Shedding Rate Research Articles

Environmental DNA shedding and decay rates from diverse animal forms and thermal regimes

AbstractEnvironmental DNA (eDNA) analysis from water samples is a promising new method to identify both targeted species and whole communities of aquatic organisms. However, the current literature regarding eDNA shedding rates primarily focuses on fish and most decay rate constants are reported for warm sunlit waters. Here, we conducted experiments to investigate how eDNA shedding differs between animal forms and how long eDNA can persist in waters of varying temperature and light conditions. We designed quantitative PCR assays for one fish (mummichog, Fundulus heteroclitus), one crustacean (grass shrimp, Palaemon spp.), and two scyphomedusae (moon jelly, Aurelia aurita and nettle, Chrysaora spp.) to estimate eDNA shedding and decay rates. We found that shedding rates were highly variable for all organisms, but grass shrimp had the lowest shedding rate. We quantified eDNA decay rate constants at 6, 15, and 23°C and found that decay rate constants of mummichog and grass shrimp were larger at higher temperatures, while those of scyphomedusae did not show clear temperature dependence. We also found that higher‐order decay models with tails fit the data better than first‐order log‐linear models, suggesting temporal variability in eDNA decay rates. Results indicate that different animal forms shed different types of eDNA, impacting both shedding and decay rates. These findings fill critical knowledge gaps regarding variation in eDNA shedding and decay across animal forms under a range of realistic marine temperature conditions. These data will be useful for interpreting field studies that utilize eDNA to investigate ocean habitats that are otherwise difficult to access.

Infection dynamics and shedding kinetics of nervous necrosis virus in juvenile seven band grouper using an intraperitoneal infection-cohabitation model

Nervous necrosis virus (NNV) affects more than 177 species of cultured fish including seven band grouper. The present study evaluated the shedding and transmission kinetics of NNV in seven band groupers using a cohabitation challenge model. Initial infection studied by intraperitoneal (IP) injection showed shedding of NNV was acute and peaked within 3–5 days after exposure. This was followed by a post peak period of diminished viral shedding and afterwards an increase in shedding was noticed at 12–16 days in half of the infected fish. In the cohabitation challenge viral shedders used at 10%, 30%, and 50% yielded 20%, 30%, and 100% mortality respectively in naïve fish while within-host viral load during the onset of mortality did not show any significant difference across the groups. Peak viral shedding rate was 5.76 × 105 TCID50/L in the 10% shedder group. The other two groups shed at significantly higher than the 10% group with similar rates: 5.82 × 106 TCID50/L in the 30% and 6.1 × 106 TCID50/L in the 50% shedder groups. The empirically derived lowest effective infectious dose of NNV for the bath challenge was 105 TCID50/L. The observations from the present study provide an understanding of NNV shedding kinetics and its transmissibility that assist in better decision making in management of VNN outbreaks.

Metagenomic sequencing of stool samples in Bangladeshi infants: virome association with poliovirus shedding after oral poliovirus vaccination

The potential role of enteric viral infections and the developing infant virome in affecting immune responses to the oral poliovirus vaccine (OPV) is unknown. Here we performed viral metagenomic sequencing on 3 serially collected stool samples from 30 Bangladeshi infants following OPV vaccination and compared findings to stool samples from 16 age-matched infants in the United States (US). In 14 Bangladeshi infants, available post-vaccination serum samples were tested for polio-neutralizing antibodies. The abundance (p = 0.006) and richness (p = 0.013) of the eukaryotic virome increased with age and were higher than seen in age-matched US infants (p < 0.001). In contrast, phage diversity metrics remained stable and were similar to those in US infants. Non-poliovirus eukaryotic virus abundance (3.68 log10 vs. 2.25 log10, p = 0.002), particularly from potential viral pathogens (2.78log10 vs. 0.83log10, p = 0.002), and richness (p = 0.016) were inversely associated with poliovirus shedding. Following vaccination, 28.6% of 14 infants tested developed neutralizing antibodies to all three Sabin types and also exhibited higher rates of poliovirus shedding (p = 0.020). No vaccine-derived poliovirus variants were detected. These results reveal an inverse association between eukaryotic virome abundance and poliovirus shedding. Overall gut virome ecology and concurrent viral infections may impact oral vaccine responsiveness in Bangladeshi infants.

Modelling the effects of the contaminated environments on tuberculosis in Jiangsu, China

Tuberculosis (TB) is still an important public health issue in Jiangsu province, China. In this study, based on the TB transmission routes and the statistical data of TB cases, we formulate a novel TB epidemic model accounting for the effects of the contaminated environments on TB transmission dynamics. The value of this study lies in two aspects. Mathematically, we define the basic reproduction number, R0, and prove that R0 can be used to govern the threshold dynamics of the model. Epidemiologically, we find that the annual average R0 is 1.13,>1 and TB in Jiangsu is an endemic disease. Therefore, in order to control the TB in Jiangsu efficiently, we must decrease the virus shedding rate or increase the recovery rates, and increase the environmental clearance rate.

Temperature and light effects on Trichobilharzia szidati cercariae with implications for a risk analysis

BackgroundCercarial dermatitis (swimmer’s itch) caused by bird schistosome cercariae, released from intermediate host snails, is a common disorder also at higher latitudes. Several cases were observed in the artificial Danish freshwater Ringen Lake frequently used by the public for recreational purposes. The lake may serve as a model system when establishing a risk analysis for this zoonotic disease. In order to explain high risk periods we determined infection levels of intermediate host snails from early spring to late summer (March, June and August) and elucidated the effect of temperature and light on parasite shedding, behavior and life span.ResultsField studies revealed no shedding snails in March and June but in late summer the prevalence of Trichobilharzia szidati infection (in a sample of 226 pulmonate Lymnaea stagnalis snails) reached 10%. When investigated under laboratory conditions the cercarial shedding rate (number of cercariae shed per snail per day) was positively correlated to temperature raising from a mean of 3000 (SD 4000) at 7 °C to a mean of 44,000 (SD 30,000) at 27 °C). The cercarial life span was inversely correlated to temperature but the parasites remained active for up to 60 h at 20 °C indicating accumulation of cercariae in the lake during summer periods. Cercariae exhibited positive phototaxy suggesting a higher pathogen concentration in surface water of the lake during daytime when the public visits the lake.ConclusionThe only causative agent of cercarial dermatitis in Ringen Lake detected was T. szidati. The infection risk associated with aquatic activities is low during spring and early summer (March-June). In late summer the risk of infection is high since the release, behavior and life span of the infective parasite larvae have optimal conditions.

The impact of infected T lymphocyte burst rate and viral shedding rate on optimal treatment scheduling in a human immunodeficiency virus infection

We consider a mathematical model of human immunodeficiency virus (HIV) infection dynamics of T lymphocyte (T cell), infected T cell, and viral populations under reverse transcriptase inhibitor (RTI) andprotease inhibitor (PI) treatment. Existence, uniqueness, and characterization of optimal treatment profiles which minimize total amount of drug used, viral, and infected T cell populations, while maximizing levels of T cells are determined analytically. Numerical optimal control experiments are also performed to illustrate how burst rate of infected T cells and shedding rate of virions impact optimal treatment profiles. Finally, a sensitivity analysis is performed to detect how model input parameters contribute to output variance.

Simulating phase transitions and control measures for network epidemics caused by infections with presymptomatic, asymptomatic, and symptomatic stages.

We investigate phase transitions associated with three control methods for epidemics on small world networks. Motivated by the behavior of SARS-CoV-2, we construct a theoretical SIR model of a virus that exhibits presymptomatic, asymptomatic, and symptomatic stages in two possible pathways. Using agent-based simulations on small world networks, we observe phase transitions for epidemic spread related to: 1) Global social distancing with a fixed probability of adherence. 2) Individually initiated social isolation when a threshold number of contacts are infected. 3) Viral shedding rate. The primary driver of total number of infections is the viral shedding rate, with probability of social distancing being the next critical factor. Individually initiated social isolation was effective when initiated in response to a single infected contact. For each of these control measures, the total number of infections exhibits a sharp phase transition as the strength of the measure is varied.

Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells

SummaryCirculating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.

Extended-Spectrum β-Lactamase-Producing Enterobacterales Shedding by Dogs and Cats Hospitalized in an Emergency and Critical Care Department of a Veterinary Teaching Hospital.

Extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) gut shedding in human medicine is considered as a major reservoir for ESBL-associated infections in high risk patients. In veterinary medicine, data regarding ESBL-PE gut shedding on admission to emergency and critical care department is scarce. We aimed to determine ESBL-PE shedding rates by dogs and cats in this setting and to determine the risk factors for shedding, at two separate periods, three-years apart. Rectal swabs were collected from animals, on admission and 72 h post admission, enriched and plated on Chromagar ESBL plates, followed by bacterial identification. ESBL phenotype was confirmed and antibiotic susceptibility profiles were determined (Vitek 2). Medical records were reviewed for risk factor analysis (SPSS). Overall, 248 animals were sampled, including 108 animals on period I (2015–2016) and 140 animals on period II (2019). In both periods combined, 21.4% of animals shed ESBL-PE on admission, and shedding rates increased significantly during hospitalization (53.7%, p-value < 0.001). The main ESBL-PE species were Escherichia coli and Klebsiella pneumoniae, accounting for more than 85% of the isolates. In a multivariable analysis, previous hospitalization was a risk factor for ESBL-PE gut shedding (p-value = 0.01, Odds ratio = 3.05, 95% Confidence interval 1.28–7.27). Our findings demonstrate significant ESBL-PE gut shedding among small animals in the emergency and critical care department, posing the necessity to design and implement control measures to prevent transmission and optimize antibiotic therapy in this setting.

Shedding of bovine alphaherpesvirus-1 in bovine extended frozen semen in Indian semen stations: A longitudinal analysis

Infectious bovine rhinotracheitis (IBR) caused by bovine alphaherpesvirus 1 (BoHV-1) is an economically important disease of cattle and buffaloes. Following acute infection, the virus usually attains latency in the sensory neurons. Stress-induced reactivation of latency can cause the infected animals to intermittently shed the virus in body secretions including semen. A longitudinal analysis was carried out to study BoHV-1 shedding in the semen of IBR seropositive cattle and buffaloes. The study involved data generated from the screening of 119,850 extended frozen semen (EFS) batches, collected from 1,229 IBR seropositive bulls, over a period of four years (April 2015 to March 2019). A TaqMan based real-time PCR assay was employed to detect the gB gene BoHV-1 DNA in the EFS batch samples. Each sample was tested in duplicate and amplification in any of the replicates at or below the threshold cycle (Ct ≤ 40) was considered positive. The overall positivity of BoHV-1 in EFS batches was 1.18%. About 41% of the bulls (509 of 1,229) were found to have excreted the virus in semen at least once during the study period. The frequency of viral shedding in buffaloes (0.96%) was significantly lower than that of cattle (1.3%) (p < 0.001). No significant difference was noted in the rate of shedding between the first and the second ejaculates collected on the same day (p = 0.607). The rate of shedding also did not vary among various breeds of cattle (p = 0.454) or with the age of the bulls (p = 0.054). No significant variation in the shedding rate was observed in cattle across different seasons (p = 0.101); while in buffaloes, the rate was higher in autumn (1.2%) than in winter (0.7%) (p = 0.037). The difference in positivity among semen stations was statistically significant (p < 0.001). Analysis of data revealed that ≥100 EFS batch samples/bull were screened from 361 of the 1,229 bulls included in the study. None of the EFS batches screened from 39 of these 361 bulls were found positive during the four years, suggesting they were non-shedders. Further research is warranted to delineate the underlying features of the seropositive non-shedders; following which an adequate risk assessment may be made for the maintenance of infected but non-shedding bulls in semen production.

PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: Faster PEG shedding attenuates anti-PEG IgM production

PEGylation-modification with polyethylene glycol (PEG)-is useful for stabilizing lipid nanoparticles (LNPs). However, such PEGylation can prevent small interfering RNA (siRNA) encapsulated in LNPs from exerting its gene-silencing effects by disrupting the interaction of LNPs with target cells and by inducing the accelerated blood clearance phenomenon via anti-PEG IgM. PEG-lipids with short acyl chains can be used to address these issues because they are quickly shed from LNPs after administration; however, there are few reports on the relationships among PEG shedding rate, anti-PEG IgM production, and the gene-silencing activity of siRNA upon repeated LNP administration. Here, in mice, we found that LNPs conjugated to a fast-shedding PEG-lipid (short acyl chain) induced less anti-PEG IgM compared with LNPs conjugated to a slow-shedding PEG-lipid (long acyl chain). Moreover, pretreatment of mice with LNPs conjugated to the slow-shedding PEG-lipid caused loss of RNA interference activity after subsequent LNP administration because the payload siRNA was delivered primarily to Kupffer cells rather than to hepatocytes. Together, these findings imply that manipulating PEG shedding rate and anti-PEG antibody production is enormously important in the development of RNA interference-based therapeutics utilizing LNP technology.

Ovarian morphological features and proteome reveal fecundity fitness disadvantages in β-cypermethrin-resistant strains of Blattella germanica (L.) (Blattodea: Blattellidae)

To evaluate whether the development of β-cypermethrin resistance in Blattella germanica (L.) (Blattaria: Blattellidae) affects the fecundity fitness of this insect and to determine the underlying mechanism, we compared fecundity differences between β-cypermethrin-resistant (R) and sensitive (S) strains of B. germanica, observed the physiological structural changes of ovaries from an visual perspective, and analyzed differences in the ovarian proteome using proteomic methods. The results showed that, compared with the S strain of B. germanica, the R strain of B. germanica had a significantly higher ootheca shedding rate, a significantly lower number of hatched and surviving nymphs, a significantly higher female proportion in the population and defective ovarian development. Ovarian proteomic analysis showed a total of 64 differentially expressed proteins in the R strain, including 18 upregulated proteins and 46 downregulated proteins. Twenty-four significantly differentially expressed proteins were further studied, and 14 were successfully identified, which were mainly classified into the following categories: immunity-related proteins, development-related proteins, structural proteins, energy metabolism-related proteins and proteins with unknown functions. The differential expression of these proteins reflects the overall changes in cell structure and metabolism associated with β-cypermethrin resistance and explains the possible molecular mechanism of fecundity fitness disadvantages. In summary, β-cypermethrin resistance can cause fecundity fitness disadvantages in B. germanica. The metabolic deviations needed to overcome the adverse effects of insecticides may result in an energy exchange that affects energy allocation and, ultimately, the basic needs of the insect. The fitness cost due to insecticide resistance is critical to the delay of the evolution of resistance.

Pediatric Enteroviral Central Nervous System Infections in Bialystok, Poland: Epidemiology, Viral Types, and Drivers of Seasonal Variation.

Enteroviruses are common causes of infections of the central nervous system (CNS) that in temperate climates tend to peak in the summer. The aim of the study was to describe epidemiology, drivers of seasonality, and types of enteroviruses causing infections of the CNS in children in Northeastern Poland. We prospectively collected data on children hospitalized with infection of the CNS attributed to enteroviruses in Bialystok, Poland, from January 2015 to December 2019. In total, 224 children were included. Nineteen different enterovirus types were identified in isolates collected from 188 children. Coxsackie B5 (32%), echovirus 30 (20%), and echovirus 6 (14%) were the three most common types. Enteroviruses were more prevalent during the summer–fall season. Infections caused by echovirus 30 peaked early in June and coxsackievirus B5 in July, whereas echovirus 6 peaked late in October. Phylogenetic analyses of these three enterovirus types showed multiple lineages co-circulating in this region. Mean air temperatures and precipitation rates were independently associated with monthly number of cases. Considering lack of effective treatment or vaccine, easy transmission of enteroviruses between susceptible individuals, their high mutation rate and prolonged time of viral shedding, continued monitoring and surveillance are imperative to recognize enteroviral infections of the CNS and the changes in circulation of enteroviruses in Poland.

Clinical predictors and timing of cessation of viral RNA shedding in patients with COVID-19.

Molecular detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is key in the diagnosis of coronavirus disease 2019 (COVID-19) and has been widely used for followup of cases as a proxy for contagiousness. The persistence of SARS-CoV-2 RNA shedding in the context of clinical features and comorbidities is understudied. We retrospectively reviewed laboratory-confirmed COVID-19 adult symptomatic cases at Mayo Clinic, eventually achieving cessation of viral RNA shedding (CVS), defined as two consecutive negative SARS-CoV-2 PCR results on nasopharyngeal swabs collected at least 24 h apart. A total of 251 patients were included, median age was 53 years and 59 % female. The most common symptoms at diagnosis were cough, myalgia, dyspnea, fever and chills. Myalgia, cough, anosmia, ageusia and sore throat were common at CVS, but fever and dyspnea were not observed. The median time from symptom onset to CVS was 23 days, and did not differ by symptoms. The weekly cumulative CVS rate was 2, 14, 44, 73, 91 and 95 % at 1-6 weeks from symptom onset, respectively. Cough and fever were associated with a positive PCR test if tested within 2 weeks of symptoms (P < 0.05). Patients with asthma or immunosuppression were less likely to achieve CVS if tested 3 weeks into symptoms (P < 0.04). The cumulative CVS rate at 3 weeks from symptom-onset is 44 % in our entire cohort. The findings of our study highlight the low yield of repeating a SARS-CoV-2 NP PCR test within 21 days of a laboratoryconfirmed COVID-19 diagnosis.

Influenza A (H3) illness and viral aerosol shedding from symptomatic naturally infected and experimentally infected cases.

BackgroundIt has long been known that nasal inoculation with influenza A virus produces asymptomatic to febrile infections. Uncertainty persists about whether these infections are sufficiently similar to natural infections for studying human‐to‐human transmission.MethodsWe compared influenza A viral aerosol shedding from volunteers nasally inoculated with A/Wisconsin/2005 (H3N2) and college community adults naturally infected with influenza A/H3N2 (2012‐2013), selected for influenza‐like illness with objectively measured fever or a positive Quidel QuickVue A&B test. Propensity scores were used to control for differences in symptom presentation observed between experimentally and naturally infected groups.ResultsEleven (28%) experimental and 71 (86%) natural cases shed into fine particle aerosols (P < .001). The geometric mean (geometric standard deviation) for viral positive fine aerosol samples from experimental and natural cases was 5.1E + 3 (4.72) and 3.9E + 4 (15.12) RNA copies/half hour, respectively. The 95th percentile shedding rate was 2.4 log10 greater for naturally infected cases (1.4E + 07 vs 7.4E + 04). Certain influenza‐like illness‐related symptoms were associated with viral aerosol shedding. The almost complete lack of symptom severity distributional overlap between groups did not support propensity score–adjusted shedding comparisons.ConclusionsDue to selection bias, the natural and experimental infections had limited symptom severity distributional overlap precluding valid, propensity score–adjusted comparison. Relative to the symptomatic naturally infected cases, where high aerosol shedders were found, experimental cases did not produce high aerosol shedders. Studying the frequency of aerosol shedding at the highest observed levels in natural infections without selection on symptoms or fever would support helpful comparisons.

Aquatic macrophytes and macroinvertebrate predators affect densities of snail hosts and local production of schistosome cercariae that cause human schistosomiasis.

BackgroundSchistosomiasis is responsible for the second highest burden of disease among neglected tropical diseases globally, with over 90 percent of cases occurring in African regions where drugs to treat the disease are only sporadically available. Additionally, human re-infection after treatment can be a problem where there are high numbers of infected snails in the environment. Recent experiments indicate that aquatic factors, including plants, nutrients, or predators, can influence snail abundance and parasite production within infected snails, both components of human risk. This study investigated how snail host abundance and release of cercariae (the free swimming stage infective to humans) varies at water access sites in an endemic region in Senegal, a setting where human schistosomiasis prevalence is among the highest globally.Methods/Principal findingsWe collected snail intermediate hosts at 15 random points stratified by three habitat types at 36 water access sites, and counted cercarial production by each snail after transfer to the laboratory on the same day. We found that aquatic vegetation was positively associated with per-capita cercarial release by snails, probably because macrophytes harbor periphyton resources that snails feed upon, and well-fed snails tend to produce more parasites. In contrast, the abundance of aquatic macroinvertebrate snail predators was negatively associated with per-capita cercarial release by snails, probably because of several potential sublethal effects on snails or snail infection, despite a positive association between snail predators and total snail numbers at a site, possibly due to shared habitat usage or prey tracking by the predators. Thus, complex bottom-up and top-down ecological effects in this region plausibly influence the snail shedding rate and thus, total local density of schistosome cercariae.Conclusions/SignificanceOur study suggests that aquatic macrophytes and snail predators can influence per-capita cercarial production and total abundance of snails. Thus, snail control efforts might benefit by targeting specific snail habitats where parasite production is greatest. In conclusion, a better understanding of top-down and bottom-up ecological factors that regulate densities of cercarial release by snails, rather than solely snail densities or snail infection prevalence, might facilitate improved schistosomiasis control.

Dynamics of HSV-2 infection with a therapeutic vaccine

Herpes-Simplex Virus type 2 (HSV-2) is a lifelong infection, which has infected over 400 million individuals aged 15-49 years, worldwide. While the disease can be treated with episodic and suppressive antiviral drugs to reduce the rate of recurrence (i.e., symptomatic disease) and viral shedding, none of the currently available therapies can clear the virus from the body of an infected person. A number of therapeutic vaccine platforms are currently in development in order to achieve similar effects to treatment. Due to the inadequate data from clinical trials of therapeutic vaccines, modeling efforts to quantify the impact of vaccination have been limited. In this study, we propose a compartmental deterministic model for the dynamics of HSV-2 to evaluate the effect of a potential vaccine candidate with the inclusion of a booster dose. Despite its simplicity that may not address the complexity of HSV-2 disease, the model shows that targeting symptomatic infection for vaccination is the most effective strategy in the long-term. This conclusion is based on the assumption of an optimal vaccine efficacy, conferring immunity levels that prevent viral shedding and recurrence transiently. Our model provides a framework for developing a computational system to include more heterogeneous characteristics of the disease and individuals, and investigate effectiveness and cost-effectiveness of vaccination scenarios when clinical data become available.

Liquid jet disintegration memory effect on downstream spray fluctuations in a coaxial twin-fluid injector

This paper intends to investigate the influence of unsteadiness in the liquid jet disintegration process on downstream fluctuations of spray characteristics in a coaxial twin-fluid injector. Time-resolved high-speed shadowgraphic imaging of the spray was obtained for different axial locations downstream of the injector exit at z = 0, 8Dl, and 30Dl, where Dl is the central liquid tube diameter. The primary jet breakup unsteadiness close to the injector exit was characterized by measuring both shear-driven Kelvin–Helmholtz (KH) instability and flapping instability in addition to jet breakup length fluctuations. Downstream of the liquid jet core region, the liquid shedding rate (fshed) was measured at z = 8Dl. The power spectrum of time series data of instantaneous volume mean diameter (VMD) measured at z = 30Dl indicated periodic variation of the droplet size. The corresponding frequency (fVMD) was obtained. It was found that for lower range of gas-to-liquid momentum flux ratio (M &amp;lt; 4), both fshed and fVMD are larger than the frequency of KH instability. Also, for such conditions, larger temporal variation of the droplet size is realized, and this leads to higher fluctuations of the local liquid mass flux. Proper orthogonal decomposition analysis of the shadowgraph images for different axial locations identified similar topology of the dominant mode that corresponds to flapping instability. The results suggest that even far downstream of the injector exit, some memory of the upstream unsteady jet breakup process is retained, which strongly influences spatio-temporal evolution of droplet characteristics, thereby contributing to local spray fluctuations.

Clinical observation on the effect of Chinese medicine-“TCM formula” intervention on recurrence and metastasis of triple negative breast cancer

ObjectiveThis study used a prospective cohort study to observe the effect of triple-negative breast cancer on the 2-year disease-free survival rate with or without “TCM formula”. MethodsFrom November 1 st, 2016, the first patient was enrolled in the cohort study. A total of 356 patients were enrolled on January 30, 2019. Among them, 154 cases were followed up for 2 years. During the follow-up, there were 6 cases of shedding, so 6 cases were affected. A total of 148 cases were included in the analysis, including 73 in the exposed group and 75 in the non-exposed group. The exposed group was given “TCM formula” on the basis of standardized treatment, and the non-exposed group was treated with simple triple-negative breast cancer. The two groups visited each of the three months. The interview included safety examination (hematology and imaging). The endpoint was the difference in 2-year invasive disease-free survival between the exposed and non-exposed groups and the safety of the “TCM formula”. ResultsThere were 6 cases of shedding during the experiment and the shedding rate was 3.9 %. The 2-year rate of invasive disease-free survival in the exposed team was 88.7 % and the non-exposed group was 82.5 %. Logistic multivariate regression analysis predicted that “TCM formula” could reduce the disease-related recurrence and metastasis rate by 11 % (OR = 0.89, 95 % CI 0.37−0.956, P＜0.05). Through K–M survival analysis, TNBC patients with age ≤35 years and regional lymph node stage N1 may be the benefit group of “TCM formula”(P＜0.05). During the study, the incidence of total adverse events was 8.2 % in the exposed group, mainly manifested as stomach discomfort, diarrhea, and hepatocyte damage. Conclusion1. In the exposed group, the two-year rate of invasive disease-free survival increased by 6.2 % compared with the non-exposed group(P＞0.05). 2. According to K–M survival analysis, TNBC patients with age ≤35 years and regional lymph node metastasis to N1 may be potential beneficiaries of “TCM formula”. 3. “TCM Formula” is safe and tolerable to most patients.

Gene-Dose Effect of MEFV Gain-of-Function Mutations Determines ex vivo Neutrophil Activation in Familial Mediterranean Fever.

Familial Mediterranean fever (FMF) is caused by mutations within the Mediterranean fever (MEFV) gene. Disease severity depends on genotype and gene dose with most serious clinical courses observed in patients with M694V homozygosity. Neutrophils are thought to play an important role in the initiation and perpetuation of inflammatory processes in FMF, but little is known about the specific characteristics of these cells in FMF patients. To further characterize neutrophilic inflammatory responses in FMF and to delineate gene–dose effects on a cellular level, we analyzed cytokine production and activation levels of isolated neutrophils derived from patients and subjects with distinct MEFV genotypes, as well as healthy and disease controls. Serum levels of interleukin-18 (IL-18) (median 11,485 pg/ml), S100A12 (median 9,726 ng/ml), and caspase-1 (median 394 pg/ml) were significantly increased in patients with homozygous M694V mutations. Spontaneous release of S100A12, caspase-1, proteinase 3, and myeloperoxidase (MPO) was restricted to ex vivo cultured neutrophils derived from patients with two pathogenic MEFV mutations. IL-18 secretion was highest in patients with two mutations but also increased in neutrophils from healthy heterozygous MEFV mutation carriers, exhibiting an ex vivo gene–dose effect, which was formerly described by us in patients' serum. CD62L (l-selectin) was spontaneously shed from the surface of ex vivo cultured neutrophils [median of geometric mean fluorescence intensity (gMFI) after 5 h: 28.8% of the initial level]. While neutrophils derived from healthy heterozygous mutation carriers again showed a gene–dose effect (median gMFI: 67.1%), healthy and disease controls had significant lower shedding rates (median gMFI: 83.6 and 82.9%, respectively). Co-culture with colchicine and/or stimulation with adenosine triphosphate (ATP) and lipopolysaccharide (LPS) led to a significant increase in receptor shedding. Neutrophils were not prevented from spontaneous shedding by blocking IL-1 or the NLRP3 inflammasome. In summary, the data demonstrate that ex vivo cultured neutrophils derived from FMF patients display a unique phenotype with spontaneous release of high amounts of IL-18, S100A12, MPO, caspase-1, and proteinase 3 and spontaneous activation as demonstrated by the loss of CD62L. Neutrophilic activation seems to be independent from IL-1 activation and displays a gene–dose effect that may be responsible for genotype-dependent phenotypes.