of atherosclerosis. We measured the gene expression of tumor necrosis factor-alpha (TNFalpha), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6) in adipose tissue (AT) of living kidney donors (LKD) and patients with peripheral arterial disease (PAD). Quantitative polymerase chain reaction (qPCR) and flow cytometry analyses were performed in subcutaneous (SAT), visceral (VAT), and perivascular adipose tissue (PVAT). Data of PAD patients showed significantly higher expression in VAT in all three genes (TNFalpha 5-fold, p<0.05; MCP-1 3.6-fold, p<0.05; IL-6 18.8-fold, p<0.001). The differences in PVAT and SAT were less significant. Total body pro-inflammatory status was documented by higher TNFalpha concentration in patients (4.86+/-1.4 pg/ml) compared to LKDs (2.14+/-0.9 pg/ml; p<0.001), as was hsCRP (11.8+/-7.0 in PAD; 1.5+/-0.48 in LKDs; p=0.017). We found no age-dependent relationship between gene expression vs. TNFalpha and hsCRP concentrations in both compared groups. No effect of the atherosclerosis score on gene expression and circulating inflammatory markers within the PAD group was observed. Our results suggest that the AT of PAD patients infiltrated with macrophages produces more cytokines involved in the development of inflammation and atherosclerosis.

Human adipose tissue accumulation is associated with pro-inflammatory changes in subcutaneous rather than visceral adipose tissue.

The importance of the involvement of adipose tissue macrophage subpopulations in obesity-related disorders is well known from different animal models, but human data are scarcer. Subcutaneous (n=44) and visceral (n=52) adipose tissues of healthy living kidney donors were obtained during living donor nephrectomy. Stromal vascular fractions were isolated and analysed by flow cytometry using CD14, CD16, CD36 and CD163 antibodies. Total macrophage numbers in subcutaneous adipose tissue increased (P=0.02) with body mass index (BMI), with a similar increase seen in the proportion of phagocytic CD14+CD16+CD36high macrophages (P<0.01). On the other hand, there was an inverse correlation between anti-inflammatory CD14+CD16-CD163+ macrophages (P<0.05) and BMI. These correlations disappeared after excluding obese subjects (BMI ⩾30 kg m-2) from the analysis. Interestingly, none of these subpopulations were significantly related to BMI in visceral adipose tissue. Obesity per se is associated with distinct, highly phagocytic macrophage accumulation in human subcutaneous adipose tissue.

The effect of ectopic fat on graft function after living kidney transplantation.

Renal transplantation is associated with a large number of risk factors that can have an influence on early renal graft function (ERGF). One of these factors could be the increasing number of obese kidney donors. The mechanisms of reduced ERGF in obese kidney donors are still poorly understood. To that end, we compared ERGF in recipients with body mass index (BMI), perivascular fat and plasma inflammation markers of live kidney donors. We hypothesized that the BMI of donors would negatively correlate with an average increase of glomerular filtration rate (GFR) and that it would also be associated with increased perivascular and plasma inflammation markers in the first seven days after transplantation. Between January 2013 and December 2014, some 58 living kidney transplantation pairs were included in the study. Donor and recipient demographic data, preoperative BMI, blood C-reactive protein (CRP) and adiponectin levels, perivascular adipose tissue (PAT) samples and recipient blood creatinine levels were analyzed. The median CRP of donors was 0.68 mg/l (max: 8.66 mg/l, min: 0.33 mg/l), the median of M1 macrophages (CD14+CD16+) in one gram of PAT was 5940 (max: 41 100, min: 248) and the median of adiponectin was 411 930 pg/ml (max: 14 217 000, min: 167 300) in plasma. We did not find any association between early renal graft function and the percentage of M1 macrophages in donor perirenal adipose tissue (p=0.83, r=0.03, n=58), adiponectin (p=0.65, r=0.06, n=58) or CRP (p=0.16, r=0.2, n=58) in plasma. The obesity level of donors, expressed as BMI, did not correlate with early renal graft function in the first seven days after transplantation. The associations between ERGF and plasma and perivascular fat inflammation markers were not significant. We confirmed a negative correlation between the BMI of recipients and an average increase of GFR in the first seven days after transplantation (p<0.02, r=-0.325, N=58). We confirmed a negative correlation of adiponectin plasma concentration to the BMI of donors.

Macrophage phenotypes in the adipose tissue of postmenopausal women.

Atherosclerosis pathology is the interplay between high intravascular LDL particle concentration and monocyte/macrophage presence within the sub-endothelial space of the artery. In this project, phenotypes of macrophages connected with subclinical inflammation in adipose tissue of living kidney donors were studied. Samples of subcutaneous adipose tissue of living kidney donors (n=36) were exposed to collagenase. Stromal vascular fraction (SVF) was eluted from the samples, then labeled with monoclonal antibodies (anti-CD14 and anti-calprotectin), conjugated with fluorochromes and analyzed by flow cytometry. The positive correlation between the number of total macrophages and calprotectin-positive macrophages with BMI in the subcutaneous adipose tissue of postmenopausal women was demonstrated (p<0.05; R=0.43 and p<0.01; R=0.60), whereas no positive correlation in premenopausal women and men was shown. In conclusion, we documented a significant effect of BMI increase on the presence of total macrophages in adipose tissue of postmenopausal women, in contrast to premenopausal women. This difference was much more pronounced when proinflammatory macrophages with membrane-bound calprotectin were analyzed.

Laparoscopic adjustable gastric band: first experience in Slovakia.

BACKGROUND: Until recently, surgery for obesity was not done in Slovakia. After preparation in workshops, the authors began to perform laparoscopic adjustable gastric banding. Their laparoscopic work is based on a 7-year experience in laparoscopy. METHODS: From December 1997 to May 1998, 14 procedures were done. The laparoscopic adjustable gastric band was used in all procedures. The group consisted of 8 women and 6 men. Their ages ranged from 30 to 53 years (mean 43), the body mass index was 37-56 (average 46.2), the hospital stay was 3-7 days (4.8), and the operating time was 75-285 minutes (145.3). A five-trocar technique was used. RESULTS: Weight loss in the first month ranged from 9 to 15 kg. In follow-up, the weight loss averaged 3-4 kg monthly. Up to the time of writing, no band had been adjusted. Intraoperative hemorrhage occurred in two patients. No conversion was done. One patient underwent reoperation and removal of the band because of obstruction of the stoma caused by profuse vomiting after enormous intake of food. CONCLUSIONS: Despite a small early series, the procedure seems to be safe and well tolerated by patients, with sufficient early weight loss.

Complications in the first year of laparoscopic gastric banding: is it acceptable?

BACKGROUND: From December 1997 to December 1998, 25 laparoscopic adjustable silicone gastric banding (LASGB) procedures were done without previous experience in bariatric surgery. Body mass index (BMI) ranged from 37 to 57 kg/m2 (average 45.5 kg/m2). METHODS: Retrospective analysis of the 1-year experience was done. Operating time was measured, and BMI and complications were reviewed. RESULTS: Five complications were observed. There was a complication rate of 20%. On two occasions, it was gastric wall slippage, and both were corrected laparoscopically. In one patient, the intususception of the gastric wall through the band resulted after profuse vomiting. Removal of the band was necessary, with conversion to an open procedure. On two occasions, the infection of the port-site was observed, in one of these patients, port removal was necessary. No antibiotic prophylaxis was used. CONCLUSION: Despite lack of experience in bariatric surgery in these laparoscopic surgeons, the complications with LASGB appear to be acceptable. Although prior bariatric surgical experience is preferable.

30% complications with adjustable gastric banding: what did we do wrong?

BACKGROUND: The authors analyzed the complications in patients following laparoscopic adjustable gastric banding (LAGB) for morbid obesity. METHODS: Retrospective analysis of the 36 LAGB patients was done. The operations were performed from December 1997 to the December 1999 using the Lap-Band. RESULTS: 11 complications occurred. Most common was port-site infection or port migration (5 patients). These complications were termed minor. In 3 patients, slippage of the gastric wall was observed, all corrected laparoscopically. In another 3 patients, removal of the band was necessary for poor patient compliance (1), for intussusception of the gastric wall through the band with occlusion of the stoma (1), and for infection of the band (1). These complications were termed major. The overall complication rate was 30.5%. CONCLUSION: Compared to the literature, our complication rate was rather high. Based on our analysis, the following measures are recommended: 1) antibiotic prophylaxis; 2) drainage of the port-site; 3) proper band and port placement and fixation; 4) closer psychological evaluation and follow-up. By these measures, hopefully we can obtain better results in the future.

Immunological aspects of atherosclerosis.

Atherosclerosis is a degenerative inflammatory disease of the vascular wall, which is characterized by the formation of atherosclerotic plaques that contain lipids, activated smooth muscle cells, immune cells, foam cells, a necrotic core and calcified sites. In atherosclerosis pathology, monocytes and macrophages play the most important role by accumulating redundant LDL particles in their oxidized form and producing proinflammatory cytokines. Atherosclerotic plaque macrophages reveal distinct phenotypes that are distinguished into M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. Numerous environmental signals (cytokines, microbial cell molecules) that are received by macrophages drive their polarization, but it must be determined whether this classification reflects different macrophage subtypes or plasticity and phenotypic tissue changes, but the balance between subsets is crucial. M1 macrophages are dominant in symptomatic atherosclerotic plaques, while M2 macrophages are more frequent in asymptomatic plaques. Nevertheless, a positive correlation of both M1 and M2 macrophages with atherosclerotic lesion severity was also observed.

Co-cultivation of human aortic smooth muscle cells with epicardial adipocytes affects their proliferation rate.

The abnormal proliferation of vascular smooth muscle cells (VSMC) is thought to play a role in the pathogenesis of atherosclerosis. Adipocytes produce several bioactive paracrine substances that can affect the growth and migration of VSMCs. Our study focuses on the direct effect of the bioactive substances in conditioned media (CM) that was obtained by incubation with primary adipocyte-derived cell lines, including cell lines derived from both preadipocytes and from more mature cells, on the proliferation rate of human aortic smooth muscle cells (HAoSMCs). We used a Luminex assay to measure the adipokine content of the CM and showed that there was a higher concentration of monocyte chemoattractant protein-1 in renal preadipocyte-CM compared with the HAoSMC control (p<0.5). The addition of both renal preadipocyte- and epicardial adipocyte- CM resulted in the elevated production of vascular endothelial growth factor compared with the control HASoSMC CM (p<0.001). The adiponectin content in renal adipocyte-CM was increased compared to all the remaining adipocyte-CM (p<0.01). Moreover, the results showed a higher proliferation rate of HAoSMCs after co-culture with epicardial adipocyte-CM compared to the HAoSMC control (p<0.05). These results suggest that bioactive substances produced by adipocytes have a stimulatory effect on the proliferation of VSMCs.

Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR.

The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.

Characterization of the replicon from plasmid pAC1 from Acetobacter pasteurianus.

A panel of recombinant plasmids pACK5 and pACT7 was prepared by introducing kanamycin and tetracycline resistance into the partially split plasmid pAC1 which contained replicon isolated from Acetobacter pasteurianus. The replicon in plasmid pAC1 is compatible with the ColE1 replicon. Compared to pBR322, the plasmid had more than 30 copies per chromosome in Escherichia coli cells. Plasmids were transformed into E. coli DH1, Acetobacter pasteurianus 3614, Acetobacter aceti 3620, Shigella, Citrobacter, and Brevibacterium flavum cells, and the stability of plasmid DNA was tested after cultivation in nonselective conditions.