Functional evaluation of hTM, CD46 and HLA-E transgenes in vitro and in pig leg xenoperfusion experiments

Background Besides α1,3 galactosyltransferase (Gal) gene knockout several transgene combinations to prevent pig-to-human xenograft rejection are being investigated. hCD46/HLA-E double transgenic pigs were tested for prevention of xenograft rejection in an ex vivo pig-to-human xenoperfusion model. In addition, expression of human thrombomodulin (hTM-) on wild-type and/or multi-transgenic (GalTKO/hCD46) background was evaluated to overcome pig-to-human coagulation incompatibility. Methods hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for deposition of complement proteins as well as E-selectin and VCAM-1 expression. Serial blood cell counts were performed to analyze changes in human blood cell populations. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. In addition, a biochemical assay was performed using hTM-only and multi-transgenic (GalTKO/hCD46/hTM) pig aortic endothelial cells (PAEC) to evaluate the ability of hTM to generate activated protein C (APC). Subsequently, the anti-coagulant properties of hTM were tested in a microcarrier based coagulation assay with PAEC and human whole blood. Results No hyperacute rejection was seen in the ex vivo perfusion model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL1β (P < 0.0001), and IL-8 (P = 0.019) as well as human C3a, C5a and soluble C5b-9 were significantly (P < 0.05–<0.0001) lower in blood perfused through hCD46/HLA-E transgenic as compared to wild-type limbs. C3b/c, C4b/c, and C6 deposition as well as E-selectin and VCAM-1 expression were significantly (P < 0.0001) higher in tissue of wild-type as compared to transgenic limbs. Preliminary immunofluorescence staining results showed that the expression of hCD46/HLA-E is associated with a reduction of NK cell tissue infiltration (P < 0.05). A rapid decrease of platelets was observed in all xenoperfusions. In vitro findings showed that PAEC express ASGR1 and suggest that this molecule is involved in human platelet phagocytosis. In vitro, we found that the amount of APC in the supernatant of hTM transgenic cells increased significantly (P < 0.0001) with protein C concentration in a dose-dependent manner as compared to control PAEC lacking hTM, where the turnover of the protein C remained at the basal level for all of the examined concentration. In further experiments, hTM also showed the ability to prevent blood coagulation by three- to four-fold increased (P < 0.001) clotting time as compared to wild-type PAEC. The formation of TAT complexes was significantly lower when hTM-transgenic cells (P < 0.0001) were used as compared to wild-type cells. Conclusions Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since the terminal pathway of complement, endothelial cell activation, inflammatory cytokine production and NK-cell tissue infiltration were all down-regulated. We also found ASGR1 expression on the vascular endothelium of pigs, and this molecule may thus be involved in binding and phagocytosis of human platelets during pig-to-human xenotransplantation. In addition, use of the hTM transgene has the potential to overcome coagulation incompatibilities in pig-to-human xenotransplantation.

Double arterial perfusion strategy for extensive thoracic aortic surgery to avoid lower body hypothermic circulatory arrest

OBJECTIVE To analyse our results of using a double arterial perfusion strategy to avoid lower body hypothermic circulatory arrest after extensive thoracic aortic surgery. METHODS We analysed the intra- and perioperative courses of 10 patients (median age 58 years, median logistic EuroSCORE 14.6) who underwent extensive thoracic aortic surgery with a double arterial perfusion strategy. The main goal of double arterial perfusion is to separate myocardial and supra-aortic from systemic perfusion. Aortic repair starts at the most distal level of the descending aorta, followed by reinsertion of the supra-aortic vessels, and ends with completion of the proximal anastomosis or by any kind of root repair as needed. RESULTS Seven of 10 patients had prior surgery of the thoracic aorta. Indications for surgery were post-dissection aneurysm in 4 patients, true aneurysm in 3, anastomotic aneurysms in 2 and Type B aortic dissection with pseudo-coarctation in 1. Surgical access was performed through median sternotomy with left hemi-clamshell extension in all cases. There was no in-hospital mortality, but perioperative neurological symptoms occurred in 2 patients. These 2 patients developed delayed stroke (after awaking) after an initial uneventful clinical course, and in 1 of them, neurological symptoms resolved completely during follow-up. The median follow-up was 7 (±13) months. There was no death and no need for additional redo surgery during this observational period. CONCLUSIONS Extensive surgery of the thoracic aorta using a double arterial perfusion technique in order to avoid lower body hypothermic circulatory arrest is an attractive option. Further refinements of this technique may enable the safe and effective simultaneous multisegmental treatment of thoracic aortic pathology in patients who would otherwise have to undergo a two-step surgical approach.

Use of lower gastrointestinal endoscopy and fecal occult blood test in the 2007 Swiss Health Interview Survey respondents aged 50 years and older

BACKGROUND AND STUDY AIMS Colorectal cancer (CRC) incidence ranks third among all cancers in Switzerland. Screening the general population could decrease CRC incidence and mortality. The aim of this study was to analyze the use of the fecal occult blood test (FOBT) and lower gastrointestinal endoscopy in a representative sample of the Swiss population aged ≥ 50 years. METHODS Data were analyzed from the 2007 Swiss Health Interview Survey and the prevalence estimates and 95 % confidence intervals were calculated based on all instances of lower gastrointestinal endoscopy and FOBT use, as well as on their use for CRC screening. Uni- and multivariate logistic regression analyses were performed to describe the association between screening use and sociodemographic characteristics, indicators of healthcare system use, and lifestyle factors. RESULTS In 2007, approximately 36 % of the surveyed people who were aged ≥ 50 years had previously undergone FOBT and approximately 30 % had previously undergone lower gastrointestinal endoscopy. CRC screening use was 7.7 % for FOBT (within the past year) and 6.4 % for lower gastrointestinal endoscopy (within the past 5 years). CRC screening by either method was 13 %. The major determinants of participation in CRC screening were found to be sex (male), physician visits during the past year (one or more), type of health insurance (private), and previous screening for other cancer types. CONCLUSIONS The results of the 2007 Swiss Health Interview Survey indicate rather low levels of FOBT and lower gastrointestinal endoscopy use. Furthermore, the results suggest disparities in the use of CRC screening.

The lower quality of preventive care among forced migrants in a country with universal healthcare coverage

OBJECTIVE To assess the association between socio-demographic factors and the quality of preventive care and chronic care of cardiovascular (CV) risk factors in a country with universal health care coverage. METHODS Our retrospective cohort assessed a random sample of 966 patients aged 50-80years followed over 2years (2005-2006) in 4 Swiss university primary care settings (Basel/Geneva/Lausanne/Zürich). We used RAND's Quality Assessment Tools indicators and examined recommended preventive care among different socio-demographic subgroups. RESULTS Overall patients received 69.6% of recommended preventive care. Preventive care indicators were more likely to be met among men (72.8% vs. 65.4%; p<0.001), younger patients (from 71.0% at 50-59years to 66.7% at 70-80years, p for trend=0.03) and Swiss patients (71.1% vs. 62.7% in forced migrants; p=0.001). This latter difference remained in multivariate analysis adjusted for gender, age, civil status and occupation (OR 0.68; 95% CI 0.54-0.86). Forced migrants had lower scores for physical examination and breast and colon cancer screening (all p≤0.02). No major differences were seen for chronic care of CV risk factors. CONCLUSION Despite universal healthcare coverage, forced migrants receive less preventive care than Swiss patients in university primary care settings. Greater attention should be paid to forced migrants for preventive care.

Ankle dorsiflexion arthrodesis to salvage Chopart's amputation with anterior skin insufficiency

BACKGROUND In Chopart-level amputations the heel often deviates into equinus and varus when, due to the lack of healthy anterior soft tissue, rebalancing tendon transfers to the talar head are not possible. Consequently, anterior and lateral wound dehiscence and ulceration may occur requiring higher-level amputation to achieve wound closure, with considerable loss of function for the patients. METHODS Twenty-four consecutive patients (15 diabetes, 6 trauma, and 3 tumor) had Chopart's amputation and simultaneous or delayed additional ankle dorsiflexion arthrodesis to allow for tension-free wound closure or soft tissue reconstruction, or to treat secondary recurrent ulcerations. Percutaneous Achilles tendon lengthening and subtalar arthrodesis were added as needed. Wound healing problems, time to fusion and full weight-bearing in the prosthesis, complications in the prosthesis, and the ambulatory status were assessed. Satisfaction and function were evaluated by the AmpuPro score and the validated Prosthesis Evaluation Questionnaire scale. RESULTS Five patients had successful soft tissue healing and fusions but died of their underlying disease 2 to 46 months after the operation. Two diabetic patients required a transtibial amputation. The other 17 patients were followed for 27 months (range, 13-63). The average age of the 4 women and 13 men was 53.9 years (range, 16-87). Postoperative complications included minor wound healing problems in 8 patients, wound breakdown requiring revision in 4, phantom pain in 3, residual equinus in 1, and adjacent scar carcinoma in 1 patient. The time to full weight-bearing in the prosthesis ranged from 6 to 24 weeks (mean 10). The mean AmpuPro score was 107 points (of 120), and the mean Prosthesis Evaluation Questionnaire scale was 147 points (of 200). No complications occurred with the prosthesis. Twelve patients lost 1 to 2 mobility classes (mean 0.9). The arthrodeses all healed within 2.5 months (range, 1.5 to 5 months). CONCLUSION Adding an ankle arthrodesis to a Chopart's amputation either immediately or in a delayed fashion to treat anterior soft tissue complications was a successful salvage in most patients at this amputation level. It enabled the patients to preserve the advantages of a full-length limb with terminal weight-bearing. LEVEL OF EVIDENCE Level IV, retrospective case series.

Obesity and overweight associated with lower rates of colorectal cancer screening in Switzerland

Screening for colorectal cancer (CRC) is associated with reduced CRC mortality, but low screening rates have been reported in several settings. The aim of the study was to assess predictors of low CRC screening in Switzerland. A retrospective cohort of a random sample of 940 patients aged 50-80 years followed for 2 years from four Swiss University primary care settings was used. Patients with illegal residency status and a history of CRC or colorectal polyps were excluded. We abstracted sociodemographic data of patients and physicians, patient health status, and indicators derived from RAND's Quality Assessment Tools from medical charts. We defined CRC screening as colonoscopy in the last 10 years, flexible sigmoidoscopy in the last 5 years, or fecal occult blood testing in the last 2 years. We used bivariate and multivariate logistic regression analyses. Of 940 patients (mean age 63.9 years, 42.7% women), 316 (33.6%) had undergone CRC screening. In multivariate analysis, birthplace in a country outside of Western Europe and North America [odds ratio (OR) 0.65, 95% confidence interval (CI) 0.45-0.97], male sex of the physician in charge (OR 0.67, 95% CI 0.50-0.91), BMI 25.0-29.9 kg/m2 (OR 0.66, CI 0.46-0.96) and at least 30.0 kg/m2 (OR 0.61, CI 0.40-0.90) were associated with lower CRC screening rates. Obesity, overweight, birthplace outside of Western Europe and North America, and male sex of the physician in charge were associated with lower CRC screening rates in Swiss University primary care settings. Physician perception of obesity and its impact on their recommendation for CRC screening might be a target for further research.

Endoscopically assisted nerve decompression of rare nerve compression syndromes at the upper extremity

BACKGROUND Besides carpal tunnel and cubital tunnel syndrome, other nerve compression or constriction syndromes exist at the upper extremity. This study was performed to evaluate and summarize our initial experience with endoscopically assisted decompression. MATERIALS AND METHODS Between January 2011 and March 2012, six patients were endoscopically operated for rare compression or hour-glass-like constriction syndrome. This included eight decompressions: four proximal radial nerve decompressions, and two combined proximal median nerve and anterior interosseus nerve decompressions. Surgical technique and functional outcomes are presented. RESULTS There were no intraoperative complications in the series. Endoscopy allowed both identifying and removing all the compressive structures. In one case, the proximal radial neuropathy developed for 10 years without therapy and a massive hour-glass nerve constriction was observed intraoperatively which led us to perform a concurrent complementary tendon transfer to improve fingers and thumb extension. Excellent results were achieved according to the modified Roles and Maudsley classification in five out of six cases. All but one patient considered the results excellent. The poorest responder developed a CRPS II and refused post-operative physiotherapy. CONCLUSION Endoscopically assisted decompression in rare compression syndrome of the upper extremity is highly appreciated by patients and provides excellent functional results. This minimally invasive surgical technique will likely be further described in future clinical studies.

Conception Rate Using the Select-Synch Protocol in Combination with a Lower Dose Progesterone-Releasing Intravaginal Insert (1.38 g) in Swiss Dairy Cows

Background: Synchronization programs have become standard in the dairy industry. In Switzerland, these programs are used but newly. The objective of this study was A) to estimate the pregnancy rate after a Select-Synch protocol in- cluding a low dosage of progesterone in CIDR (1.38 g). As a second step B) this pregnancy rate should be compared to cows from another Swiss study that used a Select-Synch protocol with the 1.9 g insert (Rudolph et al., 2011). Methods: A) 196 cows were included in the study. Cows received a CIDR 1.38 g and 2.5 ml of buserelin i.m. on d 0. On d 7, the CIDR insert was removed and 5 ml of dinoprost was administered i.m. On d 0 a milk sample for progesterone analysis was taken. Pregnancy was determined at or more than 35 days after artificial insemination. B) The 1.38 g group and the 1.9 g group were compared as to cow and farm factors, number of preceding AI’s, gynecological and uterine pretreat- ment and treatment itself. A forward selection procedure was used (test result considered significant if p-value  0.05). Results: A) The pregnancy rate, using the Select-Synch protocol with the CIDR 1.38 g was 44.4%. B) The CIDR 1.9 g Select-Synch group revealed a pregnancy rate of 50.4% (Rudolph et al., 2011). Significant differences between the groups were not found. Conclusion: The 1.38 g CIDR-Select-Synch protocol may be recommended for multiparous dairy cows. The pregnancy rate compared to the 1.9 g CIDR-Select-Synch protocol was 8% lower, but this difference was not significant.

Blood lead level association with lower body weight in NHANES 1999-2006

BACKGROUND Lead exposure is associated with low birth-weight. The objective of this study is to determine whether lead exposure is associated with lower body weight in children, adolescents and adults. METHODS We analyzed data from NHANES 1999-2006 for participants aged ≥3 using multiple logistic and multivariate linear regression. Using age- and sex-standardized BMI Z-scores, overweight and obese children (ages 3-19) were classified by BMI ≥85 th and ≥95 th percentiles, respectively. The adult population (age ≥20) was classified as overweight and obese with BMI measures of 25-29.9 and ≥30, respectively. Blood lead level (BLL) was categorized by weighted quartiles. RESULTS Multivariate linear regressions revealed a lower BMI Z-score in children and adolescents when the highest lead quartile was compared to the lowest lead quartile (β (SE)=-0.33 (0.07), p<0.001), and a decreased BMI in adults (β (SE)=-2.58 (0.25), p<0.001). Multiple logistic analyses in children and adolescents found a negative association between BLL and the percentage of obese and overweight with BLL in the highest quartile compared to the lowest quartile (OR=0.42, 95% CI: 0.30-0.59; and OR=0.67, 95% CI: 0.52-0.88, respectively). Adults in the highest lead quartile were less likely to be obese (OR=0.42, 95% CI: 0.35-0.50) compared to those in the lowest lead quartile. Further analyses with blood lead as restricted cubic splines, confirmed the dose-relationship between blood lead and body weight outcomes. CONCLUSIONS BLLs are associated with lower body mass index and obesity in children, adolescents and adults.

Lower-tropospheric humidity: climatology, trends and the relation to the ITCZ

The tropical region is an area of maximum humidity and serves as the major humidity source of the globe. Among other phenomena, it is governed by the so-called Inter-Tropical Convergence Zone (ITCZ) which is commonly defined by converging low-level winds or enhanced precipitation. Given its importance as a humidity source, we investigate the humidity fields in the tropics in different reanalysis data sets, deduce the climatology and variability and assess the relationship to the ITCZ. Therefore, a new analysis method of the specific humidity distribution is introduced which allows detecting the location of the humidity maximum, the strength and the meridional extent. The results show that the humidity maximum in boreal summer is strongly shifted northward over the warm pool/Asia Monsoon area and the Gulf of Mexico. These shifts go along with a peak in the strength in both areas; however, the extent shrinks over the warm pool/Asia Monsoon area, whereas it is wider over the Gulf of Mexico. In winter, such connections between location, strength and extent are not found. Still, a peak in strength is again identified over the Gulf of Mexico in boreal winter. The variability of the three characteristics is dominated by inter-annual signals in both seasons. The results using ERA-interim data suggest a positive trend in the Gulf of Mexico/Atlantic region from 1979 to 2010, showing an increased northward shift in the recent years. Although the trend is only weakly confirmed by the results using MERRA reanalysis data, it is in phase with a trend in hurricane activity�a possible hint of the importance of the new method on hurricanes. Furthermore, the position of the maximum humidity coincides with one of the ITCZ in most areas. One exception is the western and central Pacific, where the area is dominated by the double ITCZ in boreal winter. Nevertheless, the new method enables us to gain more insight into the humidity distribution, its variability and the relationship to ITCZ characteristics.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

The Pneumococcal Polysaccharide Capsule and Pneumolysin Differentially Affect CXCL8 and IL-6 Release from Cells of the Upper and Lower Respiratory Tract

The polysaccharide capsule and pneumolysin toxin are major virulence factors of the human bacterial pathogen Streptococcus pneumoniae. Colonization of the nasopharynx is asymptomatic but invasion of the lungs can result in invasive pneumonia. Here we show that the capsule suppresses the release of the pro-inflammatory cytokines CXCL8 (IL-8) and IL-6 from the human pharyngeal epithelial cell line Detroit 562. Release of both cytokines was much less from human bronchial epithelial cells (iHBEC) but levels were also affected by capsule. Pneumolysin stimulates CXCL8 release from both cell lines. Suppression of CXCL8 homologue (CXCL2/MIP-2) release by the capsule was also observed in vivo during intranasal colonization of mice but was only discernable in the absence of pneumolysin. When pneumococci were administered intranasally to mice in a model of long term, stable nasopharyngeal carriage, encapsulated S. pneumoniae remained in the nasopharynx whereas the nonencapsulated pneumococci disseminated into the lungs. Pneumococcal capsule plays a role not only in protection from phagocytosis but also in modulation of the pro-inflammatory immune response in the respiratory tract.