Biological safety concepts of genetically modified live bacterial vaccines

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

Percutaneous autologous venous valve transplantation: short-term feasibility study in an ovine model

BACKGROUND: Limited experience with bioprosthetic venous valve percutaneously inserted into femoral veins in 15 patients has been promising in short-term results only to show disappointing long-term results. Percutaneous autogenous venous valve (PAVV) transplantation was explored in an ovine model as a possible alternative treatment. METHODS: PAVV consisted of a vein segment containing a valve that was attached to a stent template. The stent templates (n = 9) were designed and hand made in our research laboratory. They consist of two stainless steel square stents 13 or 15 mm in diameter to fit the ovine jugular veins (JV), which ranges from 10 to 15 mm in diameter. A valve-containing segment of JV was harvested and attached with sutures and barbs inside the stent template (n = 9). The valve devices were then manually folded and front loaded inside the 4 cm chamber of the 13F delivery sheath and delivered into the contralateral JV by femoral vein approach. Transplanted PAVVs were studied by immediate and 3 months venograms. Animals were euthanized at 3 months, and jugular veins harvested to perform angioscopic evaluations in vitro. RESULTS: PAVV transplantation was successful in all nine animals. Good valve function with no reflux was observed on immediate and 3 months venograms in eight valves. The transplanted maximal JV diameter ranged from 10.2 mm to 15.4 mm (mean 13.1 +/- 1.5 mm). Venoscopic examination revealed intact, flexible, nonthickened valve leaflets in eight specimens. One PAVV exhibited normal function of one leaflet only; the other cusp was accidentally cut during the transplantation procedure. All transplanted autologous valves were free of thrombus and incorporated into the vein wall of the host vessel. CONCLUSION: This study demonstrated that autogenous valve transplants remained patent and competent without long-term anticoagulation for up to 3 months. The percutaneous autogenous venous valve may provide in future minimally invasive treatment for patients with chronic deep venous insufficiency, but long-term studies need to be done to document its continued patency and function.

Retention of canopy trees as biological legacies for balancing woody biomass production and biodiversity in managed aspen forests of the Great Lakes Region

Green-tree retention under the conceptual framework of ecological forestry has the potential to provide both biomass feedstock for industry and maintain quality wildlife habitat. I examined the effects of retained canopy trees as biological legacies (“legacy trees”) in aspen (Populus spp.) forests on above-ground live woody biomass, understory plant floristic quality, and bird diversity. Additionally, I evaluated habitat quality for a high conservation priority species, the Golden-winged Warbler (Vermivora chrysoptera). I selected 27 aspen-dominated forest stands in northern Wisconsin with nine stands in each of three legacy tree retention treatments (conifer retention, hardwood retention, and clearcuts or no retention) across a chronosequence (4-36 years post-harvest). Conifer retention stands had greater legacy tree and all tree species biomass but lower regenerating tree biomass than clearcuts. Coniferous but not hardwood legacy trees appeared to suppress regenerating tree biomass. I evaluated the floristic quality of the understory plant assemblage by estimating the mean coefficient of conservatism (C). Mean C was lower in young stands than in middle-age or old stands; there was a marginally significant (p=0.058) interaction effect between legacy tree retention treatment and stand age. Late-seral plant species were positively associated with stand age and legacy tree diameter or age revealing an important relationship between legacy tree retention and stand development. Bird species richness was greatest in stands with hardwood retention particularly early in stand development. Six conservation priority bird species were indicators of legacy tree retention or clearcuts. Retention of legacy trees in aspen stands provided higher quality nest habitat for the Golden-winged Warbler than clearcuts based on high pairing success and nesting activity. Retention of hardwoods, particularly northern red oak (Quercus rubra), yielded the most consistent positive effects in this study with the highest bird species richness and the highest quality habitat for the Golden-winged Warbler. This treatment maintained stand biomass comparable to clearcuts and did not suppress regenerating tree biomass. In conclusion, legacy tree retention can enhance even-aged management techniques to produce a win-win scenario for the conservation of declining bird species and late-seral understory plants and for production of woody biomass feedstock from naturally regenerating aspen forests.

Prevalence of nephrolithiasis in patients with ileal bladder substitutes

OBJECTIVES: To assess the effect of ileal bladder substitutes with preservation of the ileocecal valve and distal 25 cm of ileum on nephrolithiasis. METHODS: We reviewed a consecutive series of 518 patients (44 women and 474 men) with ileal orthotopic bladder substitution in whom 55 to 65 cm of ileum was resected but with preservation of the ileocecal valve and distal 25 cm of ileum, to determine prevalence of nephrolithiasis as well as bicarbonate, base excess, creatinine levels, and urinary pH at time of stone diagnosis and 2 years before it. RESULTS: Four male patients with a median age of 66 years (range, 50 to 70 years) developed nephrolithiasis after ileal bladder substitute, for a total of five calculi. The prevalence of nephrolithiasis in this retrospective cohort is thus 1% (5 of 518). They developed the calculi after a median follow-up of 8 years (range, 4 to 17 years). The four patients were diagnosed with calculi at 2.3, 3, 10, 10.3, and 14 years after bladder substitute. Two of the stones were uric acid calculi; the remaining three were calcium oxalate. None of our patients were acidotic or had elevated serum creatinines at time of stone formation. Urinary pH determined once in spontaneously voided urine at the time of stone diagnosis was pH 6.0 for the two uric acid calculi and pH 7.0 for the remaining calculi. CONCLUSIONS: The present study demonstrates a low prevalence of calculi in our cohort.

Temporary neurological dysfunction after surgery of the thoracic aorta: a predictor of poor outcome and impaired quality of life

BACKGROUND: Transient neurological dysfunction (TND) consists of postoperative confusion, delirium and agitation. It is underestimated after surgery on the thoracic aorta and its influence on long-term quality of life (QoL) has not yet been studied. This study aimed to assess the influence of TND on short- and long-term outcome following surgery of the ascending aorta and proximal arch. METHODS: Nine hundred and seven patients undergoing surgery of the ascending aorta and the proximal aortic arch at our institution were included. Two hundred and ninety patients (31.9%) underwent surgery because of acute aortic dissection type A (AADA) and 617 patients because of aortic aneurysm. In 547 patients (60.3%) the distal anastomosis was performed using deep hypothermic circulatory arrest (DHCA). TND was defined as a Glasgow coma scale (GCS) value <13. All surviving patients had a clinical follow up and QoL was assessed with an SF-36 questionnaire. RESULTS: Overall in-hospital mortality was 8.3%. TND occurred in 89 patients (9.8%). As compared to patients without TND, those who suffered from TND were older (66.4 vs 59.9 years, p<0.01) underwent more frequently emergent procedures (53% vs 32%, p<0.05) and surgery under DHCA (84.3% vs 57.7%, p<0.05). However, duration of DHCA and extent of surgery did not influence the incidence of TND. In-hospital mortality in the group of patients with TND compared to the group without TND was similar (12.0% vs 11.4%; p=ns). Patients with TND suffered more frequently from coronary artery disease (28% vs 20.8%, p=ns) and were more frequently admitted in a compromised haemodynamic condition (23.6% vs 9.9%, p<0.05). Postoperative course revealed more pulmonary complications such as prolonged mechanical ventilation. Additional to their transient neurological dysfunction, significantly more patients had strokes with permanent neurological loss of function (14.6% vs 4.8%, p<0.05) compared to the patients without TND. ICU and hospital stay were significantly prolonged in TND patients (18+/-13 days vs 12+/-7 days, p<0.05). Over a mean follow-up interval of 27+/-14 months, patients with TND showed a significantly impaired QoL. CONCLUSION: The neurological outcome following surgery of the ascending aorta and proximal aortic arch is of paramount importance. The impact of TND on short- and long-term outcome is underestimated and negatively affects the short- and long-term outcome.

Genotype 3 is associated with accelerated fibrosis progression in chronic hepatitis C

BACKGROUND/AIMS: While several risk factors for the histological progression of chronic hepatitis C have been identified, the contribution of HCV genotypes to liver fibrosis evolution remains controversial. The aim of this study was to assess independent predictors for fibrosis progression. METHODS: We identified 1189 patients from the Swiss Hepatitis C Cohort database with at least one biopsy prior to antiviral treatment and assessable date of infection. Stage-constant fibrosis progression rate was assessed using the ratio of fibrosis Metavir score to duration of infection. Stage-specific fibrosis progression rates were obtained using a Markov model. Risk factors were assessed by univariate and multivariate regression models. RESULTS: Independent risk factors for accelerated stage-constant fibrosis progression (>0.083 fibrosis units/year) included male sex (OR=1.60, [95% CI 1.21-2.12], P<0.001), age at infection (OR=1.08, [1.06-1.09], P<0.001), histological activity (OR=2.03, [1.54-2.68], P<0.001) and genotype 3 (OR=1.89, [1.37-2.61], P<0.001). Slower progression rates were observed in patients infected by blood transfusion (P=0.02) and invasive procedures or needle stick (P=0.03), compared to those infected by intravenous drug use. Maximum likelihood estimates (95% CI) of stage-specific progression rates (fibrosis units/year) for genotype 3 versus the other genotypes were: F0-->F1: 0.126 (0.106-0.145) versus 0.091 (0.083-0.100), F1-->F2: 0.099 (0.080-0.117) versus 0.065 (0.058-0.073), F2-->F3: 0.077 (0.058-0.096) versus 0.068 (0.057-0.080) and F3-->F4: 0.171 (0.106-0.236) versus 0.112 (0.083-0.142, overall P<0.001). CONCLUSIONS: This study shows a significant association of genotype 3 with accelerated fibrosis using both stage-constant and stage-specific estimates of fibrosis progression rates. This observation may have important consequences for the management of patients infected with this genotype.

Graft coronary vasculopathy in cardiac transplantation--evaluation of risk factors by multivariate analysis

The development of coronary vasculopathy is the main determinant of long-term survival in cardiac transplantation. The identification of risk factors, therefore, seems necessary in order to identify possible treatment strategies. Ninety-five out of 397 patients, undergoing orthotopic cardiac transplantation from 10/1985 to 10/1992 were evaluated retrospectively on the basis of perioperative and postoperative variables including age, sex, diagnosis, previous operations, renal function, cholesterol levels, dosage of immunosuppressive drugs (cyclosporin A, azathioprine, steroids), incidence of rejection, treatment with calcium channel blockers at 3, 6, 12, and 18 months postoperatively. Coronary vasculopathy was assessed by annual angiography at 1 and 2 years postoperatively. After univariate analysis, data were evaluated by stepwise multiple logistic regression analysis. Coronary vasculopathy was assessed in 15 patients at 1 (16%), and in 23 patients (24%) at 2, years. On multivariate analysis, previous operations and the incidence of rejections were identified as significant risk factors (P < 0.05), whereas the underlying diagnosis had borderline significance (P = 0.058) for the development of graft coronary vasculopathy. In contrast, all other variables were not significant in our subset of patients investigated. We therefore conclude that the development of coronary vasculopathy in cardiac transplant patients mainly depends on the rejection process itself, aside from patient-dependent factors. Therapeutic measures, such as the administration of calcium channel blockers and regulation of lipid disorders, may therefore only reduce the progress of native atherosclerotic disease in the posttransplant setting.

How accurately can the acetabular rim be trimmed in hip arthroscopy for pincer-type femoral acetabular impingement: a cadaveric investigation

PURPOSE: The purpose of this study was to evaluate the precision of central hip arthroscopy in the assessment and treatment of pincer-type femoroacetabular impingement (FAI) avoiding the posterolateral portal, with its close proximity to the main arterial blood supply of the femoral head, the medial circumflex femoral artery. METHODS: Seven human cadaveric hips underwent arthroscopic trimming of the acetabular labrum and rim along a preoperatively defined 105 degrees arc of resection for treatment of a presumed pincer-type lesion. After the arthroscopic procedure, all specimens were dissected and measured for evaluation of the location, quantity, and quality of the area undergoing resection. RESULTS: The difference between the actual and planned arc of resection was 18.7 degrees +/- 4.7 degrees (range, 2 degrees to 34 degrees). This was mainly because of a lack of accuracy in the presumed posterior starting point (PSP), with a mean deviation of 19 degrees +/- 3.4 degrees (range, 10 degrees to 36 degrees). Correlation analysis showed that variance in the arc of resection was mainly dependent on the PSP (r = 0.739, P = .058). CONCLUSIONS: Central hip arthroscopy is a feasible option in treating anterosuperior pincer-type FAI by use of the anterior and anterolateral portals only. This cadaveric study showed that there is a significant risk of underestimating the actual arc of resection compared with the planned arc of resection for posterosuperior pincer-type lesions because of the modest accuracy in determining the PSP of the resection. CLINICAL RELEVANCE: Accurate preoperative planning and arthroscopic identification of anatomic landmarks at the acetabular side are crucial for the definition of the appropriate starting and ending points in the treatment of pincer-type FAI. Whereas anterosuperior pincer-type lesions can be addressed very precisely with our technique, the actual resection of posterosuperior lesions averaged 19 degrees less than the planned resection, which may have clinical implications.

Blunt traumatic thoracic aortic injuries: early or delayed repair-results of an American Association for the Surgery of Trauma prospective study

BACKGROUND: The traditional approach to stable blunt thoracic aortic injuries (TAI) is immediate repair, with delayed repair reserved for patients with major associated injuries. In recent years, there has been a trend toward delayed repair, even in low-risk patients. This study evaluates the current practices in the surgical community regarding the timing of aortic repair and its effects on outcomes. METHODS: This was a prospective, observational multicenter study sponsored by the American Association for the Surgery of Trauma. The study included patients with blunt TAI scheduled for aortic repair by open or endovascular procedure. Patients in extremis and those managed without aortic repair were excluded. The data collection included demographics, initial clinical presentation, Injury Severity Scores, type and site of aortic injury, type of aortic repair (open or endovascular repair), and time from injury to aortic repair. The study patients were divided into an early repair (< or = 24 hours) and delayed repair groups (> 24 hours). The outcome variables included survival, ventilator days, intensive care unit (ICU) and hospital lengths of stay, blood transfusions, and complications. The outcomes in the two groups were compared with multivariate analysis after adjusting for age, Glasgow Coma Scale, hypotension, major associated injuries, and type of aortic repair. A second multivariate analysis compared outcomes between early and delayed repair, in patients with and patients without major associated injuries. RESULTS: There were 178 patients with TAI eligible for inclusion and analysis, 109 (61.2%) of which underwent early repair and 69 (38.8%) delayed repair. The two groups had similar epidemiologic, injury severity, and type of repair characteristics. The adjusted mortality was significantly higher in the early repair group (adjusted OR [95% CI] 7.78 [1.69-35.70], adjusted p value = 0.008). The adjusted complication rate was similar in the two groups. However, delayed repair was associated with significantly longer ICU and hospital lengths of stay. Analysis of the 108 patients without major associated injuries, adjusting for age, Glasgow Coma Scale, hypotension, and type of aortic repair, showed that in early repair there was a trend toward higher mortality rate (adjusted OR 9.08 [0.88-93.78], adjusted p value = 0.064) but a significantly lower complication rate (adjusted OR 0.4 [0.18-0.96], adjusted p value 0.040) and shorter ICU stay (adjusted p value = 0.021) than the delayed repair group. A similar analysis of the 68 patients with major associated injuries, showed a strong trend toward higher mortality in the early repair group (adjusted OR 9.39 [0.93-95.18], adjusted p value = 0.058). The complication rate was similar in both groups (adjusted p value = 0.239). CONCLUSIONS: Delayed repair of stable blunt TAI is associated with improved survival, irrespective of the presence or not of major associated injuries. However, delayed repair is associated with a longer length of ICU stay and in the group of patients with no major associated injuries a significantly higher complication rate.

Stent thrombosis is associated with an impaired response to antiplatelet therapy

OBJECTIVES: We sought to evaluate the response to antiplatelet therapy in patients with stent thrombosis (ST). BACKGROUND: Stent thrombosis is associated with considerable morbidity and mortality. An impaired response to antiplatelet therapy might be related to an increased risk for ST. METHODS: Eighty-two patients were included in the present study: 23 patients with previous ST, 50 matched controls (coronary stenting without ST), and 9 healthy volunteers. Platelet aggregation (PA) was studied (optical aggregometry) under monotherapy with acetylsalicylic acid (ASA) 100 mg daily for one month, followed by dual therapy with ASA 100 mg and clopidogrel 75 mg daily (loading dose 300 mg) for another month. RESULTS: Maximal (5 and 20 micromol) adenosine diphosphate-induced PA was significantly higher in patients with ST compared with controls (5 micromol, p < 0.005; 20 micromol, p < 0.05) and volunteers (5 micromol, p < 0.005; 20 micromol, p < 0.05). Resistance to ASA (>20% aggregation with 0.5 mg/ml arachidonic acid) was more prevalent in patients with ST (48%) compared with control patients (32%, p = ns) and volunteers (0%, p = 0.01). Clopidogrel significantly reduced PA in all three groups, but intergroup differences persisted. Clopidogrel resistance (<10% relative change) was similar in patients with ST, control patients, and volunteers (4%, 6%, and 11%, respectively, all p = NS). However, combined ASA and clopidogrel resistance was more prevalent in patients with ST (52%) compared with controls (38%, p = NS) and volunteers (11%, p < 0.05). CONCLUSIONS: Patients with previous ST show an impaired response to antiplatelet therapy with ASA compared with controls and volunteers. Additional treatment with clopidogrel is not able to overcome these differences in PA. Acetylsalicylic acid but not clopidogrel resistance appears to be associated with ST.

Does homeopathically potentized antimony stimulate coagulation? A summary of previous findings and results of an in vitro pilot study by means of thrombelastography

BACKGROUND: Potentized antimony is traditionally used in anthroposophic medicine to enhance hemostasis in bleeding disorders, but evidence of its effectiveness is scarce. On the other hand, non-toxic and economic additional therapeutic options for hemostatic disorders are desirable. OBJECTIVES: We examined all available literature on the subject and performed a controlled pilot in vitro study to test the procoagulatory potency of antimony D 5. DESIGN: Freshly drawn citrated whole blood of 12 healthy volunteers and 12 patients with bleeding disorders was equally distributed into 344 portions, after which it was mixed with antimony D 5, or its potentized vehicle (lactose D 5) as control solution and tested with thrombelastography. The paired t-test and the Wilcoxon signed rank test were used for statistical analysis. In 5 of the 12 healthy donors, a second blood sample was drawn to assess individual variability and increase the total number of replicates. Thus three separate calculations were performed: for the 12 patients, the 12 healthy donors, and the 5 later samples from the same donors. The analysis was exploratory, and no Bonferroni correction was applied. RESULTS: In the antimony D5 samples of the 12 healthy subjects, but not the patients, there was a tendency toward a shorter clotting time (CT) (p = 0.074) and a trend for an increased clot firmness, expressed as maximal amplitude (MA) (p = 0.058). The increase of MA was significant (p = 0.011) when the later samples were included. No statistical difference was detected for the clot formation time and the clot lysis index. CONCLUSION: The exploratory results of this pilot study are inconclusive as to whether antimony D5 has a procoagulatory effect in vitro, although the results suggest an effect on MA and possibly CT. More research is warranted.