Alzheimer’s disease (AD) is pathologically characterized by extracellular beta-amyloid (Aβ) plaques and intraneuronal tau tangles in the brain. A therapeutic strategy aims to prevent or clear these Aβ plaques and the Aβ-degrading enzyme neprilysin is a potent drug to degrade plaques. The major challenge is to deliver bioactive neprilysin into the brain via the blood-brain barrier. The aim of the present study is to explore if intranasal delivery of neprilysin can eliminate plaques in a transgenic AD mouse model (APP_SweDI). We will test if collagen or platelets are useful vehicles to deliver neprilysin into the brain. Using organotypic brain slices from adult transgenic APP_SweDI mice, we show that neprilysin alone or loaded in collagen hydrogels or in platelets cleared cortical plaques. Intransasal delivery of neprilysin alone increased small Aβ depositions in the middle and caudal cortex in transgenic mice. Platelets loaded with neprilysin cleared plaques in the frontal cortex after intranasal application. Intranasal delivery of collagen-loaded neprilysin was very potent to clear plaques especially in the middle and caudal parts of the cortex. Our data support that the Aβ degrading enzyme neprilysin delivered to the mouse brain can clear Aβ plaques and intranasal delivery  is a fast and easy application. However, it must be considered that intranasal neprilysin may also activate more plaque production in the transgenic mouse brain as a side effect. The life expectancy of humans has markedly increased over the last 100 years. As age is the main risk factor for Alzheimer’s disease (AD), the number of patients suffering from AD and mixed forms of dementia will dramatically increase over the next 50 years. It is expected that there will be about 80 million AD patients worldwide in 2050. AD is characterized by severe β-amyloid (Aβ) deposition in brain (extracellular plaques), tau pathology , cell death of cholinergic neurons (loss of the neurotransmitter acetylcholine), astroglial and microglial activation, inflammation and cerebrovascular damage.Amyloid-precursor protein (APP) is a large transmembrane protein and secretases process different Aβ peptides. Especially the 42 amino acid Aβ peptide is toxic and can aggregate and form the typical plaques in the AD brain. The plaques consist of a central core of highly aggregated Aβ peptides and a halo around the plaques , which c3/Phe4, Gly9/Tyr10 (human only), Phe19/Phe20, Ala30/Ile31, and Gly33/Leu34. NEP has been shown to cleave at these sites in a phosphoramidon-sensitive manner with a Km of 2.8 μM for Aβ42  zinc metalloendo-peptidase family expressed by neurons and cerebrovascular smooth muscle cells and is the most potent Aβ-degrading enzyme . NEP is for Aβ42 . Intracefor Aβ42 . Furtherfor Aβ42 . Miners for Aβ42 ,b.in vivo in AD mouse models. One approach uses intracranial injection directly into the brain  and do not find their targets easily. Thus, different strategies need to be tested, to deliver stable neprilysin directly into the brain, especially he brain  with manhe brain  can be ehe brain  and migrhe brain . Howeverhe brain , or GDNFhe brain  or FGF-2he brain  can be lAlready in 1933 the first report was published on intranasal instillation , which ein vivo mouse models and test if intranasal applications can clear plaques in transgenic AD mice.The aim of the present study was (1) to strengthen our previous findings  that nepIn this study we used wildtype (C57BL/6N) and transgenic APP_SDI (expressing amyloid precursor protein (APP) harboring the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations; C57BL/6-Tg (Thy1-APPSwDutIowa)BWevn/Mmjax) mice, which were housed at the Medical University of Innsbruck animal facility providing open access to food and water under 12 h/12 h light-dark cycles. The transgenic mice produce Aβ plaques at an age >6 months  and we h2 with 1.2 ml/well of the following culture medium: 50% MEM/HEPES (Gibco), 25% heat inactivated horse serum , 25% Hanks’ solution (Gibco), 2 mM NaHCO3 , 6.5 mg/ml glucose , 2 mM glutamine , pH 7.2. In order to study clearance of plaques, neprilysin alone, or neprilysin loaded into collagen hydrogels, or neprilysin loaded into platelets were applied to the brain slices and incubated for 2 weeks, then postfixed 3 h with 4% paraformaldehyde and then stained by immunohistochemistry.For brain slice cultures 9 month old adult mice were used and organotypic brain slices were performed as described in detail ,b, 2018.Recombinant human neprilysin/CD10 was purchased from R&D (Nr. 1182-ZNC-010), dissolved in 100 μl sterile a.d. (100 μg/ml), aliquoted and stored frozen at minus 80°C; at the day of use, aliquots were thawed and directly used for the experiment.®, United Kingdom) was linked with 0.8 mg 4S-StarPEG in phosphate buffered saline (PBS) at pH 7.4, obtaining a final hydrogel of 2 mg/ml collagen and 1:2 molar ratio of coll:4S-StarPEG. Drops of 2 μl were pipetted onto Teflon tapes and gelled for 1 h at 37°C.In order to guarantee a slow, stable and local application of substances, neprilysin was loaded in collagen-loaded hydrogels. We have extensive experience in applying substances onto brain slices using non-toxic collagen hydrogels, which degrade over time and release the factor of interest , 2021. BWhen loading NEP, 10 μl of a peptide stock (1 μg) was added to the collagen hydrogels before crosslinking. Hydrogels (2 μl drops) have a size of approx. 1–2 mm; these hydrogels can be easily handled (see g for 10 min to obtain platelet rich plasma (PRP). All centrifugation steps were performed at room temperature. PGI2  was added to prevent platelet activation during processing. Platelets were separated from PRP by centrifugation at 400×g for 10 min and were either dissolved in 10 μl PBS or 10 μl neprilysin stock solution (1 μg neprilysin/10 μl). To load neprilysin into platelets, platelets were then incubated with neprilysin in an ultrasound path  for 3 h on ice as reported by us  were anesthetized with ketamine (100 mg/kg) and Xylazine (10 mg/kg) and blood was directly drawn from the heart by using a 21 gauge butterfly blood collection system . The blood was collected in ethylenediaminetetraacetate (EDTA) tubes  and gently mixed. Platelets were isolated as described in detail  and chared by us . Then 20 ca. 30% . In each2 stream, cryosectioned (40 μm) and stained by immunohistochemistry. As a control and to optimize the intranasal delivery (a) blue fluorescent Aβ  was infused and analyzed after 24 h or (b) a cresyl violet saturated solution was infused and analyzed after 3 h. Brains were taken, frozen in a CO2 stream, cryosectioned and visualized under the fluorescence microscope (Aβ) or light microscope (cresyl violet) or counterstained with blue DAPI .Mice were narcotized with Ketamine (100 mg/kg) and Xylazine (10 mg/kg). Ten μl substances were applied 5 mm deep bilaterally into the nose via the nostril (naris) using magnification glasses and a bright light using a 10 μl pipette (white tips) see . The subCollagen hydrogels, neprilysin standards (20-50-100 ng/lane) or medium with released neprilysin (20 μl per lane) were denatured (10 min 70°C with reducing agent) and loaded onto 10% Bis-Tris polyacrylamide gel (Invitrogen) and electrophoresis was performed for 35 min at 200 V. Samples were electrotransferred onto PVDF membranes for 20 min at 25 V in a semi-dry transfer cell (Thermo Fisher Scientific). Blotting was done by using WesternBreeze Chemiluminescent immunodetection system (Invitrogen). Blots were blocked with blocking buffer for 30 min and incubated overnight on a shaker at 4°C with primary antibodies against neprilysin . Subsequently, blots were briefly washed and incubated with alkaline phosphatase-conjugated secondary mouse (neprilysin) for 30 min at room temperature. Afterward, the blots were briefly washed again, incubated for 15 min in CDP-Star chemiluminescent substrate solution (Roche) and visualized with a cooled CCD camera .2O2/5% methanol. After incubation, the sections were then blocked in T-PBS/20% horse serum (GIBCO Invitrogen)/0.2% BSA (SERVA) for 30 min at 20°C shaking. Following blocking with mouse IgG blocking reagent (Vector MKB-2213), brain sections were incubated with primary antibody against beta-amyloid  or neprilysin  in T-PBS/0.2% BSA overnight at 20°C. The sections were then washed and incubated with a biotinylated secondary mouse antibody  in T-PBS/0.2% BSA for 1 h at 20°C shaking. Following secondary antibody incubation, sections were rinsed with PBS and incubated in avidin-biotin complex solution  for 1 h at 20°C shaking. Finally, the sections were washed with 50 mM Tris-buffered saline (TBS) and then incubated in 0.5 mg/ml 3,3’-diaminobenzidine /TBS/0.003% H2O2 at 20°C in the dark until a signal was detected. Once DAB staining was visible, the reaction was stopped by adding TBS to the sections. The brain sections were rinsed with TBS, inserted into a 6-well plate (slice down to well surface) directly onto a drop of Vectashield (Vector), cover-slipped and then evaluated under an inverse microscope (Leica DM IRB). For neprilysin the stainings were visualized using anti-mouse Alexa-488 antibody and brain slices counterstained with nuclear blue fluorescent DAPI. In addition, plaques were also stained by thioflavin S  and counterstained with nuclear DAPI.Immunohistochemistry was performed as previously described under free-floating conditions . The sli2 area.Brain sections at the cortical level were analyzed by a blinded evaluator as reported previously . Brieflypost hoc test where p < 0.05 represents significance.Statistical analysis was performed by a One Way ANOVA with a subsequent Fisher LSD Collagen hydrogels were visualized by loading fluorescent Alexa-488 antibodies and were found as ca. 2 mm round pieces . In ordeAdult organotypic brain slices were cultured for 2 weeks on semipermeable membrane inserts . The hydIn the next step we wanted to show if neprilysin loaded to platelets can also clear the plaques in the slices taken from adult transgenic mice. In this experiment we quantified small, medium and large plaques, because we expected less effects. In control slices 75 small, 26 middle sized and 12 large plaques were found per field . This nuin vivo mice via intransal infusion  did not affect the number of small and large plaques compared to controls  as analyzed by computer-assisted analysis .In order to see if intransal delivery of neprilysin can clear plaques in the brain, neprilysin was infused directly into the nose of anesthetized mice (repeated on day 1-2-3) and analyzed after 6 days. No small and large plaques were seen in wildtype mice . In tranin vivo.In the present study, we show that the Aβ-degrading enzyme neprilysin can clear plaques in an organotypic brain slice model of adult transgenic AD mice. We show that neprilysin can be loaded into collagen hydrogels and that intranasal delivery of the neprilysin-loaded collagen hydrogels eliminates plaques in the caudal cortex ex vivo AD model  are 3–5 μm small anuclear cells and play a central role in the blood clotting process and repair of vessels but are also affected in AD or cerebral amyloid angiopathy  were infused via the left and right nostril in anesthetized mice using a 10 μl pipette. Twenty four hours after infusion of blue fluorescent labeled Aβ several blue-labeled cells were seen in the bulbus but not in deeper brain areas. These stainings were visualized intracellular and we conclude that the Aβ peptide was incorporated by phagocyting cells . Not many papers have been published demonstrating intranasal delivery of Aβ. The most prominent one is the study of In the second step, we infused now neprilysin alone using our optimized intranasal delivery system. As a delivery matrix we used PBS-EDTA-heparin, which prevents blood clotting in case we damage the nose or cause some bleedings. When neprilysin alone was infused then we observed that the number of Aβ depositions increased in the middle and caudal cortex. This was unexpected and it is not easy to explain. While we expected that neprilysin should eliminate the plaques it had the opposite effects. We suggest that intranasal neprilysin rapidly diffused to the brain and rapidly cleared plaques, but this clearance process activated mechanisms in the brains of the transgenic mice to compensate and produce more plaques. These activation mechanisms are not known now. It would be interesting to see if neprilysin could clear the plaques at earlier time points.As shown, we succeded to load neprilysin into platelets. As platelets are very small anuclear cells, we wanted to take this as an advantage to deliver platelets as a vehicle to allow migration into the brain. In previous studies we delivered platelets via the intravenous route, but failed to find many platelets in the brain, as most of them were trapped in the periphery  . Thus inIn the final step we wanted to take advantage of the collagen hydrogels as a stable delivery tool. Collagen hydrogels were applied directly into the nose on both sides and we expected that they are stable and degrade within a few days and release neprilysin. This application was repeated on 3 consecutive days. In the present study it was not possible to follow up the collagen degradation in the nose and directly proof that bioactive stable neprilysin was released from the collagen. However, our immunostainings clearly show that neprilysin loaded to collagen hydrogels and placed in the nostrils, massively cleared Aβ plaques in the middle and caudal cortex. This effect was not seen in the hippocampal formation. It was surprising that the effect was quite strong and shows for the first time, that collagen-loaded hydrogels are potent intranasal delivery tools. It was also interesting to note that the plaque density in the frontal cortex was not affected. This is not easy to explain, but we suggest a time-dependent process. We hypothesize that neprilysin is slowly released in the nose from collagen hydrogels and in contrast to the pure infusion of neprilysin, it slowly diffuses into the brain. We think that at the observed time point (day 9), neprilysin is active in the caudal cortex, while in the frontal parts again new plaques are produced by the transgenic mouse brain via yet unknown mechanisms. Again an earlier time point could be interesting to test.in vitro intranasal AD model has definitively several limitations. (1) The most severe limitation is the low neuronal viability of the adult slices, however, as we do not study neurons and only plaques, this issue can be neglected in the present study. (2) As mentioned, the difference between the frontal and caudal parts is not clear. While is may make sense that platelets do not migrate deeper, it is unclear why pure neprilysin enhances plaques in the caudal parts, while collagen-loaded neprilysin massively clears the plaques. It seems to be reasonable that the brain has a very high plasticity and activates (yet unknown) processes to produce more plaques. (3) In our present study we did not follow up degradation of collagen and also not the bioactivity of neprilysin in the nose. We also did not test any negative side effects in the nose , and we also cannot say how the neprilysin enters the brain and which pathways are involved. (4) In our present study, we clearly show that neprilysin has potent stable activity to clear plaques in vitro. We also clearly show that intranasal application of neprilysin is very potent to eliminate plaques, and a slow releasing collagen hydrogel has many advantages. Definitely, it would be interesting to see if intranasal delivery of neprilysin clears plaques in human AD patients. (5) If our data are correct and the interpretations point to a compensating mechanism, then the intranasal delivery activates plaque formation after terminating the neprilysin effect. This is of course contra productary and not helpful. Our data first provide evidence that indeed the elimination of plaques can be followed by direct new production of plaques, but it may also indicate that a neprilysin therapy may need a continuous delivery all 2 weeks for several months. This indeed would be an interesting therapeutic approach.This in vivo. In conclusion, we provide further evidence that neprilysin is a potent drug to clear plaques and that intranasal delivery is a potent method to deliver neprilysin into the brain, however, compensating enhanced plaque production must be considered.Taken together, in the present study we show that the Aβ-degrading enzyme neprilysin can clear plaques in an organotypic brain slice model of adult transgenic AD mice. We show that neprilysin can be loaded into collagen hydrogels and that intranasal delivery of the neprilysin-loaded collagen hydrogels clears plaques in the caudal cortex The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Austrian Ministry of Science.CH designed and analyzed the data, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiac troponin concentrations differ in women and men, but how this influences risk prediction and whether a sex-specific approach is required is unclear. We evaluated whether sex influences the predictive ability of cardiac troponin I and T for cardiovascular events in the general population.High-sensitivity cardiac troponin (hs-cTn) I and T were measured in the Generation Scotland Scottish Family Health Study of randomly selected volunteers drawn from the general population between 2006 and 2011. Cox-regression models evaluated associations between hs-cTnI and hs-cTnT and the primary outcome of cardiovascular death, myocardial infarction, or stroke.P < 0.001 for both), and both were more strongly associated with cardiovascular events in women than men. For example, at a hs-cTnI concentration of 10 ng/L, the hazard ratio relative to the limit of blank was 9.7 (95% CI 7.6–12.4) and 5.6 (95% CI 4.7–6.6) for women and men, respectively. The hazard ratio for hs-cTnT at a concentration of 10 ng/L relative to the limit of blank was 3.7 (95% CI 3.1–4.3) and 2.2 (95% CI 2.0–2.5) for women and men, respectively.In 19 501  participants, the primary outcome occurred in 2.7% (306/11 375) of women and 5.1% (411/8126) of men during the median follow-up period of 7.9  years. Cardiac troponin I and T concentrations were lower in women than men (Cardiac troponin concentrations differ in women and men and are stronger predictors of cardiovascular events in women. Sex-specific approaches are required to provide equivalent risk prediction. Cardiovascular disease remains the main cause of death worldwide with 17.6 million people dying each year . The devCardiac biomarkers may provide an unbiased aproach toward the prediction of cardiovascular events in women and men. Increasingly, high-sensitivity cardiac troponin is considered a useful marker of risk outwith the setting of acute coronary syndromes to evaluate asymptomatic individuals and guide therapeutic approaches to prevent the onset of cardiovascular disease . Recent It remains unclear in practice how best to harness this prognostic information to guide the use of primary and secondary prevention, and whether sex-specific troponin thresholds should be considered. The use of high-sensitivity assays has identified important differences in troponin concentrations between men and women, with the 99th centile upper reference limits used for diagnosis of myocardial infarction up to 2-fold higher in men . In our The Generation Scotland Scottish Family Health Study is a well-phenotyped family-based cohort that enrolled 24 090 participants aged between 18 and 98 years and has been described previously , 16, 19.Participants completed a health questionnaire, and had physical characteristics and clinical characteristics measured according to a standardized protocol . Past mei1000SR high-sensitivity cardiac troponin I assay (Abbott Diagnostics) and cardiac troponin T was measured on Cobas e411 high-sensitivity cardiac troponin T (Roche Diagnostics) assay. During the conduct of this study, we participated in the National External Quality Assurance Scheme (UKNEQAS) for these biomarkers. Both assays were calibrated and quality controlled using the manufacturer’s reagents. Coefficient of variations for cardiac troponin I were 6.2%, 6.0%, and 4.6% for the low, intermediate, and high control, respectively. Coefficients of variation for cardiac troponin T were 5.0% and 3.4% for the low and high control, respectively. Cardiac troponin T has a limit of blank (LoB) of 3 ng/L and limit of detection (LoD) of 5 ng/L. Cardiac troponin I has a LoB of 1.2 ng/L and LoD of 1.9 ng/L (Serum cardiac troponin I was measured on ARCHITECT 1.9 ng/L .a) cardiovascular death (I00 to I99), (b) myocardial infarction , and (c) stroke . Secondary outcomes were cardiovascular death, noncardiovascular death, and all-cause death.We used the Information Services Division National Health Service record linkage for Scotland to collect clinical outcome data until the end of September 2017. Information on cause of death was obtained using the National Health Service Central Register. Clinical outcomes were classified using the 10th revision of the International Classification of Diseases (ICD-10). The primary outcome was a composite endpoint of cardiovascular events including the following component endpoints: (Continuous variables are presented as mean (SD) or median (25th–75th percentile), as appropriate. Categorical variables are presented as absolute numbers (%). For continous analyses, troponin values below the LoB were set to the LoB value divided by 2. The correlation between cardiac troponin I and T was assessed by Spearman correlation. Sex-specific incidence rates were calculated per 1000 person-years for clinical outcomes.Unadjusted and adjusted multiple fractional polynomial Cox proportional hazard regression analysis were conducted to quantify the relationship between cardiac troponin as a continuous variable with the primary outcome, stratified by sex. The multivariable model is adjusted for age, total cholesterol, high-density lipoprotein cholesterol, systolic blood pressure, cigarettes smoked per day, rheumatoid arthritis, diabetes mellitus, Scottish Index of Multiple Deprivation score, family history of cardiovascular disease, use of blood pressure medications, and use of cholesterol-lowering medications. Each continuous variable was chosen through backward-stepwise selection of the best fractional polynomial transformation. We created hazard ratio (HR) plots for the primary outcome at 5 years of the unadjusted and adjusted cardiac troponin I and T models for women and men, and evaluated the HR relative to the LoB. Due to the low proportion of missing covariates (<6%) and the large number of available samples we did not use imputation techniques, focusing on complete case analysis instead. We constructed receiver operating characteristic (ROC) curves and determined the area under the curve (AUC) to assess discrimination of cardiac troponin for predicting the primary outcome at 5 years in women and men. Individuals with no events by the 5-year mark were censored. Comparisons between unpaired AUCs were tested according to the DeLong method. In secondary analyses, we evaluated the HR relative to the LoD, and we evaluated the additional outcomes of cardiovascular death, noncardiovascular death, and all-cause death. All statistical analysis was performed using R v.3.6.2.P < 0.001 for both; r = 0.351, 95% CI 0.334 to 0.370 versus r = 0.446, 95% CI 0.428 to 0.463; P < 0.001).Our study population included 19 501 individuals  years, and a total number of 717 (3.7%) individuals experienced a primary outcome event. In those participants with an incident cardiovascular event , women wBased on our unadjusted and adjusted regression models we illustrate the HR of a cardiovascular event at 5 years according to cardiac troponin I and troponin T concentrations in men and women . For estP = 0.080), and for cardiac troponin T  there was a trend toward better discrimination in women than in men.Overall, cardiac troponin I and T concentrations had a good discriminative ability to predict 5-year cardiovascular risk . For carP < 0.001 for both) and all-cause death (P < 0.001 for both) in women and men in fully adjusted models. Cardiac troponin I was not associated with noncardiovascular death in either women (P = 0.597) or men (P = 0.364), whereas cardiac troponin T was for both sexes  study, the Nord-Trøndelag Health Study (HUNT) study and the Activity and Function in the Elderly in Ulm (ActiFE) study, showing an interaction between troponin and sex in relation to future cardiovascular events across different ethnicities and age groups , 22, 23.What are the implications of these observed sex differences? Women tend to be undertreated for cardiovascular risk , and carIn contrast to the acute care setting, the general population contains a high proportion of individuals with cardiac troponin values below the LoD. As our study was not designed to develop a risk prediction tool, but rather to evaluate sex differences between troponins in this setting, we have used cardiac troponins over their entire concentration range. We therefore cannot exclude that the imprecision profile of these assays in individuals with very low cardiac troponin concentrations may have affected the accuracy of our results. When using the LoD rather than the LoB as the reference, differences between women and men were less pronounced. Women have lower troponin concentrations than men and therefore a greater proportion of women have undetectable cardiac troponin concentrations. Our analyses suggest that discrimination in the modeling of future cardiovascular events is partly dependent on being able to identify those individuals who are very low risk with the lowest cardiac troponin values. The clinical implications of this are important. For example, in the USA, cardiac troponin values below the LoD are not reported because of concerns about assay imprecision. While precision is greater in those with higher values and therefore the user can be more confident in actioning the results of those identified as higher risk, it is less clear that based on current analytical precision (and reporting requirements) we are fully harnessing the potential of these tests to identify those who are lower risk. Those developing clinical tools to guide primary prevention approaches that incorporate cardiac troponin should be aware that including troponin values below the LoD may affect the accuracy of prediction and limit the future application of these tools in practice.Another important observation in our study is that cardiac troponin T, but not cardiac troponin I predicts noncardiovascular death in both women and men. This extends our previous finding that cardiac troponin I has a greater specificity for future cardiovascular risk , 16. AltSeveral limitations merit attention. First, no cardiac imaging data were available and studying the possible structural microvascular cardiac differences between women and men in relation to troponin and outcome was not possible, although this is of secondary value as we have incident cardiovascular outcomes. Second, most of the Generation Scotland Family Health Study participants are Caucasian and generalizing our findings to other ethnic groups should be done with caution. Third, although high-sensitivity testing was used, still a high proportion of individuals had undetectable cardiac troponin concentrations, particularly for cardiac troponin T and particularly in women. Imprecision in those with very low cardiac troponin concentrations might have influenced the accuracy of our model estimates. Finally, cardiac troponin I was only measured using one manufacturer’s assay, which precludes the direct extrapolation of our findings to other cardiac troponin I assays.In conclusion, cardiac troponin I and T are independent predictors of cardiovascular events in both women and men in the general population. Cardiac troponin concentrations differ in women and men and are stronger predictors of cardiovascular events in women. Sex-specific approaches are required to provide equivalent risk prediction when using high-sensitivity cardiac troponin testing in isolation for risk prediction and the prevention of cardiovascular disease. Ideally, cardiac troponin would be used as a continuous measure in a cardiovascular risk prediction tool that incorporates sex and other clinical features.Clinical Chemistry onlinehvab109_Supplementary_DataClick here for additional data file.


The effect of smoking on coronary artery plaques examined by coronary computed tomography angiography (CCTA) in type 2 diabetes mellitus (DM) patients is not fully understood. This study explored the effect of smoking on coronary artery plaques by comparing the characteristics of plaques between diabetes patients with and without a smoking history and among those with different smoking durations.In total, 1058 DM patients found to have coronary plaques on CCTA were categorized into the smoker (n=448) and nonsmoker groups (n=610). Smokers were stratified by smoking duration . The plaque types, luminal stenosis [obstructive (<50%) or nonobstructive (≥50%) stenosis], segment involvement score (SIS), and segment stenosis score (SSS) of the CCTA data were compared among groups.Compared to nonsmokers, smokers demonstrated increased odds ratios (ORs) of any noncalcified plaques , obstructive plaques , multivessel disease , SIS≥4 , and SSS≥7 . Compared to diabetes patients with a smoking duration ≤20 years, those with a smoking duration of 20~40 years and >40 years had higher OR of any mixed plaques , obstructive plaques , multivessel disease , and SSS≥7 . Diabetes with a smoking duration >40 years had a higher OR of SIS≥4 .Smoking is independently associated with the presence of noncalcified, obstructive, and more extensive coronary artery plaques in diabetes patients, and a longer smoking duration is significantly associated with a higher risk of mixed, obstructive, and more extensive plaques. Diabetes seriously threatens global health, and the number of adults with diabetes has more than tripled over the past 20 years . For typCoronary computed tomographic angiography (CCTA), a noninvasive diagnostic technique, has played an important role in detecting the presence of coronary artery plaques and assessing the severity of coronary artery stenosis for a long time, and treatment decision-making in coronary artery disease based solely on CCTA is potentially feasible because of its strong agreement with decisions derived from conventional coronary angiography . Previou2), severe liver failure. Consequently, 1058 patients were enrolled in our study. The clinical data of the patients consisted of age, sex, body mass index (BMI), duration of DM, presence of complications of DM, duration of smoking, hypertension history, dyslipidemia history, statins use, diabetes treatment, systolic blood pressure, and diastolic blood pressure. The laboratory data included fasting blood glucose, HbA1c, triglycerides, cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).This retrospective study was approved by the institutional ethics committee of our hospital, and the need for informed consent was waived. CCTA images of type 2 DM patients acquired between January 2018 and December 2020 in our hospital were reviewed. The inclusion criteria were as follows: clinical diagnosis of type 2 DM; patients with complete clinical data; and patients with coronary artery plaques on CCTA. The exclusion criteria were as follows: the image quality of CCTA was too poor to identify the state of the coronary artery; patients with a history of percutaneous coronary intervention or coronary artery bypass grafting; and patients with growth diseases, concomitant neuroendocrine tumors, severe renal failure  or a Siemens dual-source CT scanner . Beta-blocker was not used for the heart rate reduction. Patients were examined in the supine position. A 70-90 ml (tailored to body weight) bolus of iodinated contrast agent  was injected into an antecubital vein at a flow rate of 5 ml/s followed by a 30 ml saline flush at the same flow rate. The coverage of the CT scan was from the tracheal bifurcation to 20 mm below the inferior cardiac apex. For the Revolution CT system, the tube voltage and tube current were set automatically by kV Assist and Smart-mA based on the scout image of the patients, collimation was 256×0.625 mm, and the rotation time was 0.28 seconds. For the SOMATOM Definition system, the tube voltage was 100~120 kV, the tube current was 220 mAs, the collimation was 64/128×0.6 mm, and the rotation time was 0.33 s. Retrospective electrocardiographic gating was used to eliminate artifacts of cardiac motion. Subsequently, the initial data set was reconstructed upon completion of the scan, and images were transferred to image-processing workstations  for image analysis. Alternative image reconstruction methods, including multiplanar reconstruction, maximum intensity projection, volume reconstruction, and curvature plane reconstruction, were used for the evaluation of coronary artery plaques.Two cardiovascular radiologists blinded to the clinical information of the patients analyzed the images independently. The two observers reached a consensus by discussion when there were disagreements. In this study, coronary arteries were divided into 4 branches, including the left main (LM), left anterior descending (LAD), left circumflex (LCX), and right coronary artery (RCA), and into 16 segments according to a modified standard of the American Heart Association Figure 1t-test. Continuous variables with nonnormal distributions were compared using the Wilcoxon rank-sum test. The associations of the mean number of diseased vessels, SIS, and SSS with the subgroups mentioned before were analyzed. The smoker group was further divided into three subgroups according to the duration of smoking  to examine the effect of smoking duration on the coronary artery plaque type and burden. A two-tailed P value of less than 0.05 was considered to indicate a significant difference.Statistical analysis was performed using SPSS software (version 25.0). Baseline clinical data were stratified based on smoking history (whether there was a history of smoking). Baseline clinical and laboratory data, the number of involved coronary vessels and segments, the number and types of plaques, and the extent of luminal narrowing caused by plaques were analyzed statistically in each patient. Categorical variables are expressed as numbers and percentages, and continuous variables are expressed as the mean ± standard deviation. The chi-square test was used for categorical variables. Multivariate logistic regression was used to analyze the associations of plaque characteristics between the nonsmoker and smoker groups. Continuous variables with normal distributions were compared using Student’s A total of 1058 patients with DM who were found to have coronary artery plaques on CCTA were evaluated. The baseline clinical information is shown in In total, 1181 diseased coronary vessels with 2128 diseased segments and 1563 diseased coronary vessels with 2738 diseased segments were found in the smoker and nonsmoker groups, respectively. The anatomical distribution of the plaques is shown in vs. 1.7 ± 2.1 and 0.6 ± 1.0 vs. 0.4 ± 0.9 per patient, respectively; both P-values < 0.05). Mixed plaques and noncalcified plaques were more common, and calcified plaques were less frequently observed in the smoker group than in the nonsmoker group . DM patients in the smoker group had more obstructive plaques than those in the nonsmoker group . Regarding the grade of luminal stenosis, obstructive plaques were more frequently observed in the smoker group than in the nonsmoker group .A total of 4870 coronary plaques were analyzed. The types of coronary artery plaque and coronary artery stenosis stratified by smoking history are shown in vs. 9.7 ± 9.1, P < 0.05). There was no significant difference in multivessel disease, coronary vessels, or SIS between the two groups .The extents of coronary artery plaque and coronary artery stenosis stratified by smoking history are shown in As shown in As shown in The present study had four main findings. First, compared to those in the nonsmoker group, DM patients in the smoker group showed a higher prevalence of mixed plaques and noncalcified plaques and a lower prevalence of calcified plaques. Second, compared to those in the nonsmoker group, DM patients in the smoker group showed a higher prevalence of obstructive plaques. Third, smoking was an independent risk factor for noncalcified plaques, obstructive coronary artery disease, and more extensive lesions in DM patients after adjustment for confounding factors. Fourth, DM patients with longer smoking durations had a higher likelihood of mixed plaques and obstructive coronary artery disease and more extensive lesions than those with shorter smoking durations.DM patients already have a high coronary plaque burden, which indicates active progression of atherosclerosis , 16, andFor the types of plaques, the present data showed that the proportion of mixed plaques and noncalcified plaques was higher and that of calcified plaques was lower in the smoker group than in the nonsmoker group; when adjusted for confounding factors, smoking was independently associated with an increased adjusted odds ratio of the presence of noncalcified plaques, and a longer smoking duration was correlated with mixed plaques. Noncalcified plaques and mixed plaques show relatively high incidences of major adverse cardiovascular events, and noncalcified plaque is associated with an increased risk of atherosclerotic cardiovascular disease , 23. SimIt has been reported that the percent of low-density plaque content and mild calcification could predict late plaque events in asymptomatic patients with DM, and lumen narrowing caused by plaques increases the probability of an acute event . CalcifiAccording to the present data, DM patients with a smoking history had a higher risk of more extensive coronary artery disease than nonsmokers after adjustment for confounding factors. Patients with extensive coronary artery disease can benefit from surgical revascularization. The evaluation of the extent of coronary artery plaque assessment could be important for treatment. For DM patients with three-vessel coronary artery disease, coronary artery bypass grafting results in better long-term survival than percutaneous coronary intervention . A studyA previous study indicated that with increasing smoking duration, the adjusted hazard ratio for coronary heart disease also increased . It has There are some limitations of our research. First, since this is a single-center study, selection bias is inevitable in this study. Therefore, the results of this study remain to be verified by multicenter studies. Second, traditional coronary angiography results of the extent of coronary stenosis were not correlated with our CCTA findings, as CCTA can distinguish different types of plaques, and the assessment of coronary plaques by CCTA has been widely accepted. Third, as a retrospective study, the evolution of the disease remains to be discussed in further follow-up studies. Finally, nondiabetic patients were not enrolled in our study. Thus, further exploration is required to investigate the potential difference in characteristics of coronary artery disease between patients with and without DM.CCTA detected a heavy plaque burden in DM patients. Smoking is independently associated with the presence of noncalcified plaques, obstructive plaques, and more extensive coronary artery plaques in DM patients, and a longer smoking duration is significantly associated with a higher risk of mixed plaques, obstructive plaques, and more extensive coronary artery plaques than a shorter smoking duration.yangzg666@163.com.The datasets presented in this article are not readily available because of the privacy of patient information. Requests to access the datasets should be directed to Z-GY, The studies involving human participants were reviewed and approved by the Biomedical Research Ethics Committee, West China Hospital of Sichuan University . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.YJ, TP, YL, and Z-GY contributed to the conception and design of the study. YJ, TP, W-LQ, and W-FY collected the data. YJ, TP, and RS participated in the data analysis and interpretation. YJ wrote the manuscript with contributions from all authors. All authors contributed to the article and approved the submitted version.This study was financially supported by the 1·3·5 project for disciplines of excellence of West China Hospital, Sichuan University (ZYGD18013).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The TT only group did not receive any intervention throughout the detraining period (no‐TT group). Body composition was tested using anthropometrics, dual energy X‐ray absorptiometry, and magnetic resonance imaging. After an overnight fast, basal metabolic rate (BMR), lipid panel, serum testosterone, inflammatory biomarkers, glucose effectiveness, and insulin sensitivity were measured. Finally, peak isometric and isokinetic torques were measured only in the TT+RT group. All measurements were conducted at the beginning and at the end of DD. Absolute thigh muscle cross‐sectional areas (CSAs) demonstrated interaction effects (p < 0.05) between the TT+RT  and no‐TT  groups. Similarly, absolute knee extensor muscle CSA demonstrated interaction effects (p < 0.05) between the TT+RT  and no‐TT  groups. There was a trend (p = 0.07) of increasing visceral adipose tissue (VAT) CSAs in the TT+RT (18%) and in the no‐TT (16% cm2) groups. There was an interaction (p = 0.005) between TT+RT (decreased by 3.7%) and no‐TT groups (increased by 9.0%) in BMR. No interactions were evident between groups over time for biomarkers related to carbohydrate, lipid metabolism, or inflammation. Finally, there were no changes (p > 0.05) in peak isometric or isokinetic torques and rise time following 16 weeks of the DD period in the TT+RT group. TT+RT during 16 weeks of DD was minimally effective at preventing detraining relative to no‐TT on muscle size, BMR, and VAT. However, neuromuscular gains were successfully maintained.The dose de‐escalation (DD) effects of testosterone and evoked resistance training (RT) on body composition, cardiometabolic, and neuromuscular variables were investigated. Thirteen men with chronic complete spinal cord injury (SCI) were followed for additional 16 weeks after receiving either testosterone treatment only (TT) or TT+RT. During the 16‐week DD period, the TT+RT group underwent a program of once weekly electrical stimulation with gradually decreasing ankle weights and testosterone patches of 2 mg day Sixteen weeks of dose de‐escalation program did not adequately maintain the gain in muscle size and basal metabolic rate. However, it prevents detrimental changes in neuromuscular adaptations. A limited number of studies have focused on investigating the effects of DD . Reducing the frequency of training has previously been recommended as an effective strategy to enhance adherence and compliance to a longitudinal exercise programs  in the TT+RT group and ceased administration of testosterone in the TT group (no‐TT) similar to earlier work  on parameters of body composition, cardiometabolic profiles, and neuromuscular parameters compared to no‐TT  in men with chronic complete SCI. In the current study, the TT+RT group underwent a decrease in the dose of testosterone and the volume of NMES‐RT during the DD period. For the TT+RT, the DD program was designed by reducing the training volume (four sets of 10 to three sets of 10) and the training frequency (twice weekly to once weekly) from the original 16‐week training  that were alternated between left and right shoulders at bedtime. Additionally, the TT+RT group received 16 weeks of supervised progressive RT, twice weekly using surface NMES and ankle weights  and no‐TT (n = 6) on cardiometabolic risk factors and neuromuscular parameters. Two participants withdrew from the TT+RT group , was set to deliver biphasic rectangular pulses of 30 Hz, 450 µs pulses at a current amplitude (mA) sufficient to evoke full leg extension against gravity. The current was manually increased to evoke full leg extension (three sets of 10 repetitions) with a 2–3‐min rest between sets as previously described  as well as measuring peak isometric and isokinetic torques. Additionally, magnetic resonance imaging (MRI) scans were obtained for whole thigh, individual skeletal muscles and IMF CSAs, trunk VAT, and subcutaneous adipose tissue (SAT). On the day of body composition assessment, participants were reminded to consume adequate fluids to stay hydrated and to eat a light meal 2–3 h prior to testing . Fat‐free mass (FFM), fat mass (FM), %FM, and LM for total body, trunk, legs, arms, android, and gynoid regions were measured by DXA : Magnetic resonance imaging was performed at the VA Medical Center using a General Electric Signa 1.5‐T magnet as previously described  to capture multiaxial slices of the trunk region , between kidneys and umbilicus (VATK‐U), between iliac crests and femoral heads (VATIC‐FH), and total VAT . The amount of testosterone in each sample was calculated from a linear plot generated by purified testosterone standards ranging from 2.5 to 5000 ng/dl.Fasting lipid panel  was determined using enzymatic colorimetric assays. Inflammatory biomarkers C‐reactive protein (CRP), IL‐6, Tumor necrosis factor (TNF)‐α, and free fatty acids were determined by commercially available enzyme‐linked immunosorbent assay kits (ALPCO).After fasting blood samples, an intravenous line was placed to facilitate infusion of glucose and blood sampling , data were then log‐transformed prior to any statistical analysis. Outliers were detected using normal Q–Q plots at different time points (DD0 and DD16) for each group. Mixed‐model ANOVA tests were performed to examine main time effects (DD0 and DD16) and between‐group (TT+RT vs. no‐TT) differences as well as interactions between groups on the primary (muscle CSAs) and secondary outcome variables over the course of the 16‐week DD period. If there was an interaction effect, post hoc analyses were then followed using independent t‐tests. To further dissociate a main time effect (DD0 and DD16), paired t‐tests were conducted within each group separately. Intent‐to‐treat analysis approach was adopted for those who experienced withdrawal during the trial by maintaining their group assignments. The approach was used to ensure the current study was not underpowered. Statistical analyses were performed using IBM‐SPSS version 26.0 (SPSS) and all values are presented as mean ± SD.All data were tested for normality using the Shapiro–Wilk tests and if necessary (3n = 9) withdrew from the training, because of conflict with either personal commitments or work. However, one out of the three participants agreed to participate in the DD0 and DD16 measurements without receiving testosterone patches or participating in the NMES‐RT (his data were included). The second and third participants withdrew after completion of week 1 and week 7, respectively . Seven participants were considered for analysis in the TT+RT group. In the TT+RT group (n = 6), adherence to the progressive RT protocol was 96 ± 5% over 16 weeks.Physical and SCI characteristics were not different between the two groups during the 16‐week DD period  and repetitions (30 repetitions/per week) on both legs. Progression of the training for both lower extremities in the TT+RT group is listed in Table p < 0.05) and were log‐transformed before conducting further statistical analyses.Fasting insulin, IL‐6, and CRP data at DD0 and DD16 did not meet the assumption of normality  in both TT+RT  and no‐TT  groups, but not a significant group*time interaction (p = 0.25) for serum testosterone concentrations. Follow‐up paired t‐tests indicated that there was no difference in the TT+RT group (p = 0.100) and there was a trend (p = 0.064) within DD0 and DD16 in the no‐TT group.Four days after removal of the patches, there was a significant main effect of time (3.1.1p = 0.065) sagittal diameter in both groups  that indicated decline in both groups (Table p = 0.018); a follow‐up independent t‐tests revealed differences in leg lean mass at DD0 (p = 0.005) and a trend at DD16 (p = 0.058).Arm lean mass and FFM showed a significant time effect (ps Table . A betwep = 0.09).There was a trend of interaction in total body FM between TT+RT and no‐TT groups (Table p = 0.02), mid right slices , and proximal left slices . There was also a trend (p = 0.056) of an interaction in the average absolute thigh muscle CSA between TT+RT and no‐TT groups (Table p = 0.081–0.057), mid four slices (p = 0.04–0.037), distal four slices (p = 0.038–0.058), and the average (p = 0.048–0.0043) absolute thigh muscle CSA between TT+RT and no‐TT groups. Follow‐up independent t‐tests revealed that the proximal slices (p = 0.03), mid slices (p = 0.05–0.23), distal (p = 0.039 −0.050), and average (p = 0.034–0.035) absolute thigh absolute muscle CSA were significantly different between TT+RT and no‐TT groups at DD0 but not at DD16.Absolute thigh muscle CSAs  showed interaction effects in the proximal right slices  and proximal left , right‐mid  and left‐mid , and right average  and left  absolute knee extensor muscle CSA. There were also between‐group effects in the proximal slices (right: p = 0.012 and left: p = 0.012), mid slices (right: p = 0.004 and left: p = 0.012), distal slices (right: p = 0.004 and left: p = 0.008), and average (right: p = 0.006 and left: p = 0.01) absolute knee extensor CSAs .There was a trend of an interaction between TT+RT and no‐TT groups in left adductor muscle CSA . There were no differences between TT+RT and no‐TT groups. SATL‐k, k‐U, IC‐FH remained unchanged (p > 0.05) in both the TT+RT (184±121 to 191±107 cm2) and no‐TT (155 ± 67 to 149 ± 66.5 cm2) groups.Figure 3.1.3ps = 0.845 or 0.109, respectively), resting diastolic , and resting heart rate  during the 16‐week DD period or any interaction between groups (p > 0.05).There were no main effect of time or between‐group differences in resting systolic  or between‐groups effect (p = 0.178) in BMR. Contrary, there was an interaction (p = 0.005) between TT+RT  and no‐TT  groups in BMR. A follow‐up independent t‐tests revealed that there was a difference between TT+RT and no‐TT groups at DD0 (p = 0.015) but not at DD16.There was no main time effect (p > 0.05) in fasting insulin, fasting glucose, insulin sensitivity (Si), glucose effectiveness (Sg), HBA1c (%), lipid panel, CRP, TNF‐α, IL‐6, or free fatty acids.Carbohydrate, lipid, and inflammatory biomarker profiles are presented in Table 3.1.4n = 7), there were no changes (p > 0.05) in peak isometric torques at 50 mA or 100 mA following 16 weeks of the DD period. The slowness in the rise time was also maintained at both 50 mA  and 100 mA . Isokinetic peak torques were also maintained (p > 0.05) at 60, 90, and 180 deg s−1 in the TT+RT group.In the TT+RT group  and reciprocally regressed the weightlifting program by decreasing the frequency of NMES‐RT from twice to once weekly and decreasing ankle weights by 2 lbs. per week. At the end of week 12, participants in the TT+RT performed 30 reps per leg and decreased weights to 2 lbs. and continued training for additional 4 weeks with the same weights. Similar to previous work  with longer carryover effects in men with SCI , data analysis, and drafting manuscript; M. Rehan Khan: Facilitate MRI capturing and oversee completion of the radiological scans, data collection and management; Robert A Adler: Medical monitor person, prescription of testosterone, oversee subjects' across the trial, data analysis, and drafting manuscript.Table S1Click here for additional data file.


Primary leiomyosarcoma in testis is an uncommon tumor with few cases reported. It generally develops after radiotherapy or long-term taking anabolic steroid medication. We report a 53-year-old male patient with primary testis leiomyosarcoma who presented with painless testicular enlargement without any known predisposing factors. Ultrasound revealed a large heterogeneous left testicular solid lesion. Alpha-fetoprotein (AFP) and beta-human chorionic gonadotrophin (beta-HCG) levels in serum were normal. Left radical orchidectomy following with histology assessment established a diagnosis of primary leiomyosarcoma of testis. No data of cancer metastasis was established. The patient didn’t receive any adjuvant therapy. There wasn’t any evidence of recurrence after 1 year follow-up. Leiomyosarcoma must be one of the differential diagnoses of seronegative tumors in testis. The motivation for this paper is the extreme infrequency of the situation and the differential diagnosis by all expansive inguinoscrotal tumors. Malignant spindle cell neoplasms have low frequency and are commonly sarcomas. Leiomyosarcoma accounts for 5%–10% of sarcoma . LeiomyoA 53-year-old male referred to the urology unit in Fajr hospital in Mariwan, Iran, with a six months’ history of left testis painless enlargement without any known predisposing factors. Outpatient examination findings were normal. Family history and past medical history of the patient was unremarkable. General examination was insignificant except for a mass (about 4 cm in greatest diameter) in the left testis. No other masses were detected anywhere else. Routine laboratory investigations were normal. Alpha-fetoprotein (AFP), lactate dehydrogenase (LDH) and Beta-human chorionic gonadotropin (B-HCG) levels in serum were 2.61 ng/mL, 321 IU/L and <0.1 mIU/mL, respectively which were in normal limit for the age and gender. The patient underwent ultrasound imaging, which showed a solid heterogeneous mass in the left testis. The patient was treated by inguinal high ligation radical orchiectomy due to suspicion of testicular malignancy. He passed the procedure without complications. The received specimen for histopathologic assessment consisting of left testis was measured to be 6.5 × 4.3 cm and attached spermatic cord was measured 10 cm of length and 2 cm of diameter. Sections showed an intratesticular whitish lesion with whorling pattern in upper pole measuring 4 × 3 × 3cm . HistoloThe reason of primary testis leiomyosarcoma is controversial. Its source has been recognized from smooth muscle cells from tunica propria or tunica albugínea or seminiferous tubules or blood vessel walls . PrimaryThere are limited information in the literature about the leiomyosarcoma of testis in terms of nature, histomorphology appearance, prognostic indicators, survival rate, and treatment/management due to its rare occurrence. Previous studies reveal that these tumors may be treated if diagnosed early as well as these tumors need to be differentiated from other conventional tumors of the spermatic cord origin and those related to the germ cell tumors.


Crenosoma striatum was found to be associated with bronchial lesions. This study contributed to the knowledge of parasitic infections of the European hedgehog, the most frequently hospitalized animal in wildlife rescue centers.The European hedgehog is a very common animal in Europe. This animal usually lives in woods but often it is possible to find it in gardens and parks in urban areas. Few studies describe parasites infecting the European hedgehog in Italy. In the present study, endoparasite infections and associated lesions were investigated in hedgehogs from two different regions of central Italy (Latium and Tuscany), where no investigations had been performed before. Several helminth and protozoan species were identified in the intestine and respiratory tract of the animals analyzed. The presence of the respiratory worm n = 40) from central Italy (Latium and Tuscany regions) were investigated, along with concurrent gross and histopathological lesions. The most prevalent identified endoparasites were Crenosoma striatum (45%), Capillaria erinacei (42.5%) and Brachylaemus erinacei (22.5%), in accordance with previous reports from hedgehogs in southern Italy. In few subjects, Physaloptera clausa, Acanthocephalans and Cystoisospora rastegaeivae coccidia were also identified. The infection by the lungworm C. striatum was found to be significantly associated (p < 0.01) with bronchial hyperplasia and peribronchiolitis upon histopathological examination. Awareness of the most common parasitic infections in the hedgehog and of their effects on the health of these animals is extremely important, especially in wildlife rescue centers, where European hedgehog represents the most frequently hospitalized mammal species.The European hedgehog is a synanthropic mammal, widely distributed in Europe. This species usually inhabits the edges of deciduous or mixed woods, but it is also very common in private gardens and public parks. Despite its popularity and frequency of contacts both with humans and with wild and domestic animals, few studies have examined the endoparasitic fauna of the hedgehog in Italy. In the present study, endoparasites of naturally deceased hedgehogs ( Erinaceus europaeus, is a nocturnal synanthropic mammal, widely distributed in Europe [The European hedgehog, n Europe ,2. In ItThe natural habitat of this species is represented by the edges of deciduous or mixed woods, but it is also very common in suburban and rural areas, mainly in gardens and public parks . For thiThe foraging area of the European hedgehog usually extends over a radius of 200–300 m around the nest, but it can also cover distances of several kilometers, with a considerable risk of being killed by vehicles or dying of unnatural causes ,5,6,7. APhysaloptera clausa, Crenosoma striatum, Brachylaemus erinacei or Hymenolepis erinacei [Cryptosporidium spp. [Physaloptera clausa, may act as vectors for human pathogens such as Leptospira spp. [Hedgehogs are omnivorous in diet, and they mainly feed on invertebrates, such as slugs, earthworms, beetles, caterpillars and other insects. These invertebrates can act as intermediate or paratenic hosts for several parasites of the hedgehog, which can cause morbidity or mortality in this species, for instance erinacei ,17. Addiium spp. ,18, whilira spp. .Eucoleus aerophilus [The ecological and feeding habits of the European hedgehogs, along with their high population densities, synanthropic attitudes and common contacts with both wild and domestic animals, implicate the potential involvement of this species in the maintenance and spread of parasites with zoonotic potential or also infecting other wild and domestic animal species, such as rophilus ,20,21,22rophilus ,24,25, cErinaceus europaeus), found dead in Central Italy, were necropsied. The hedgehogs in this study were either provided by wildlife rescue centers or were collected as victims of predation  or road traffic accidents. All specimens were in good conservation conditions. After the collection of fecal samples, the carcasses were frozen at −20 °C, and later thawed overnight at room temperature before the analyses were carried out. At necropsy, subjects were weighed, and the sex and age class were determined. Animals were classified as juveniles or adults based on physical development, bodyweight and season of finding.Between January and August 2021, 40 hedgehogs  were cut open and examined by a dissecting microscope for the presence of adult parasites. Recovered adult helminths were examined soon after collection or preserved in 70% ethanol until identified at the species level based on their morphologic and metric features ,22,26,27g, and the sediment was examined by the flotation test by using a zinc sulphate (ZnS) solution with a specific gravity of 1.350. Fecal samples positive for coccidia were dissolved in 2.5% K2Cr2O7 solution at 22 ± 1 °C and checked daily until sporulation of oocysts, that were examined for evaluating their morphometrical features. Fecal smears from the intestinal content were also performed and stained by Kinyoun’s acid-fast method for Cryptosporidium spp. detection [Individual fecal samples were qualitatively examined. Approximately two grams of feces were homogenized in 10 mL of water and filtered through gauze. The obtained fecal homogenate was centrifuged in a 15 mL tube for 5 min at 1600× etection .The identification of the parasitic stages found at parasitological examination of fecal samples  was based on morphological and metrical features ,29,30.Internal organs were examined for the presence of gross lesions. Regardless of the presence of infection and/or macroscopically evident lesions, tissue samples from the lung, stomach and intestine were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 4 μm, stained with Hematoxylin and Eosin (HE) and examined for histopathological lesions.p-value was less than 0.05. Prevalence and the 95% confidence interval (95% CI) were also calculated.All statistical analyses were conducted in R Language . Fisher’The 40 deceased hedgehogs examined in this study were identified as 22 males and 18 females, and 15 juveniles and 25 adults. Twenty-one animals were collected after natural death from Centro Recupero Fauna Selvatica Lipu, Rome, Latium , eight from Parco Regionale Riviera di Ulisse, Latina, Latium , four from Giardino Faunistico Pian dell’Abatino, Rieti, Latium , three from Riserva Naturale “Lago di Vico”, Viterbo, Latium , and four from Grosseto, Tuscany .From the parasitological examination of the respiratory and digestive tract and fecal samples, 22 hedgehogs out of 40 resulted positive for almost one parasite, with an overall infection prevalence of 55%.Crenosoma striatum    (Cystoisospora rastegaivae (5%)   b, follow.2–57.8) a and Bra.6–35.4) , while avae (5%) b, Physala (2.5%) c and AcaB. erinacei and C. striatum, respectively. No statistical difference was observed between male and female prevalence, while the prevalence in adults was statistically higher (p < 0.01) than in juveniles, especially for C. striatum and C. erinacei. All examined fecal smears were negative for the presence of Cryptosporidium spp. oocysts.Among positive hedgehogs, only 18.2% (4/22) of animals were found infected by a single parasite species, while multiple parasite species were identified in most infected hedgehogs . Specifically, two different parasites were observed in thirteen animals (59.1%), three in four animals (18.2%) and four in one animal (4.5%). The highest prevalence was observed in male (59.1%) and adult (80%) hedgehogs, while juveniles showed a very low prevalence (13.3%), with only two hedgehogs being positive for The prevalence and 95% confidence interval for each parasite found in the different sex and age groups are reported in C. striatum, bundles of adult nematodes protruded from the cut surface of lungs at necropsy. Histological examination of lungs in C. striatum-infected animals showed the presence of worms in the lumen of the bronchi and bronchioles in 13 out of 18 animals (72.2%), along with hyperplasia of the bronchial epithelium , with a higher significance for bronchial hyperplasia and peribronchiolitis (p < 0.01) . In three subjects found heavily infected by ithelium  and mild < 0.01) .Sections of worms and nematode eggs were frequently observed microscopically in the lumen of the intestine, usually not associated with significant gross or histopathological lesions in the intestine wall. In some cases, a mild inflammatory infiltrate, consisting of lymphocytes, plasma cells and rare eosinophils, was detected in the lamina propria of the intestinal mucosa. In a single sample, a section of an adult Acanthocephalan parasite was evidenced , while tp < 0.01). No hoglets (very young hedgehogs under 100 g in body weight) were examined in the present study, but in the available literature, there is agreement upon this age class being generally not frequently positive for parasites [The prevalence of infected animals in the present study (55%) is lower than in studies conducted both in Italy 100%) [00% 1,2424 and inarasites ,32. Gaglarasites  suggestearasites ,36.C. striatum, which is a known cause of verminous pneumonia in hedgehogs. The observed prevalence in this study (45%) is consistent with data from Portugal [C. striatum is a meta-strongyloid nematode characterized by an indirect lifecycle, with the hedgehog as the final host. Animals acquire infection by ingestion of third-stage larvae contained in intermediate hosts such as snails and slugs [Bordetella bronchiseptica or Pasteurella multocida, lung abscessation and pleuropneumonia [C. striatum infection and the presence of lung lesions upon histopathological examination, which was highly significant for bronchial hyperplasia and peribronchiolitis (p < 0.01). However, more animals should be analyzed to draw definitive conclusions on C. striatum pathogenicity.The most prevalent species in this study was Portugal  and GreePortugal , but lowPortugal ,24, whicnd slugs ,25,37,38nd slugs ,40. Vermneumonia , and morneumonia . In the C. striatum adults were identified among respiratory nematodes, while no Eucoleus aerophilus was identified [E. aerophilus infection seems to occur less frequently than C. striatum infection, with reported prevalence rates of under 40% in various countries  is a species frequently reported for infecting the hedgehog worldwide, including in Italy [Data obtained in this study confirm the low prevalence of Acanthocephalan infection in the hedgehog reported by previous studies ,43,46. Iin Italy ,24,60C. rastegaievae is considered a common protozoan species infecting hedgehogs, and it may contribute to the development of clinical signs such as dark-green droppings and hemorrhagic enteritis in hedgehogs of all ages [all ages ,61.E. europaeus from central Italy. Included among prevalent parasites were species considered highly pathogenic for the hedgehog, such as C. striatum. Lung lesions associated with this parasite were also highlighted in this study based on pathological findings. Awareness of parasitic infections in hedgehogs is particularly important in wildlife rescue centers [In conclusion, data from this study evidenced a high prevalence of multiple endoparasite infections in  centers , both to


RMS amplitude of the 30Hz/2mm (ICC = 0.89–0.99) and 40Hz/4mm vibration (ICC = 0.95–0.99) indicated substantial reproducibility. The intraday reliability of high-pass filter at 100-450Hz for these parameters was also substantial . The interday reliability (session 1 vs. session 3) of the mean normalized sEMGRMS amplitude for band-stop filtered means of 40 Hz/4mm and 30Hz/2mm vibration recordings was substantial (ICC = 0.82 and 0.93). However, ICCs of the mean and median frequency were indicative of fair reliability (ICC of 0.43 to 0.59). The interday reliability of mean normalized sEMGRMS amplitude for high-pass filter at 100-450Hz was substantial  for the 30Hz/2mm S-WBV and moderate (ICC = 0.73) for the 40/4mm S-WBV. The ICCs for mean and median sEMG frequency ICCs indicated slight to fair reproducibility (ICC of 0.16 to 0.56). The intraday reliability of the strongest MVC contraction and average MVC turned out substantial (ICC = 0.91–0.98). The interday reliability coefficients of the strongest MVC contraction and average MVCs were 0.91 and 0.82, respectively. Concluded, the intraday reliability proved satisfactory for all variables; however, the interday comparison showed sufficient ICC levels only for the mean amplitude. We therefore recommend this parameter should be used when analyzing PFM sEMG recorded during vibration. ICCs of the mean and median frequency for both signal processing methods were indicative of insufficient reliability and did not reach the threshold for usefulness. Our study showed similar reliability of PFM sEMG during S-WBV in case of the two filtering methods used.The primary aim of the study was to assess intraday and interday reliability of surface electromyography (sEMG) reflex activity of the pelvic floor muscles during synchronous whole-body vibration (S-WBV) of two intensities  using band-stop filter and high-pass filter signal processing. The secondary aim of the study was to assess intraday and interday (test-retest) reliability of sEMG obtained from maximal voluntary contraction (MVC) test. We evaluated the intraday reliability of sEMG recordings obtained during sessions 1 and 2 performed on the same day. The sessions consisting of maximal voluntary pelvic floor muscle contraction and synchronous vibration sets with 1-hour rest in-between sessions 1 and 2 in healthy nulliparous women. The next intraday reliability was evaluated between the results of sessions 3 and 4 performed on the same day but followed at an interval of 4 weeks. to include the entire menstrual cycle. The interday reliability was determined based on the results of sessions 1 and 3 using the intraclass correlation coefficient . The intraday ICCs for band-stop filtered mean and median sEMG frequency and mean normalized sEMG There is a growing interest of researchers and practitioners in the effect of whole body vibration (WBV) exercise in training . WBV hasPelvic floor muscles (PFM) are striated muscles that include muscle spindles  thereforConsidering the risk of motion artifact and reflex activity contributions to EMG amplitude, surface EMG activity recording during vibration exercise is difficult , 16. SebThe present study focuses on testing the intraday and interday reliability of PFM sEMG during synchronous whole body vibration (S-WBV) of two intensities using two signal processing methods described in literature. A wider insight into PFM sEMG response to vibration gained from measurement reliability assessment may help interpret the effects of vibration on voluntary and reflexive PFM activity in healthy women and women with pelvic floor dysfunction.The primary aim of the study was to assess intraday (between two sessions within the same day) and interday (test-retest) reliability of reflex sEMG activity of the pelvic floor muscles during 60 seconds of synchronous whole-body vibration (S-WBV) of two intensities . The secondary aim of the study was to assess intraday and interday (test-retest) reliability of sEMG obtained from MVC test. In our experiment, sEMG activity was processed using two filtering methods . We hypothesized that the reliability of sEMG signals recorded during vibration might differ depending on the signal filtering method.The study protocol was approved by the Bioethics Committee at the Academy of Physical Education in Katowice, Poland (1/2017). The recruited women received comprehensive information on the study aim and methods, and gave their informed written consent to participate as required by the Declaration of Helsinki". The present study constitutes a phase of a project designed to determine the effect of whole body vibration on reflexive PFM activity and voluntary contraction (ACTRN12618000531213).The participants were recruited out of students of our Academy by the flyers which contained information about the study aim and procedure. The flyers were posted inside the Academy building. The recruitment process lasted from February 2019 to June 2019. The recruitment was carried out by a person not involved in the investigations. The participants were included based on the following inclusion criteria: nulliparous women without PF dysfunction, regular menstruation, good general health and no history of previous vibration platform exercises. Exclusion criteria included history of (or current) stress urinary incontinence, pregnancy, childbirth(s), pelvic surgery, diabetes, hypertension, neurological abnormalities, urinary tract infection, elevated temperature, practicing professional sport, spinal pain, pelvic organ prolapse, unhealed fracture, nephrolithiasis. Ten healthy adult women were invited to participate. One woman was excluded as she did not meet the inclusion criteria; another one could not participate in the second measurement session due to urinary tract infection. Ultimately, 8 healthy nulliparas entered the study . During To consider the study sample representative of a larger population, the sample size was determined based on Walter et al. recommendations . We assuThe women enrolled in the study reported for testing to the Electromyography and Pelvic Floor Muscles Laboratory at the Institute of Physiotherapy and Health Sciences, at the Jerzy Kukuczka Academy of Physical Education in Katowice.The study procedure consisted of four sessions of sEMG recording . Two sesParticipants were scheduled for the examination sessions just after menstruation but no later than two days after the end of their menstrual bleeding to assure that they all were in the follicular phase of their cycles. The reexamination was performed following one menstrual cycle in the next follicular phase (no later than two days after the end of the next menstruation) which was in about four weeks. Performing both the examination and reexamination in the same phase of the menstrual cycle allowed to eliminate the cycle-related effect of hormonal fluctuation on PFM activity .The participants were asked not to take up intensive physical exercises 24 hours before each session. Prior to PFM sEMG, they were also asked to empty their bladder. The mode of experiment administration was one-to-one and face-to-face. During each sEMG session the subjects were wearing socks. The recording took place while standing on the vibration platform in semi-squat  testing position  without Bioelectric potentials generated by the PMFs were recorded with pear-shaped and tapered vaginal probes . This design of vaginal electrode might be less prone to recording motion artifacts than cylindrical probes . Each stPFM electrical activity was recorded using Myo Trace 400 (Noraxon U.S.A. Inc.) sEMG with a preamplifier . A 16-bit analog to digital (A/D) converter with a sampling frequency of 1000Hz.During each session, the sEMG MVC recording comprised of three 5-second attempts at maximum contraction of the PFM in the testing position, with a 60-second rest in between each attempt.To record reflex activity of PFM the sEMG was performed during S-WBV on a vibration platform . The vibration set comprised of three sEMG recordings during vibration with frequency of 30Hz and amplitude of 2mm, and three recordings during vibration with frequency of 40Hz and amplitude of 4mm (applied at random order). Each vibration exposure lasted 60 seconds; a 5-minute rest was allowed between the recordings to eliminate potential PFM fatigue. The participants were not informed about vibration intensity.RMS parameter used to measure the amplitude.MVC sEMG data were filtered at 20-450Hz, after filtering signal was rectified and the root mean square value was calculated using a 100 ms sliding window. EMGWe employed two methods of raw sEMG signal processing: band-pass filter by Hazell et al.  and bandth-order Chebyshev type II with a band-stop of ±1Hz, transition band of ± 1.5Hz, minimum band-stop attenuation of 100dB and maximum 0.01dB ripple was used. The filter was centered at the frequency of the fundamental vibration frequency  and the harmonics of up to 450 Hz. In all sEMG records, the power line frequency of 50Hz and its harmonics 100 Hz were filtered. The data were full-wave rectified and the root mean square (sEMGRMS) was calculated within a 100-ms window.In order to delete sEMG signal spikes that might be considered vibration artifacts in fundamental frequency and its harmonics, the sEMG signal was filtered using the filtering regimen by Abercromby et al. . The banRMS) was calculated within a 100-ms window.As a separate filtering protocol, we used regimen employed by Hazell et al. . The sEMRMS %MVC), the root mean square amplitude value of MVC, and the mean (MNF) and median (MDF) frequencies. The analyses of the median frequency (MDF) and mean frequency (MNF) of EMG signals were based on the Total Power Spectrum which had been determined using Fast Fourier Transformations (FFT).Based on the processed signal, the following sEMG parameters were calculated from each sEMG recordings, both from time and frequency domain: the mean of root mean square amplitude value next normalized to the maximal voluntary contraction value to represent the reflex PFM activity .Reliability of sEMG signals concerning reflex PFM activity during vibration was analyzed based on the means calculated from three sEMGThe Shapiro–Wilk test was used to check whether the data had normal distribution. The mean and standard deviation values were calculated for all parameters. Data skewness, kurtosis and modality were also checked.RMS amplitude, mean and median sEMG frequency) were compared with repeated measures ANOVA [repeated factor- four levels: four sessions].The mean values of MVC and vibration sEMG variables (sEMGk = 3), 2-way mixed-effects, consistency, multiple measurements model ICC  [RMS amplitude, mean and median frequency were averaged to produce each data point. The averaging of the sEMG values occurred before the entry into the ICC analysis what gave eight pairs of datapoints. The ICC  was used to analyze the intraday and interday reliability of the greatest MVC value (k = 1). The intraday and interday reliability coefficients of the average value from three MVC measurements (k = 3) were analyzed by the ICC . The following ICC intervals were chosen: a) ≤ 0.10, no reproducibility: b) 0.11–0.40, slight reproducibility; c) 0.41–0.60, fair reproducibility, d) 0.61–0.80, moderate reproducibility; e) 0.81–1.0, substantial reproducibility [Derived from the ANOVA results of the mean square of error and mean square between subjects, the reliability was assessed by the intraclass correlation coefficient (ICC). ICC estimates and their 95% confidence intervals were calculated based on a mean value of three sEMG recordings  . Three scibility . The corl/(n−1)) ; coefficIn all tests, the level of statistical significance was set at p = 0.05. The tool for data analysis was Statistica, version 13.3.RMS amplitude, mean and median sEMG frequency (mean ± SD) of the two processing methods during vibration of two intensity are shown in The sEMGRMS amplitude of the 30Hz/2mm vibration indicated substantial reproducibility; the inter-measurement variability was low, i.e., between 3.15% and 15.62%. The intraday reliability of high-pass filter at 100-450Hz was substantial with inter-measurement variability of 2.12% and 15.93%  of the mean normalized sEMGmplitude .The ICCs for high-pass filtered mean and median frequencies of the 30Hz/2mm and 40/4mm S-WBV sessions indicated slight to fair reproducibility. The variability between the measurements showed values between 5.66% and 8.14% for measured variables. The interday reliability of the mean normalized amplitude for high-pass filter at 100-450Hz was substantial for the 30Hz/2mm S-WBV and moderate for the 40/4mm S-WBV. The respective correlation coefficients were significant .The intra- and interday reliability of the greatest value and average MVC amplitude exhibited substantial reproducibility .RMS during WBV [Surface electromyography is commonly used in vibration studies. Since vibrations inevitably contaminate the surface EMG signal with motion artifacts, a number of filtering regimens are recommended to remove the vibration frequency –19. Howering WBV .The authors of the few studies exploring the effects of whole body vibration on PFM activity in healthy individuals and/or those with PFM dysfunction used sEMG signal filtering to remove artifacts. In the study of Luginbuehl et al. , the funRMS amplitude of 30Hz/2mm vibration indicated substantial reliability. The intraday reliability of band-stop filtered and high-pass filtered PFM sEMG variables of 40Hz/4mm intensity vibration was also substantial.It is still undetermined whether PFM sEMG obtained using sEMG signal filtering methods have satisfactory reliability. To our knowledge, this is the first report to evaluate reliability of PFM sEMG during vibration after signal processing with band-stop and high-pass at 100-450Hz filters. Hence, no comparisons can be made regarding the levels of reliability, CV%, SEM and MD values. We evaluated sEMG variables  during whole body vibration of two intensities. The intraday ICC for band-stop and high-pass filtered mean and median frequencies and the mean normalized sEMGRMS %MVC amplitude (ICC = 0.73). The interday reliability of the high-pass filtered 30Hz/2mm vibration recordings was somewhat higher for MNF and MDF  and substantial for the mean sEMGRMS %MVC amplitude (ICC = 0.90). The ICCs for band-stop filtered MNF and MDF of the 30Hz/2mm and 40Hz/4mm S-WBV sessions were indicative of fair reliability (ICC of 0.43 to 0.59). The interday reliability of the mean sEMGRMS %MVC amplitude for band-stop filtered 40 Hz/4mm and 30Hz/2mm vibration recordings was substantial (ICC of 0.82 and 0.93 respectively).High-pass filtered interday sEMG recordings of the 40Hz/4mm measurement were slightly reproducible for MNF and MDF , and moderately reproducible with respect to the mean sEMGRMS %MVC of the 30Hz/2mm and 40Hz/4mm S-WBV sessions was substantial while the interday (test-retest) reliability was moderate to substantial for both signal filtering methods. Our study demonstrated that the intraday sEMG reliability of the MNF and MDF ranged from moderate to substantial while the interday reliability was poor.The intraday reliability of the mean normalized amplitude EMGAn analysis of PFM MVC reliability revealed some similarities between our and other authors’ findings. Our study showed substantial intraday and interday reliability of the MVC. Grape et al.  obtainedThe parameters of synchronous vibration applied in our study had been determined in a pilot experiment. Stania et. al.  revealedThe activity of PFM depends on body positions; the level of background muscle activity increases in standing , 32. In Some limitations of the study should be noted. sEMG recordings were performed in healthy women so the reliability data cannot apply to individuals with pelvic floor muscle dysfunction. Also, three women suffered acute effects of high intensity vibration including erythema and itching; the experienced discomfort might have affected sEMG results. No studies could be found to confirm reliability of results obtained with a pear-shaped electrode which we used; it should be noted though that Scharschmidt et al.  concludeOur study was focused on the assessment of intraday and interday reliability of reflex PFM sEMG activity during 60 seconds of synchronous whole-body vibration of two intensities  using signal processing methods described in literature .RMS %MVC were not high for both filtering methods. The intraday reliability of MNF and MDF reached substantial reproducibility for band-stop and high-pass filtering. However, the ICCs for MNF and MDF cover a broad range of interday reliability (from slight to fair) while SEM and MD are high. The intraday reliability proved satisfactory for all variables; however, the interday comparison showed moderate to substantial ICC only for the mean sEMGRMS amplitude. We therefore recommend this parameter should be used when analyzing PFM sEMG recorded during vibration. The intra- and interday reliability of the greatest value and average MVC amplitude exhibited substantial reproducibility.Band-stop filtering and high-pass filtering of the mean normalized amplitude obtained from three PFM sEMG recordings made during S-WBV of two intensities yielded intraclass correlation coefficients indicating substantial intraday reliability while the interday reliability was moderate to substantial. Despite slight differences, SEM, CV% and MD of sEMGOur study showed similar reliability of PFM sEMG during S-WBV in case of the two filtering methods used. Therefore, the question arises as to whether it is possible to compare results from different research centers which use different filtering methods of sEMG recorded during WBV. Our results indicate a need for further interpretations of PFM sEMG recordings obtained during S-WBV for the needs of clinical studies in patients with PFM dysfunctions.S1 Dataset(XLSX)Click here for additional data file.


Case Presentation. Here, we report a case of successful transplantation of both kidneys from a deceased donor with active herpes zoster infection at the time of organ retrieval. Recipients were treated preemptively with acyclovir. At 4 months posttransplant, both kidney recipients experienced no infectious complications and were off dialysis with functioning transplant grafts.  The limited donor pool and increasing recipient wait list require a reevaluation of kidney organ suitability for transplantation. Use of higher infectious risk organs that were previously discarded may help improve access to transplantation and reduce patient mortality without placing patients at a higher risk of poor posttransplant outcomes. There is very little data available regarding the safe use of kidney organs from deceased donors with varicella zoster virus infection at the time of organ retrieval.  The use of kidney organs from donors with active herpes zoster infection appears to be a safe option to expand the kidney donor pool. Given the limited donor pool and increasing demand for organs, there has been an incentive to utilize higher risk organs for transplantation, including those from donors with increased infectious risk . FollowiVaricella zoster virus (VZV) is an alpha-herpesvirus with seropositivity in approximately 90% of adults in the United States . It presHZ infection in transplant recipients is a significant cause of morbidity and in rare cases may be fatal . The incThere are no reports to date regarding the use of kidney organs from donors with active HZ infection at the time of donation. We describe the first case of successful transplantation of two kidneys from a deceased donor with active VZV shingles infection at the time of organ retrieval.An older patient with severe anoxic brain injury after cardiac arrest became a donor after cardiac death. Prior to donation, the donor was noted to have a three-day history of a vesicular erythematous rash on the left flank extending into the left groin. The donor was noted to be positive for VZV IgG serology, and a swab of a vesicular lesion was positive for VZV. The infectious disease team was consulted for the donor and diagnosed active HZ infection without evidence of dissemination or immunosuppression, for which the donor received 2 days of treatment dose intravenous valacyclovir, the last dose being given the morning of organ retrieval. On collateral history, there were no preceding symptoms of encephalitis. The donor was negative for cytomegalovirus (CMV).Pneumocystis jirovecii). According to our local protocol, neither patient received CMV prophylaxis. Given active HZ in the donor, both patients received treatment dose acyclovir 10 mg/kg IV on POD 0, then were transitioned to renally adjusted treatment dose oral acyclovir for 7 days  followed by prophylactic renally dosed oral acyclovir (400 mg daily or bid based on changes in estimated GFR over time) until 3 months posttransplant. Recipients were monitored clinically for symptoms and signs of VZV infection.After a comprehensive discussion regarding potential donor-derived infection risks between the transplant nephrologist, transplant surgeon, and infectious disease team, kidneys were allocated to two recipients on dialysis, both of whom were VZV immune. The first was a 52-year-old female with panel reactive antibody (PRA) 0%, 3 human leukocyte antigen (HLA) mismatch (MM) with the donor, CMV negative, and Epstein Barr virus (EBV) immune. The second recipient was a 51-year-old female with PRA 50%, 6 HLA MM, CMV positive, and EBV immune. Both had end-stage kidney disease secondary to type 1 diabetes mellitus and were induced with Basiliximab. Maintenance immunosuppression consisted of mycophenolate mofetil (MMF) 1 g PO bid, prednisone 5 mg daily, and tacrolimus to target a trough level of 8-10 in the first 3 months, then 5-8 thereafter. Both patients received perioperative antibiotic prophylaxis with cephalexin and prophylaxis with oral sulfamethoxazole 400 mg/trimethoprim 80 mg  and disseminated or organ invasive HZ should be treated with IV acyclovir, akin to primary varicella .Pretransplant vaccination in seronegative patients and antiviral prophylaxis posttransplant with acyclovir or valganciclovir have dramatically reduced the incidence of VZV reactivation; however, the risk persists for many years posttransplant, and there is limited evidence on long-term prophylactic therapy. Given the risk of VZV reactivation in the posttransplant period, the recombinant subunit vaccine Shingrix is recommended in transplant recipients over the age of 50 years.In conclusion, the relative risk and benefit of accepting increased infectious risk donor organs including from donors with active HZ infection must be considered in the context of the increasing demand for donor organs and the corresponding risk of death or comorbidity accrual on the transplant waiting list if such organs are discarded. Our experience is the first documented report of utilizing organs from a donor with active HZ infection, and we demonstrate excellent outcomes with no complications or adverse infectious outcomes as a result.


Clozapine has a unique efficacy profile among individuals suffering from treatment-resistant schizophrenia, but is associated with hematological side effects. The use of granulocyte colony-stimulating factors (G-CSF) to allow clozapine continuation or rechallenge has emerged as a promising option, but evidence is still scarce.To describe the largest case series so far published regarding this practice.9/L since 2004 in Quebec and was cross-referenced with hospital pharmacy software to identify patients who had received at least one dose of G-CSF, such as filgrastim, while being exposed to clozapine. All data were collected retrospectively, using patients’ medical files, from January to July 2019.A national clozapine hematological monitoring database was consulted to identify all patients who had had neutrophil count <1.5 × 10Using G-CSF, three out of eight patients could maintain clozapine despite neutropenia episodes that otherwise would have required treatment discontinuation. The only side effect reported was mild short-lived back pain, over a mean 3-year follow-up period. In all but one case, filgrastim was used on an “as-needed” basis at doses of 300 mcg administered subcutaneously.These results suggest that the “as-needed” use of G-CSF is well-tolerated and may allow clozapine rechallenge in some well-selected patients, adding to the paucity of data regarding long-term safety and efficacy of this strategy. More research may help to better define potential candidates and optimal regimen of such practice. Clozapine is well known for its unique efficacy in patients with treatment-resistant schizophrenia (TRS), representing about 30% of this vulnerable population . Regardl9/L warrants for clozapine cessation and forbids its rechallenge  below 1.5 × 10hallenge . In somehallenge . In suchhallenge . In somehallenge .A brief review of the literature has highlighted two different off-label pharmacological strategies in Canada, used to re-expose a patient to clozapine. These strategies include the use of lithium to induce leukocytosis and the use of granulocyte colony-stimulating factor (G-CSF) to stimulate leukopoiesis . These sClinical Global Impression severity scale (CGI-S) was used to evaluate the disease severity during four critical turning points: before and after clozapine introduction, after a red result, and after clozapine rechallenge . Noteworthy, he had received a single dose of clozapine by mistake in his 20s, which led to an ANC drop. However, at that time, the patient was also taking sodium valproate, chlorpromazine and olanzapine. Three months after clozapine initiation, while also receiving quetiapine, a first neutropenia occurred. Clozapine was discontinued but reintroduced 8 months later with a lowered ANC surveillance threshold given his low baseline values . Despite this adjustment, the patient had nine other neutropenia episodes during which clozapine could nonetheless be maintained using a total of 13 “as-needed” doses of filgrastim over 3 months. Following a 10th neutropenia, clozapine was discontinued because of severe agranulocytosis.A 53-year-old Caucasian male diagnosed with DSM-5 schizophrenia at the age of 22 had been hospitalized for the last 30 years due to TRS. Clozapine was only introduced at the age of 53 due to a low ANC at baseline . Two months later, the patient experienced two neutropenia episodes in a row. At that time, the patient was also taking quetiapine, oxazepam, and citalopram. Two doses of filgrastim were given during the last episode, a few days apart, which only led to transitory ANC normalization. Clozapine was withdrawn shortly after as the medical team feared the undocumented innocuity of filgrastim in this off-label use.A 60-year-old Caucasian male diagnosed with DSM-5 schizophrenia and potomania at the age of 19 was receiving clozapine for 10 years when a first neutropenia occurred. Clozapine was discontinued, leading to numerous psychiatric hospitalizations during the following 2 years. As a result, clozapine was rechallenged with a reduced ANC surveillance threshold  patients remained on clozapine treatment after a mean follow-up period of 3 years using only 1-2 G-CSF doses see . There wAll patients were Caucasian and median age at first neutropenia was 48 years see . We obseA mean number of 4 (ranging from one to ten events) neutropenia events were observed for each patient. Almost every patient (7/8) had one or more subsequent ANC drop following the initial neutropenia. For these patients, as previously observed in other studies, subsequent neutropenia episodes developed quicker following clozapine rechallenge, with lower mean ANC at nadir for the third and subsequent neutropenia episode in comparison to the first .Lithium use to induce leucocytosis seems to be a common practice in Quebec as it was used at some point in 7/8 cases while it was only used in 5/30 cases in a previous systematic review .9/L to 1.5 × 109/L. Intervals between doses and number of doses received by patients varied extensively (see As for G-CSF use, 7/8 patients had an “as-needed” strategy with a pre-established ANC threshold varying from 0.5 × 10vely see . Patientvely see . Howevervely see . The “asvely see . Given tvely see . ConsequIn all cases, aside from leucocytosis, G-CSF was well tolerated, with only 1 patient reporting short-lived back pain.As for the timeline of use, G-CSF was given at the first occurrence of neutropenia solely for patient 3 resulting in a rapid ANC normalization and a quick rechallenge. In contrast, 4/8 patients did not use G-CSF at the first neutropenia episode and stopped clozapine for an average time of 16 months before a rechallenge was attempted, which is longer than the 9 months median time reported by Myles and colleagues’ . This leAlthough clozapine rechallenge following agranulocytosis seems to pose more risks and may have lower success rates according to a recent systematic review, many questions remain unanswered regarding predictive factors for clozapine rechallenge . LikewisOne thing is certain, clozapine cessation has immense consequences on psychopathology and recovery . Indeed,In conclusion, to our knowledge, there are no death reports following clozapine rechallenge using G-CSF. Furthermore, G-CSF is generally well-tolerated and very few side effects have been reported so far from its use in psychiatry . Also, p