Management of emergency general surgical conditions remains a challenge in rural sub-Saharan Africa due to issues such as insufficient human capacity and infrastructure. This study describes the burden of emergency general surgical conditions and the ability to provide care for these conditions at three rural district hospitals in Rwanda.This retrospective cross-sectional study included all patients presenting to Butaro, Kirehe and Rwinkwavu District Hospitals between January 1st 2015 and December 31st 2015 with emergency general surgical conditions, defined as non-traumatic, non-obstetric acute care surgical conditions. We describe patient demographics, clinical characteristics, management and outcomes.p < 0.001), with the shortest being urological emergencies at 1.5 days (interquartile range (IQR):1, 6) and the longest being complicated hernia at 17.5 days . Of all patients, 54% were operated on at the district hospital, either by a general surgeon or general practitioner. Patients were more likely to receive surgery if they presented to a hospital with a general surgeon compared to a hospital with only general practitioners . In addition, the general surgeon was more likely to treat patients with complex diagnoses such as acute abdominal conditions  compared to general practitioners. For patients who received surgery, 73.3% had no postoperative complications and 3.2% died.In 2015, 356 patients presented with emergency general surgical conditions. The majority were male (57.2%) and adults aged 15–60 years (54.5%). The most common diagnostic group was soft tissue infections (71.6%), followed by acute abdominal conditions (14.3%). The median length of symptoms prior to diagnosis differed significantly by diagnosis type (While acute abdominal conditions are often considered the most common emergency general surgical condition in sub-Saharan Africa, soft tissue infections were the most common in our setting. This could represent a true difference in epidemiology in rural settings compared to referral facilities in urban settings. Patients were more likely to receive an operation in a hospital with a general surgeon as opposed to a general practitioner. This provides evidence to support increasing the surgical workforce in district hospitals in order to increase surgical availability for patients. As of 2015, an estimated 5 billion people do not have access to timely, safe, and affordable surgical and anesthesia care . HoweverCurrently, there are limited studies in sub-Saharan Africa assessing the burden of emergency general surgical conditions, defined in this paper as non-traumatic, non-obstetric acute surgical conditions, and the care provided for these patients at rural district hospitals. In Rwanda, a patient with an emergency general surgical condition usually first presents to a health center for care and is referred to the district hospital if surgery is needed. While, 82.5% of operations occur in the district hospitals, most of these are cesarean sections .For non-At the district hospital, patients undergo an operation if there is a provider with adequate experience as well as appropriate equipment and infrastructure at the facility. Typically, general practitioners perform minor procedures and some major procedures such as caesarian sections. Anesthesia care is provided by anesthesia technicians and peri-operative nursing care by non-specialized nursing staff . If equiThis study was conducted at three of the 42 district hospitals in Rwanda: Butaro District Hospital located in the Northern Province with a catchment population of 340,000 people and Kirehe and Rwinkwavu District Hospitals located in the Eastern Province with catchment populations of 292,215 and 265,000, respectively. Care at these hospitals is led by the Ministry of Health with support from Partners In Health/Inshuti Mu Buzima (PIH/IMB), a US-based non-governmental organization. In 2015, each of the hospitals had two operating theaters. Butaro had one general surgeon on staff but Kirehe and Rwinkwavu had no surgeons. Occasionally, these hospitals, particularly Butaro, received external missions that provide surgical care. In addition, PIH/IMB provided economic and social support to select patients who had challenges accessing care.This retrospective cross-sectional study included patients who presented between 01 January and 31 December 2015 with an emergency general surgical condition at Butaro, Kirehe and Rwinkwavu District Hospitals and were admitted to the emergency and surgical wards of these district hospitals for management. We excluded patients admitted in other hospital wards, and those transferred directly to tertiary care with only names documented in the district hospital emergency logbook without available and completed medical files. We defined emergency general surgical conditions as non-traumatic and non-obstetric acute care surgical conditions. While there are more conditions that meet this definition, in this paper we focused on the most common conditions of:1) acute abdominal conditions , 2) complicated hernias , 3) soft tissue infections , 4) urological emergencies (acute urinary retention and testicular torsion), and5) thoracic emergencies .Data was extracted from hospital admission registers, patient files and operating room logbooks from the three district hospitals. Trained data collectors used a predesigned tool to collect patient data on paper forms. Data was entered into an electronic Access database and analyzed using Stata v13.0 . We described patient demographics, clinical characteristics, management, and outcomes using frequencies and percents for categorical data, and median and interquartile ranges for continuous data. We assessed the relationship between district hospital and whether the patient received surgery. We then compared the diagnosis type, surgical intervention, and treatment outcome by whether a patient was operated on by general surgeon or a general practitioner. We used Fisher’s exact test for categorical variables and Kruskal-Wallis Rank Sum test for continuous variables.n = 202 out of 353) and aged 15–60 years  . Among the 237 patients who had health insurance status recorded, 95.4% (n = 226) were insured.In 2015, 356 patients presented with emergency general surgical conditions at the three district hospitals. The majority of these patients were male  followed by acute abdominal conditions , complicated hernias , urological emergencies , and thoracic emergencies  (Table n = 134) followed by pyomyositis  were the most common diagnoses. Bowel obstruction  followed by volvulus  were the most common type of acute abdominal conditions. Of the 274 patients with duration of symptoms recorded, 54 (19.7%) presented within 3 days of symptom onset and the majority  presented after 7 days or more. The duration of symptoms prior to presentation differed by diagnosis type as follows: complicated hernias had a median duration of 17.5 days (interquartile range (IQR) =1, 208), soft tissue infections was 10 days , acute abdominal conditions was four days , and urological emergencies was 1.5 days  (p < 0.001). Among the 141 patients with prior surgical history documented, 25 (17.7%) had a history of previous surgery and for the 222 with past medical history recorded, 52 (23.4%) had at least one underlying medical illness such as hypertension, diabetes, HIV, or psychosis.The most common primary diagnoses were soft tissue infections , wound dressing , insertion of urethral catheter , insertion of nasogastric tube , and blood transfusion   underwent an operation at the district hospital, 185(52.0%) received surgery and non-surgical management and three patients (0.8%) received surgery alone. Another 143 patients (43.5%) received non-surgical treatment and for 13 patients (3.7%), we found no indication of receipt of surgery or non-surgical management. For the 188 patients who received surgery, 81 (43.1%) of these operations occurred at Butaro, compared to 59 (30.8%) at Rwinkwavu, and 49 (26.1%) at Kirehe (Table p < 0.001).For the 110 patients with type of anesthesia recorded, the most common was spinal/regional anesthesia  followed by general anesthesia , sedation  and local anesthesia . Type of surgical provider was recorded for 122 patients, with74 (60.7%) operated on by a general practitioner and 48(39.3%) by a general surgeon. The most common surgical procedure was incision and drainage , followed by laparotomy , debridement , hernia repair , amputation  and suprapubic catheterization .Non-surgical treatment included antibiotics  had at least one complication (Table n = 7) and wound dehiscence . For 188 patients who received surgery, 85.1% (n = 160) were discharged uneventfully, 4.8% (n = 9) were recommended for transfer and 3.2% (n = 6) died. The outcome was unknown for 6.9% (n = 13) of these patients. For patients who were discharged (with or without transfer recommendation), the median length of stay was 12 days . For the 168 patients who did not receive surgery, 39.3% (n = 66) were discharged, 35.7% (n = 60) were recommended for transfer, 7.1% (n = 12) diedand17.9%(n = 30) had an unknown outcome. The median length of stay for patients who were discharged without surgery (with or without transfer recommendation) was 8 days .Of the 145 patients with information on in-hospital post-operative complications, 33.8% (p < 0.001) and the type of operative treatment (p < 0.001) provided by the general surgeon compared to general practitioners (Table p < 0.001).However, there was no difference in presence of post-operative complication (p = 0.332), type of post-operative complication (p = 0.222), clinical outcomes (p = 0.062) and length of hospital stay (p = 0.342) for patients treated by general surgeon or general practitioners.In the bivariate analysis, there was a difference in the emergency general surgical conditions  did not receive surgery at the district hospital, of whom 57.1% (n = 96) were discharged home or their outcome was not reported, revealing potential surgical treatment gaps that largely reflect limited surgical capacity. These findings suggest that it is imperative to recruit and retain surgeons to rural district hospitals, as well as encourage task sharing for non-surgeon providers. Improving access to surgical care at district hospitals by increasing the number of surgical providers, anesthesia providers, and equipped functional theaters will help in advancement of universal health coverage [Of patients presenting with emergency general surgical diagnoses, approximately half received an operation at the district hospital with operations primarily performed by general practitioners. Due to the shortage of trained surgeons in sub-Saharan Africa, especially in rural areas, surgical procedures are often performed by general practitioners or other trained mid-level cadres , 16, 17.coverage .For patients who did receive surgery at the district hospital, our results also demonstrate that most were discharged without postoperative complications. Secondly, while a substantial portion of our patients did not receive surgery, we were not able to follow-up on their definitive outcome, particularly for those not recommended for transfer. Future studies should prospectively follow such patients to shed light on reasons for their lack of surgical treatment and outcome post discharge. The mortality rate (3.2%) and postoperative complications rate 33.8%) are comparable to studies done in other low- and middle-income settings , 8.While% are comThere are limitations to our study that should be considered when interpreting the results. As our study used retrospective chart review, data was missing for some variables due to incomplete documentation in patient charts. A routine audit of patient charts is recommended to encourage complete documentation. Secondly, while a substantial portion of our patients did not receive surgery, we were not able to follow-up on their definitive outcome, particularly for those not recommended for transfer. Future studies should prospectively follow such patients to shed light on reasons for their lack of surgical treatment and their outcomes post discharge. Another possible limitation is the generalizability to the rest of Rwanda or the region, as the hospitals studied are supported by Partners In Health/Inshuti Mu Buzima. PIH/IMB’s support might affect patients’ decision to seek care, as well as patient management in the hospitals. However, these hospitals are still managed by the Ministry of Health, which mandates the same surgical package of care across all district hospitals in Rwanda. Additionally, we do not believe that patient characteristics and disease burden across the rural districts of Rwanda would vary much from what we observed here. Therefore, these results are still relevant to the rest of Rwanda and other settings in rural sub-Saharan Africa.Management of emergency general surgery conditions in district hospitals remains a challenge. Our study found that only half of these patients receive surgery. Patients who do receive emergency general surgery at these three district hospitals have low mortality and limited post-operative complications. However, similar to other low- and middle-income countries, there is insufficient trained personnel and infrastructure to address all of the need. Improving surgical capacity at the district hospital and community education for early presentation would likely result in timely, safe and affordable emergency general surgical care at the district hospital and improved patient outcomes.


IgM and IgT were differentially regulated upon infection. IgT was highly upregulated locally upon infection with the intestinal parasite Enteromyxum leei or systemically after Nodavirus infection. Long-term intestinal parasitic infections increased the serum titer of both isotypes. Mucosal vaccination against Photobacterium damselae subsp. piscicida finely regulated the Ig response inducing a systemic increase of IgM titers in serum and a local IgT response in skin mucus when animals were exposed to the pathogen by bath challenge. Interestingly, plant-based diets inhibit IgT upregulation upon intestinal parasitic challenge, which was related to a worse disease outcome. All these results corroborate the mucosal role of IgT and emphasize the importance of a finely tuned regulation of Ig isotypes upon infection, which could be of special interest in vaccination studies.Three different immunoglobulin (Ig) isotypes can be found in teleost fish, IgM, IgD, and the teleost-specific IgT. IgM is considered to have a systemic activity, and IgT is attributed a mucosal role, similar to mammalian IgA. In this study, the complete sequence of gilthead sea bream IgM and IgT in their membrane (m) and soluble (s) forms are described for the first time in a perciform fish. Their constitutive gene expression is analyzed in different tissues, and their regulation upon viral, bacterial, parasitic, mucosal vaccination and dietary challenges are studied. GCB IgM and IgT have the prototypical structure when compared to other fish Igs. The constitutive expression of  IgT, ithelium , showingl organs –12 and il organs . IgT, del organs , 14 and Sparus aurata) is a marine species belonging to the Sparidae family (Teleostei: Perciformes). It is the main farmed fish species in the Mediterranean basin (Gilthead sea bream (GSB)  is the causative agent of pasteurellosis, an important disease affecting, among others, juvenile specimens of GSB, up to 100 g, while larger fish are resistant  unless otherwise stated. Animals were kept according to the Guidelines of the European Union Council (Directive 2010/63/EU) and the Spanish RD 53/2013.Specific pathogen-free and clinically healthy GSB juvenile specimens from commercial fish farms were kept in 5 μm-filtered and UV irradiated sea water at temperature always above 18°C and below 26°C, with natural photoperiod. Fish were fed All efforts were made to minimize suffering of the fish used for the experiments in accordance with national  and the current European Union legislation (86/609/EU), the Bioethical Committees of the University of Murcia for the use of laboratory animals , and the CSIC National Committee on Bioethics under approval number 151-2014.Dicentrarchus labrax L., at the Istituto Zooprofilattico delle Venice, Italy) by intramuscular injection . The control group (CTRL) was injected with the same volume of the supplemented MEM alone. After 24 and 48 h post-challenge (pc), animals were sacrificed and head kidney and brain tissue samples were collected in Trizol (Gibco) for subsequent RNA extraction.To perform this integrative study, five different trials, including challenge with different types of pathogens , immunization, and diets, were performed. For clarity, a summary of the timings and experimental groups can be found in Figure Phdp followed by bath challenge with the same bacterium. GSB were fed at a feeding rate of 1.5% of fish biomass with a commercial pelleted diet , alone or supplemented with an experimental oral vaccine designed for the active immunization of fish against Phdp. The vaccine priming was done by administering during 5 days 0.02 ml vaccine per fish plus 0.005 g of repeats of molecular CpG motifs obtained as phenol-extracted genomic Vibrio anguillarum DNA (vDNA)  was performed at the facilities of the Spanish Oceanographic Institute  and consisted of an oral vaccination trial against the pathogenic bacterium A (vDNA)  as adjuvreported  and anim for 5 h . Non-vacE. leei by anal intubation (T3) or by exposure to contaminated effluent mimicking the natural route of infection (T4 and T5). In effluent infections, a group of naïve GSB received water effluent (RCPT) from a donor tank holding E. leei-infected GSB, whereas another was kept unexposed (CTRL). In all E. leei challenges, in the final lethal sampling, samples were taken to determine the parasite prevalence and intensity of infection by histological analysis of intestines. In T5, additional intermediate non-lethal individual sampling was performed by probing the rectum with a cotton swab, and PCR diagnosis was carried out with primers specific for E. leei rDNA as described elsewhere  and consisted of experimental infections with lsewhere .E. leei-infected intestinal scrapings (RCPT) or with the same volume of PBS for the control (CTRL) group. At 17 and 64 days post-challenge (dpc), seven fish from each group were sacrificed and pieces of head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at −80°C, until RNA extraction. The prevalence of infection was 42.8 and 85.7% with low and high mean intensities of infection at 17 and 64 dpc, respectively.Trial 3 samples used in the current work come from the infection described in Pérez-Cordón et al. . Briefly+ cells were found in posterior intestine and head kidney of infected fish.Trial 4 samples come from the infection described in Estensoro et al. . GSB werE. leei infection. GSB were fed either a FO-based diet or a diet containing a blend of VO at 66% replacement. After the initial feeding period (9 months), fish from both dietary groups were individually tagged with passive integrated transponders and set up as CTRL or effluent RCPT. All fish were non-lethally sampled at 32, 53, and 88 dpc for PCR parasite diagnosis, and classified as early infected (EARLY), when E. leei-positive at 32 or 53 dpc, or late infected (LATE), when positive at 88 dpc or later. A final lethal sampling was performed at 102 dpc, and pieces of head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at −80°C until RNA extraction. The final global prevalence of infection was 92.3 and 80% for VO and FO, respectively. Mean intensity of infection was high in EARLY groups, and medium to low in LATE groups, and always higher in VO than in FO fish. All the intestinal samples of RCPT GSB used in the expression analyses of IgT and IgM were selected from E. leei-positive fish.Trial 5 samples came from the infection described in Estensoro et al. . It consIn all the trials, the lethal samplings involved sacrificing the fish by overexposure to the anesthetic MS-222 .Total RNA from the different tissues used in this study, with the exception of T1, was extracted using the MagMAX™-96 total RNA isolation kit  following manufacturer’s instructions. Total RNA from head kidney and brain from T1 samples was extracted using Trizol  in combination with the RNeasy Mini Kit  including a DNase treatment with RNase-free DNase set (Qiagen) all following manufacturer’s instructions. The concentration of all RNA samples was determined using a Nanodrop 2000c  and the RNA integrity was assessed on an Agilent 2100 Bioanalyzer. To avoid genomic DNA contamination, 500 ng of RNA samples were treated with DNaseI amplification grade , and reverse transcription was performed using the High-Capacity cDNA Archive Kit .-actin, which was used as a housekeeping gene, are shown in Table Real-time quantitative PCR was carried out with the CFX96 Connect™ Real-Time PCR Detection System , using a 96-well layout designed for simultaneously profiling all the genes of interest for all individuals studied in each tissue. The primers of the Ig genes studied and the βGSBIgT was produced by GenScript  using as an antigen a selected peptide tested to not cross-react with any other Ig or expressed gene in the GSB transcriptomic database. To verify the specificity of the antibody, 100 µg of skin mucus protein in lysis buffer  Triton X-100, 1 mM PMSF) from RCPT GSB (T4) were resolved in a 4–20% Mini-PROTEAN® TGX™ gel (Bio-Rad) under denaturing [0.1 M dithiothreitol (DTT) and heat treatment 10 min at 95°C] and native (no DTT and heat treatment) conditions, and transferred to nitrocellulose membranes. The membranes were blocked for 2 h with 20 mM Tris 0.5 M NaCl pH 7.4 (TBS) 5% (w/v) non-fat dry milk and incubated overnight with a1:200 dilution of rabbit anti-GSBIgT in TBS 1% non-fat dry milk at 4°C. After two 5 min washes with TBS supplemented with 0.05% (v/v) Tween-20 (TTBS) and other two washes with TBS, membranes were incubated with a 1:20,000 dilution of goat anti-rabbit-HRP (Sigma) for 1 h at RT, washed again, and visualized by chemiluminescence detection with Clarity™ Western ECL substrate (Bio-Rad) in an Amersham Imager 600 .A customized polyclonal anti-GSBIgM . For IgM detection, MaxiSorp 96-well plates  were coated with 50 µl of a 1:3,000 dilution of fish serum in carbonate/bicarbonate buffer pH 9.6 and incubated overnight at 4°C. After three washes of 5 min with 200 µl TTBS and one 5-min wash with TBS, plates were blocked with 200 µl of TBS 5% (w/v) non-fat dry milk for 2 h at 37°C. Subsequently, the washing steps were repeated and 50 µl of polyclonal rabbit anti-GSBIgM  1:40,000GSBIgT (GenScript) diluted 1:1,000 in PBST 1% BSA were added to the wells, and plates were incubated for 1.5 h at 37°C and washed again. One hundred microliters of goat anti-rabbit-HRP (Sigma) 1:10,000 in PBST 1% BSA were added for 1 h at 37°C, and after the final washes, plates were developed with TMB as described before.To measure total serum IgT, plates were coated with 100 µl of fish serum diluted 1:5 in PBS pH 7.4 overnight at 4°C, washed as described before and blocked with 200 µl PBS 0.05% (v/v) Tween-20 (PBST) 1% (w/v) BSA for 2 h at 37°C. After washing, 100 µl of a customized polyclonal rabbit anti-Phdp strain PC.435.1. Each assay run included a negative control, a monoclonal antibody against seabream IgM , and a customized polyclonal rabbit anti-GSBIgT (GenScript). Both antibodies were diluted 1:1,000 in PBS 1% BSA and further applied following a protocol as previously reported for IgM detection (An indirect ELISA was used to assess the specific amount of serum and skin mucus IgT and IgM (T2). Polystyrene microtiter flat wells were sensitized and coated with the antigen, etection . For IgTThe serum and mucus dilutions used in the different ELISA protocols were previously optimized using a range of serial dilutions of control and experimental samples.t-test. When conditions were not met, non-parametric tests (Mann–Whitney–Wilcoxon) were used. For multiple comparisons one-way ANOVA followed by the Tukey test or two-way ANOVA followed by the Bonferroni test were used. In all cases, significant differences were considered at p < 0.05.Statistical analysis was performed using GraphPad PRISM v5.03 and R statistical software v3.0.2 . The tesin silico analysis of the GSB transcriptomic database. The data mining revealed the presence of full length sequences for both, soluble (s) and membrane (m) bound, heavy chains of IgM and IgT, which were uploaded to GenBank with the following accession numbers: sigm JQ811851, mIgM KX599199, sIgT KX599200, and mIgT KX599201. For IgD, only a partial sequence, comprising three Igδ domains, transmembrane, and intracellular domains, was identified. Phylogenetic analysis, performed by alignment of the constant Ig domains, supported the classification of these three Igs as IgM, IgT, and IgD, as they clustered together with the same isotypes found in different fish species  and the salmonids and cyprinids each grouped in a separate branch within each of the isotypes analyzed, supporting the phylogenetic analysis and evolutionary conservation of these molecules. The subsequent analyses were focused only on GSB IgM and IgT due to the lack of sufficient information in the currently available database to describe the full IgD molecule, and to the complexity of the fish IgD protein.The study of the different Ig isotypes present in GSB was approached by Oncorhynchus mykiss) IgT  and the target organ for each pathogen, brain for nodavirus (Phdp (E. leei (Three infection models T1–T3), using viral, bacterial, and parasitic pathogens showed a differential expression of the different forms of odavirus  and inteus  10 dpc and only a slight upregulation of both IgM forms was observed in spleen of CTRL-CHAL  when compared to NAÏVE fish   than in spleen, but the expression of IgT was almost unchanged, with sIgT showing a marginal expression decrease when compared with NAÏVE fish.In T2, the bath challenge with ) Figure B. By conmIgM and mIgT, suggesting the presence of higher numbers of B cells. In head kidney, only upregulation of both forms of IgT was detected.In the parasite infection trial by anal intubation (T3), the prevalence of infection at 17 and 64 dpc was 42.8 and 85.7% with low and high mean intensity of infection, respectively . Almost GSBIgT antibody was developed and validated in western-blot, using skin mucus samples from RCPT GSB (T4). The antibody clearly recognized a band of the expected molecular weight for the IgT monomers (~65 kDa) in samples under denaturing conditions. Under non-denaturing conditions, there were no clear bands at the monomer level, whereas bands of higher molecular weight corresponding to IgT polymers were observed  was assessed. Figure The effect of the parasite Phdp (T2) and the levels of specific antibodies (IgM and IgT) were measured in serum and skin mucus. After bacterial challenge, the levels of specific IgM antibodies in the serum of vaccinated animals (VAC-CHAL) were significantly increased, remaining unchanged in mucus. On the contrary, the levels of specific IgT antibodies in mucus increased, and no changes were detected in serum  groups were studied (not shown).Vegetable oil replacement in fish diets has previously shown an effect on disease resistance and immune-related gene expression, including IgM , 46, 47. outcome . This in+/IgD+ cells exist in teleost, whereas the expression of IgM and IgT are mutually exclusive   . The int+ cells were the main responders when challenged with an intestinal myxozoan parasite, while IgM responses were only found in serum. In the same study, it was also shown that most intestinal bacteria were mainly coated with IgT. Since then, a plethora of studies have arisen showing the importance of IgT+ B cells in MALT. Fish skin, another important mucosal tissue in teleosts, shows an Ig pattern very similar to intestine. IgT+ B cells constitute a very important subset of the rainbow trout skin B cell population and IgT is found coating the majority of the skin microbiota specifically responding to skin parasites, whereas IgM changes are only detected in serum (IgT gene expression in the gills of grouper (Epinephelus coioides) larvae was induced upon bath immunization with a nervous necrosis virus vaccine, whereas in gut, an increase in IgT response was only observed upon oral vaccination. By contrast, IgM responses were detected systemically upon both immunization routes  and by exposure to contaminated water from an infected donor tank , which yielded different results. Anal intubation increased Igs expression only after 64 dpc, and this effect was more pronounced in the intestine than in head kidney. Although all the measured Igs were upregulated, the changes in mIgM and mIgT were more prominent. We propose that this was due to the infiltration or proliferation of intestinal IgM+ and IgT+ B cells. These findings are in agreement with those found by Estensoro et al.  showed expression patterns similar to those infected by anal intubation. By contrast, fish with well-established infections (EARLY) showed a surprisingly high modulation of sIgM and particularly sIgT. These changes were also detected in serum, where total titers of IgM and IgT were increased after long-term infections. Although the presence of resident IgT plasma cells in teleost intestines has not been demonstrated so far, our results hint toward an increase in these cells upon long-term parasite exposure. Future efforts should be directed to the development of optimized anti-GSBIgT antibodies to better characterize these local IgT responses.Parasites invading mucosal surfaces of rainbow trout [intestine  and skins in GSB . In thisnscripts  and incr B cells , 64 partsIgT expression. Although no changes were observed in fish fed VO diet before parasite exposure, VO diet completely inhibited the upregulation of sIgT induced by long-term parasitic infections. We correlate this effect with the worse disease outcome found in fish fed this diet  upon infection when compared to fish fed the FO diet. This increase appears as an attempt to compensate with IgM the lack of IgT, which seems not to have any positive effect regarding disease outcome, underlining the crucial role of IgT in mucosal responses. These measurements of IgT and IgM show the potential role of these isotypes, but functional evidence for specific protection remains to be demonstrated. In any case, we consider that this model of a dietary intervention that induced a blockade or inhibition of sIgT expression is as a very interesting tool to study the role of IgT in intestinal health.The most interesting finding of this study was the effect a VO diet on his diet . The pronfection . Of inte. The current results clearly show that the dynamics of expression of IgM and IgT are very different and depend on the types of pathogen or stimulation, immunization, challenge , tissues, and time after challenge. Finally, we propose the fish–AS-B, JP-S, MP, IE, and JC-G conceived and designed the study, analyzed the data, and wrote the manuscript. AS-B, IE, and MP performed the parasite challenges, sample manipulation, and assays associated with it, including ELISA and western-blot. JP-S and JC-G performed all gene expression experiments and analyses in the study. JG-V and VM conceived and performed the bacterial vaccination/challenge experiment and sampling and performed specific ELISA assays. BN and PP performed the viral challenge, sampling, and RNA extraction of such samples. EG-C carried out the epitope study for the antibody development. All the authors reviewed the manuscript.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.


Subjects had lower appetite following P supplementation, which was expressed as a significantly (p = 0.02) lower desire to eat a meal (4.0 ± 0.7 cm) compared with placebo (5.8 ± 0.9 cm). P supplementation recovers the blunted diet-induced thermogenesis in overweight and obese subjects and enhances their postprandial satiety.Diet-induced thermogenesis (DIT) is believed to be largely related to ATP production, which is dependent on phosphorus (P) availability. We aimed to test the effect of P addition on DIT of lean and overweight/obese healthy subjects. DIT was measured with or without P in 10 lean and 13 overweight/obese adults in a double-blind randomized cross-over pilot study with one week washout period. After 10 h overnight fast, resting metabolic rate, respiratory quotient, and substrate utilization were measured at fasting and every 30 min for 3 h after subjects drank a standardized glucose solution, with P (500 mg) or placebo pills. Subjective ratings of hunger and satiety were assessed before and after the end of each experiment using validated visual analogue scale (VAS) questionnaires. Overweight/obese subjects had a blunted DIT with placebo, while P supplementation induced a 23% increase in their DIT area under the curve ( Diet induced thermogenesis (DIT) is the increase in energy expenditure above the basal resting rate that takes place after the ingestion of food components. It accounts for 5%–15% of total energy expenditure . There is a large body of evidence that shows a blunted or reduced DIT in obese individuals ,4,5,6,7—On the other hand, obese subjects taking P supplementation in a weight reducing program had an increase in their basal metabolic rate ,19. SimiPostprandial ATP production in the liver has also been linked to signaling satiety, whereby changes in hepatic energy status are transmitted via hepatic vagal afferent activity to the central nervous system ,24,25,26More studies are warranted to investigate the relationship between P intake and DIT and satiety in the context of obesity and insulin resistance in young adults. Therefore, the objective of the present study was to test the effect of P supplementation on diet-induced thermogenesis and satiety after the ingestion of glucose solution in lean and overweight/obese healthy men and women. The experimental design is a double blind randomized cross over design, whereby each subject was studied twice  with a minimum of 1-week washout period. A total of 10 lean and 13 overweight and obese (herein referred to only as obese) adults were recruited. Subjects were healthy, non-smokers, sedentary , with st® Glucose Tolerance Beverages, Thermo Fisher Scientific Inc., Waltham, MA, USA) with 500 mg phosphorus pills  on the first experiment and placebo pills (Cellulose) on the second one . Using indirect calorimetry, diet-induced thermogenesis was assessed afterwards by measuring RMR every 30 min for the following 3 h. Respiratory quotient (RQ = VCO2/VO2) was calculated at fasting and postprandially from the volumes of oxygen consumed and carbon dioxide produced. Substrate utilization—namely carbohydrate and fat oxidation—were also calculated from non-protein RQ [After 10 h overnight fast and after signing the consent form detailing the study’s purpose and protocol, weight, height, and body composition  were measured using standard procedures. Thereafter, resting metabolic rate (RMR) was measured using indirect calorimetry . Subjects were then asked to drink a standard 75 g glucose solution  questionnaires . MoreoveThis study was approved by the Institutional Review Board of the Lebanese American University.t-test was performed to compare data between lean and obese groups. Paired t-test was used for comparisons between P and placebo experiments. Repeated measures were performed to analyze within-subject results for diet-induced thermogenesis. Pearson’s correlation was used to compute the bivariate relations between BMI and P intake. Based on the effect size and SD difference from the study by Khattab et al. (2015) [p < 0.05. Data are reported as Mean ± SEM. Two-tailed independent . (2015) , a minimSubject characteristics are presented in t-test). Obese subjects had a blunted DIT with placebo, as RMR did not change from baseline over 3 h after ingestion of glucose solution (p > 0.05 using repeated measures) (p < 0.05) (t-test) (easures) A, while  < 0.05) B. Moreov(t-test) A.Substrate utilization is presented in p < 0.05) for the “desire to eat a meal” and the “desire to eat something savory”. Subjective appetite scores measured using VAS at the end of the experiment showed higher appetite and lower hunger with phosphorus supplementation versus placebo , which wr = −0.48, p = 0.024)  assessed using the FFQ and BMI (= 0.024)  after po2 consumption and energy expenditure measured using indirect calorimetry in humans [To our knowledge, this pilot study is the first investigating the effect of P supplementation on DIT in lean and obese young healthy men and women. Addition of P to glucose solution resulted in an increase in DIT compared with placebo in both lean and obese subjects. The increase was more pronounced in obese individuals, probably because they had a blunted DIT with placebo, whereby postprandial energy expenditure did not differ from fasting baseline. Lower DIT in obese individuals compared to lean was previously found ,4,5,6,7,n humans , which vOur results are in line with those by Jaedig et al. (1994)  and KaciPostprandial subjective appetite of lean and obese subjects was suppressed compared to placebo in the present study, 3 h after the ingestion of glucose solution with the addition of phosphorus. This is in line with our previous findings, whereby phosphorus addition to different carbohydrate preloads resulted in lower subsequent ad libitum energy intake in lean individuals . Longer-Interestingly, usual dietary P intake of lean subjects assessed using FFQ was significantly higher than that of obese group. This difference could not have affected baseline P, since fasting serum P is tightly controlled in healthy humans and is not affected by diet ,44. TherTaken together, our findings support a role of P in regulating energy metabolism, and thus in the management of obesity. Indeed, an inverse correlation between P intake and BMI was observed in our study when data from lean and obese subjects were pooled together, albeit our small sample size. Consistently, this was widely reported in the literature in both animal  and humaLimitations of the present study include that it is a pilot study lacking assessments of potential mechanism(s) of action—especially regarding insulin sensitivity, glucose tolerance, hepatic ATP, and objective measures of appetite. Strengths include our original findings with respect to the P effect on DIT in lean and obese healthy adults and the cross-over double-blinded nature of the design that reduces bias. Future studies are warranted to replicate results in longer-term studies with large sample size and to investigate mechanism(s) of action. Phosphorus supplementation increases postprandial thermogenesis in lean and obese adults, with a more pronounced effect among obese subjects. Phosphorus also promotes fullness in obesity. Our findings set the stage for meal P supplementation or fortification as a promising obesity treatment at moderate levels , which are far below the upper safe level of intake (4 g) .


Rosmarinus officinalis L.), turmeric , and ginger  essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT) and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs), and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells.The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, The use of essential oils (EOs) of aromatic plants for dietary and therapeutic purposes has been a focus on research in health sciences due to their different biological properties such as antimicrobial, analgesic, anti-inflammatory, antiparasitic, antioxidant, and antitumoral effects –4. The mRosmarinus officinalis L.), Lamiaceae family, are used as a food condiment and to enhance or change the flavor of foods [Curcuma longa L.) and ginger  are members of the Zingiberaceae family. Turmeric is a native plant from India and South Asia. However, it has now been found worldwide and widely used as a spice, giving foods a characteristic flavor and color. Ginger originates from Southeast Asia and has marked characteristics of odor and hot flavor [The dried leaves of rosemary , turmeric , and ginger  essential oils in HeLa human cervical cancer cells, aiming at its determining potential anticancer activity.The antitumor property of EOs has been the source of investigation for the development of drugs to treat different types of cancer. There is the perspective that EOs can be used as a therapeutic agent and confer benefits for human health, provided their toxicity can be established. The objective of this study was to evaluate the cytotoxic activity of rosemary , turmeric (CEO), and ginger (GEO) essential oils were previously obtained by hydrodistillation of dried leaves oficinalis  and rhizC. longa  and Z. oficinale , accordificinale . The EOsμg/mL) , and 2 μg/mL amphotericin B . The cells were kept in an incubator  in a humidified atmosphere of 5% CO2 in air at 37°C [HeLa human cervical cancer and HepG2 human liver cells were obtained from the Rio de Janeiro Cell Bank . Each cell line was cultured in complete Dulbecco's Modified Eagle's Medium , supplemented with 20% (v/v) fetal bovine serum (FBS) , solution containing penicillin (100 IU/mL) and streptomycin  were seeded onto 96-well plates overnight. After that, the cells were treated with complete medium only (control cells) or seven different concentrations of REO (31.12–1192 μg/mL), CEO (32.81–2100 μg/mL), or GEO (20.12–1928 μg/mL) for 24 h. Posteriorly, the cells were washed with PBS and 100 μL of medium (DMEM + 2% SFB) containing MTT (0.5 mg/mL)  was added in each well. The microplate was incubated for 3 h. After this period the solution was discarded by inversion and 100 μL of dimethyl sulfoxide (DMSO)  was added. The plate was placed in a microplate shaker at 250 rpm for 15 minutes. Absorbance was determined at 540 nm in a spectrophotometer . This assay was performed in triplicate.MTT reduction test, adapted from Mosmann , was car6 cells/mL) were seeded onto 96-well plates overnight. Then, the cells were treated with complete medium only (control cells), REO, CEO, and GEO, at the same concentrations used for the MTT assay, for 24 h. After that, the supernatant of each well was removed and 100 μL of DMEM containing 5% (v/v) FBS and neutral red  was added. After 3 h of incubation, NR medium was discarded and the cells were washed with PBS. Posteriorly, 100 μL of NR desorb solution (50 EtOH : 1 acetic acid : 49 water) was added to the wells. The plates were shaken and the absorbance was measured at 550 nm.NRU assay  was used6 cells/mL) were seeded onto plate overnight. Then, the cells were treated with complete medium only (control cells), REO, CEO, or GEO, at five different concentrations (250–2000 μg/mL) for 24 h. After that, the cells were removed from the plate and an aliquot of cell suspension was diluted with trypan blue solution (1 : 10) . The cells were then observed under light microscopy  and the viability of the cells was estimated using a Neubauer Chamber.Trypan blue dye is used to analyze cell viability via membrane rupture, since it does not cross intact membrane of viable cells. Thus, stained cells are scored as dead. Briefly, the cells  and then the nucleus disintegrates into fragments surrounded by nuclear membrane, originating apoptotic bodies . Thus, m6 cells/mL) were seeded onto 24-well plate overnight. The cells were then treated with complete medium (control cells), camptothecin (positive control), REO, CEO, or GEO for 24 h. After that, the supernatant of each well was removed and the cells were washed with PBS. Then, 400 μL of Annexin-binding buffer  and 10 μL of Annexin V were added in each well. The plates were incubated at room temperature, protected from light, for 15 min. After incubation, the cells were analyzed by a fluorescence microscopy .Annexin V binding assay allows identifying changes in the plasma membrane, which occur during the early events of the cell death process by apoptosis . The assDifferent statistical treatments were applied for the cytotoxicity assays. For the trypan blue assay, application of Kruskal-Wallis rank sum tests was necessary. In cases where the test detected significant difference at up to 10% significance, the Bonferroni pairwise comparison test was applied. The Quadratic Logistic Regression Model was applied for the other assays using the estimated parameters on the Quadratic Logistic Regression Model. The results were analyzed on the R software program version 3.2.1 (Team R Development Core 2015) using the “RDC” package .(1)ModelThe chemical characteristics of REO, CEO, and GEO are demonstrated in The REO, CEO, and GEO were submitted to MTT and NR assays to assess cytotoxicity activity . The resα-pinene), while main components for CEO and GEO were sesquiterpenes and oxygenated monoterpenes. Both CEO and GEO showed significant cytotoxic activity in HeLa cells in comparison to HepG2 cells  in comparison to HepG2 cells (control) Figures . The maj Figures . Wang etβ-sesquiphellandrene had strong antiproliferative activity in different tumor cells and also exhibited synergism with chemotherapeutic agents. In the present study, the compound β-sesquiphellandrene was the fifth-most prevalent compound in CEO (2.4%), where this may also have an effect on the suppression of cervical cancer colonies in combinatorial action with other compounds identified.The studies conducted by Tyagi et al.  evaluateμg/mL) showed no significant difference in comparison to HepG2 cells (control). However, comparison of concentrations pairwise yielded significant differences . The significant comparisons between pairs were 2,000-1,500 (p = 0.5178) and 2,000-250 (p = 0.7496) for CEO; 2,000-1,000 (p = 0.9089) and 2,000-250 (p = 0.0423) for REO; and 2,000-250 (p = 0.036) and 1,500-250 (p = 0.4382) for GEO. Based on these results, it can be noted that GEO had greater antiproliferative activity against HeLa cervical cancer cells at a lower concentration. Our results corroborate those of the studies by Jeena et al. [ in vitro in DLA (L929) cells. The authors also highlighted the idea that GEO showed antitumor potential and may be used as an anticancer agent.The concentrations of REO, CEO, and GEO used in this experiment in HeLa cells .Our results also show the same characteristic morphological patterns of cell death by apoptosis for CEO and GEO, causing HeLa cells to exhibit changes in their morphology at the lowest concentration studied Figures . CEO andThe bioactive properties of EOs have attracted growing interest from scholars seeking to unveil the mechanisms of action of these natural products, prompting an increase in studies assessing the antiproliferative, antitumor, and anticancer activity of these compounds .C. longa) and ginger  essential oils were possibly responsible for presenting better antitumor activity. CEO and GEO showed effective cytotoxic activity against human cervical cancer cells (HeLa), inducing significant reduction in cell viability of these tumor cells. Our results clearly show that this cytotoxicity was responsible for inducing cellular death in human cervical cancer cell by apoptosis. Therefore, both CEO and GEO can be considered promising chemotherapeutic agents in the treatment of cervical cancer. Further in-depth studies determining the mechanism of action of both CEO and GEO, along with their components, must be warranted.Oxygenated monoterpene compounds present in turmeric (


A significant gender interaction was identified (p < 0.01). There was an association between %5 mC and LTL in females , but not in males. Further sensitivity analyses by race revealed similar associations in African Americans and whites . The present study, for the first time, shows that lower levels of global DNA methylation are associated with shorter telomere lengths in youth, which may decrease genome stability and augment the susceptibility to diseases. Longitudinal studies are warranted to establish the effects of global DNA methylation on LTL maintenance over time.Emerging evidence suggests that epigenetics regulates telomere dynamics in adults. However, the relationship between these pathways in children and youth remains unknown. Thus, we examined this association in 542 healthy adolescents aged 14 to 18 years old . Global DNA methylation level (%5-mC) was quantified using ELISA method. Leukocyte telomere length (LTL) was defined as relative telomere to single copy gene (T/S) ratio. Multiple linear regression models, adjusted for age, gender, ethnicity, Tanner stage, BMI, PA, and batch effect, revealed that %5 mC was associated with LTL (adjusted β = 0.17,  The downregulation of DNA methylation levels in long interspersed nuclear element-1 (LINE-1) and short interspersed nuclear elements (SINEs) have been suggested as key developmental events of cancer4. Global DNA methylation has been examined in surrogate tissues, including leukocyte DNA5. Overall methylation levels reflect overall alterations in gene expression. Low levels of leukocyte global DNA methylation have been associated with increased risks for various cancers, including head and neck squamous cell carcinoma, bladder, breast, gastric, and colorectal adenoma in adults6. Our recent research has shown that leukocyte DNA methylation alterations  are associated with obesity, hypertension, and vitamin D deficiency in adolescents11.Global DNA methylation is an important epigenetic mechanism, starting as early as fetal development, regulating gene expression, chromosomal stability, and disease susceptibility15. Mutations in telomerase and telomere components lead to a broad spectrum of disease that has presentations in adults and children. The syndromes of short telomeres, such as Dyskeratosis Congenita, is marked by vascular and degenerative components as well as by cancer predisposition17. We have previously shown that diet and physical activity influence telomere dynamics in adolescents20.Telomere shortening has been proposed as a mechanism for decreasing chromosomal stability, which increases risks for chronic diseases, cancer, cardiovascular disease, and overall mortality22. LINE-1 DNA hypomethylation, a surrogate for global DNA hypomethylation, may decrease the methylation of subtelomeric regions, which are associated with short telomere length21. Moreover, DNA methylation levels in multiple subtelomeric and imprinted loci were associated with leukocyte telomere lengths22. However, the relationships between epigenetics and telomeres remain unknown in youth. The present study, for the first time, tested the hypothesis that lower levels of %5 mC would be associated with shorter LTL.Both leukocyte global DNA methylation and telomeres are involved in aging processes and disease vulnerability. Emerging studies have suggested that global DNA methylation may help regulate the variability of telomere length (LTL) in adultsp < 0.01). Additionally as previously reported, females had fewer minutes of vigorous physical activity (PA)18 (p < 0.05). Likewise as previously reported, African Americans were heavier and had longer LTL than Whites18.The general characteristics, levels of %5 mC, and LTL of our subjects were presented by gender and ethnicity in Table p < 0.01, Table p < 0.01) .In the whole sample, multiple linear regression model adjusted for age, gender, ethnicity, Tanner stage, BMI, and batch effect was significant (p < 0.01). There was a significant association between %5 mC and LTL in females , but not in males . Participants were recruited, from local public high schools in the Augusta, Georgia area (located in Southeast US), to take part in the Lifestyle, Adiposity, and Cardiovascular Health in Youth (LACHY) study. Participants were excluded with the following self-reported conditions: taking current medications or diagnosed with chronic medical conditions that could affect growth, maturation, physical activity, nutritional status, or metabolism.The Institutional Review Board at the Medical College of Georgia, Augusta University approved this project . Flyers, approved by school superintendents and principals, were distributed to all high schools. Written informed consent was obtained from all adult subjects and from parents/guardians of those younger than 18 years old. All methods were performed in accordance with the relevant guidelines and regulations. Each participant was assigned a unique subject number. All data were anonymized and de-identified prior to analysis.2).Height and weight were obtained according to standard procedures, using a wall-mounted stadiometer  and calibrated electronic scale . Prior to testing each week, the electronic scale was checked for accuracy using known weights. BMI was calculated as weight (kg) divided by height  to 5 (fully mature) as described by TannerThe total daily minutes spent for moderate and vigorous physical activities (PA) were assessed using MTI Actigraph monitors , uniaxial accelerometers that measure vertical acceleration and deceleration. With epoch length set at 1 min and expressed as counts per minute, the accelerometers started recording when the subject left our institute after the first testing day. The subjects were instructed to 1) wear the monitor for a period of 7 days, 2) remove it for sleep, bathing, and any activity that may cause harm to either the monitor or another person (e.g. during contact sports), and 3) bring the monitor back to us after 1 week. Data from days 1 and 7 were discarded because a full day of information was not available. Movement counts were converted to average minutes per day spent in moderate  and vigorous  PA by the software accompanying the device.Genomic DNA was extracted from stored buffy coat samples using Qiagen® QiAamp® DNA Mini Kit . DNA samples with OD 260/280 and 260/230 greater than 1.8 were used for global DNA methylation measurement. Global DNA methylation level was quantified in 100 ng genomic DNA using leukocyte DNA with the MethylFlashTM Methylated DNA Quantification kit  following manufacturer’s instruction. Briefly, the methylated DNA was detected using capture and detection antibodies to 5-methylcytosine (5 mC) and then quantified colorimetrically by reading absorbance at 450 nm using Bio-Tek PowerWave HT Microplate Spectrophotometer . The amount of methylated DNA is proportional to the OD intensity measured. Relative quantification was used to calculate percentage of 5-mC (%5 mC) in total leukocyte DNA following the manufacturer’s instructions. Each sample was run in duplicate.41. The relative ratio of telomere repeat copy number (T) to single-copy gene copy number  was determined using a 7500 Fast real-time PCR System . Samples were done in triplicate. Threshold values (Ct) were obtained by averaging the triplicates. Each 96-well plate contained a five-point standard curve using the same control genomic DNA from 3 to 48 ng to control the day-to-day variations. Standard curves with linearity R2 > 0.98 were accepted. Telomere PCRs and 36B4 PCRs were performed on separate plates, with the same sample well position. T/S ratio was calculated as: the amount of telomeric DNA (T) divided by the amount of single-copy control gene DNA (S). The intra-plate and inter-plate coefficients of variation for the T/S ratio were 5.6% and 6.8%, respectively18.Mean LTL was determined from leukocyte DNA by a modified quantitative PCR-based assay as previously describedW test and Levene’s test for equality of variances. Since %5 mC and LTL deviated from normality significantly, natural log transformations were applied. Ethnicity and gender differences were checked for age, gender, BMI, PA, Tanner Stage, levels of %5 mC, and LTL, using independent samples two-tailed t-tests.Descriptive statistics for raw variables are presented as mean ± standard deviation  with the significance level set at


Hyperglycemia is associated with an increased risk of microvascular complications in patients with type 2 diabetes. The aim of the present study was to investigate whether the reduction of the levels of HbA1c by tight glycemic control (GC) decreases the rate of microvascular complications and improves the neurological measures in patients with type 2 diabetes.Detailed clinical and neurological examinations including corneal confocal microscopy (CCM) were performed in 141 Japanese patients with type 2 diabetes and 60 age-matched control subjects at baseline and follow-up with GC for 4 years. Patients were stratified according to the mean HbA1c level during follow-up into good , fair , and poor  GC groups with similar HbA1c levels at baseline (84.5–88.2 mmol/mol).At baseline, CCM revealed significant nerve fiber damage in all patients compared to that in controls. The interval changes in most corneal nerve fiber (CNF) parameters and neurophysiological functions were significantly related with the mean HbA1c levels during follow-up. Interestingly, the baseline HbA1c level did not impact on neurological functions at follow-up. Interval changes in neuropathy outcomes were associated with mean clinical factors during follow-up and hypoglycemic strategies. Good GC improved all nerve functions, including CNF branch density and bead, but not the length and main fiber density. Fair GC deteriorated some nerve functions. Poor GC compromised all neuropathy outcomes. Irrespective of GC levels, retinopathy increased after follow-up period, while nephropathy decreased.This study showed that tight GC was beneficial just for nephropathy among microvascular complications. Despite strict GC, the retinopathy progressed in patients with type 2 diabetes. Glucose control did not improve neurophysiological and corneal nerve measurements unless near-normoglycemia was reached. Hyperglycemia is associated with an increased risk of microvascular complications in patients with type 2 diabetes mellitus (T2DM). Several large clinical trials concluded that intensive glycemic control (GC) in type 2 diabetes is associated with a reduction in microvascular complications, in particular nephropathy and vascular outcomes –3.It has been shown that intensive GC has an equivocal efficacy for diabetic peripheral neuropathy (DPN) in T2DM . The earSmall nerve fibers can be assessed objectively by quantifying intraepidermal nerve fiber density in skin biopsies; however, this is an invasive procedure that requires expert laboratory assessment and has considerable variability even among control . The useDue to potential severe hypoglycemia , euglyceThe aim of the present study is to establish whether the tight control of HbA1c improves the CNF measures and neurophysiological functions along with the occurrence of nephropathy and retinopathy in patients with poorly controlled type 2 diabetes by strict GC for 4 years.At the baseline, 141 Japanese patients with poorly controlled type 2 diabetes (HbA1c > 58.5 mmol/mol) who underwent GC for 3–5 years and 60 healthy control subjects without diabetes (HbA1c < 38.8 mmol/mol) were enrolled between June 2011 and July 2012. The patients were followed up for 4 years in average (March 2015 to November 2016) at Ishibashi Clinic, Hiroshima, Japan. The patients with diabetes were divided into the following three subgroups according to baseline HbA1c levels: subgroup 1; 58.5 mmol/mol < HbA1c ≤ 74.9 mmol/mol, subgroup 2; 74.9 mmol/mol < HbA1c ≤ 91.3 mmol/mol, and subgroup 3; HbA1c > 91.3 mmol/mol, and also according to the mean HbA1c level during follow-up . The patients assigned to the insulin-sensitizing or insulin-providing strategy were treated with biguanides or pioglitazone, or with sulfonylureas or insulin, respectively  , any typin vivo CCM   corneal nerve fiber length (CNFL), the total length of all nerve fibers (mm/mm2); (3) corneal nerve branch density (CNBD), the number of branches emanating from all major nerve trunks/mm2; (4) beading frequency (BF) (/0.1 mm); and (5) bead size (BS) (μm2) determined after enlarging five times and smoothing the original image of CCM using S-Spline Max algorithm . The pixel numbers of 120 beads were counted using Photoshop Elements 8.0  and averaged (All study subjects were examined using a Heidelberg Retina Tomograph (III) Germany) . Patientaveraged . Except All healthy control subjects and patients with T2DM underwent detailed neurophysiological examinations at baseline and final follow-up visits. The neurological deficits were assessed using the modified neuropathy disability score (NDS) , which iElectrophysiology and nerve conduction velocity (NCV) studies were performed using an electromyography instrument . The motor nerve conduction velocity (MCV) of median nerve (MN) and sensory  NCVs and their action potential amplitudes were determined. Skin temperature was maintained above 32°C.R–R) was calculated from the R–R intervals of 200 samples on electrocardiogram.The vibration perception threshold (VPT) was measured at the left medial malleolus using a biothesiometer . The warm and cold perception thresholds (PTs) at the dorsum of the foot were determined using a thermal stimulator . To assess cardiac autonomic neuropathy (CAN), the coefficient of variation of R–R intervals . The measured HbA1c levels were converted to National Glycohemoglobin Standardization Program units  and subsAt baseline and end point, bilateral retinal fundus images were captured (the field of assessment: 45°) and graded according to the ETDRS scale: no apparent retinopathy, 0; mild nonproliferative diabetic retinopathy, 1; moderate nonproliferative diabetic retinopathy, 2 .p-value of <0.05 was considered to be statistically significant.Statistical analyses were performed using SPSS ver. 19 . A post hoc analysis of sample power revealed that by using a one-sided ANOVA (significance of 0.05) and Kruskal–Wallis test for CNF measures and neurophysiological tests, the present study population provided statistical power ranging from 0.71 to 0.99. All values are presented as mean ± standard error of the mean (SEM). All data sets were tested for normality using the Shapiro–Wilk test. For normally distributed variables, comparisons between controls and patients with diabetes  or between controls and each diabetic subgroups divided by the baseline or the mean HbA1c levels were performed using one-way ANOVA for continuous variables and the χ2-test for categorical variables followed by Bonferroni correction. For non-normally distributed variables, the Kruskal–Wallis test was applied followed by the Mann–Whitney U-test and Bonferroni correction for continuous variables and the McNemar test for categorical variables. The differences between baseline and end point were assessed using the paired t-test and Wilcoxon’s signed rank test for normally and non-normally distributed continuous variables, respectively. Correlations between the neuropathy outcome measures and the mean clinical factors during follow-up or treatment strategies were assessed using Spearman’s rank correlation coefficient or multiple regression analysis. The sensitivity, specificity, positive-predictive value (PPV), and negative-predictive value (NPV) of CNF measures and neurophysiological tests in differentiating between control subjects and patients with or without neuropathy were assessed at baseline and end point using receiver-operating characteristic analysis.The R–R, warm and cold PTs, and most CNF measures) were impaired in patients or subgroups compared to those in controls . In all patients and in particular subgroups 2 and 3, the retinopathy significantly increased and progressed; however, the nephropathy significantly decreased. The incidence of neuropathy did not significantly change in all subgroups. Overall, the GC was not beneficial for improving the neurophysiological functions and CNF parameters, and some neurophysiological functions rather deteriorated after GC (Table p = 0.383) and retinopathy increased from 21.3 to 35.5% (p < 0.001); however, the cumulative incidence of nephropathy reduced from 37.6 to 22.0% (p < 0.001)  and CNFL (p = 0.069) did not improve, while they were significantly higher than those under poor GC . CNBD, BF, and BS were improved under good GC, and fair GC decreased BS. The poor GC worsened all CNF measures (p = <0.001–0.027)  inhibitor was prescribed more frequently than at baseline in all subgroups and was prescribed less frequently under good GC than others Table .p = 0.059–0.960). Except for CNFD  and ulnar nerve amplitude , changes in all other neuropathy outcome measures by GC significantly associated with the magnitude of reduction in HbA1c levels during follow-up . The duration of diabetes might worsen the MN MCV. The LDL-C level was positively associated with the VPT. The insulin-providing strategy deteriorated the nerve functions. The DPP-4 inhibitor impaired the SN SCV and amplitude (Table p = 0.962–0.194) had no significant association with interval changes in any neuropathy outcomes. The interval changes in HbA1c levels were correlated with the interval changes in CNF measures and neurophysiological tests  during follow-up and the duration of diabetes (p = 0.004) were the most significant factors. None of patients in good GC subgroup developed neuropathy. Among the neuropathy outcome measures at baseline, the MN MCV (p = 0.039) and the SN SCV (p < 0.001) in patients developing neuropathy were slower than those in patients without neuropathy.In 116 patients without neuropathy at baseline, 14 patients developed neuropathy after follow-up. When the clinical factors at baseline and their mean levels during follow-up were compared between patients with or without the development of neuropathy, the mean HbA1c levels  for 24 years, the decline in nerve functions was completely prevented . AlthougIn patients with type 1 diabetes receiving simultaneous pancreas–kidney transplantation, euglycemia improved CNF measures within 12 months after transplantation . In typevia their adenosine triphosphate production in nerve fibers. We already reported that the hyperglycemia-induced expansion of beads in CNFs might be a predictor of slow NCV in patients with type 2 diabetes (As BF and BS showed better association with the mean HbA1c level than other CNF measures, the beads may be more responsible to strict GC than other CNF measures. The beads visualized by CCM are composed of the accumulated mitochondria, glycogen particles, and vesicles , and mitdiabetes . HoweverAs the GC strategy influences DPN , we examIn good GC subgroup, BMI significantly decreased after GC, and hence, good response to lifestyle intervention may improve the neuropathy outcomes . Among tBecause the performance and area under curve of most CNF parameters at baseline and end point for differentiating between controls and patients with or without neuropathy were satisfactory, the assessment of neuropathy outcome measures at baseline may predict the subsequent changes especially in those with more severe damage and those that are the subjects that may further benefit from GC. Because CNFD and CNFL excellently differentiated between controls and patients without neuropathy, these are the best measures for detecting the early DPN. Neurophysiological tests had modest sensitivity and specificity. Before and after developing mild neuropathy, high PPV and NPV were found, respectively, suggesting that the neurophysiological tests contribute to the sensitive diagnosis in the early stage and reliable diagnosis after developing neuropathy.In the current study, with optimal GC, nephropathy decreased, while the retinopathy increased. The initial extreme hyperglycemia paired with rapid and substantial HbA1c reduction might develop the retinopathy . The difThe present study measured the main modifiable risk factors including HbA1c level, blood pressure, and BMI monthly, and the lipid profile, estimated glomerular filtration rate, and ACR every 3 months. Therefore, the mean clinical factors impacting on DPN are representative, and the contribution of HbA1c levels attained by GC and other risk factors on neuropathy outcome measures and other microangiopathies was reliably evaluated. We estimated the morphological and functional measures of SNFs. The altered CNFs were found in all patients with type 2 diabetes at baseline, and near-normoglycemia improved some CNF parameters and all neurophysiological tests. The present study had some limitations. First, we assessed neuropathy outcome measures only at baseline and end point. Ideally, these examinations should be performed annually for assessing the trends of their changes. Second, the development of DPN depends on the metabolic memory . Our folIn conclusion, the HbA1c level closer to 47.5 mmol/mol achieved mainly with insulin-sensitizing agents and lifestyle modification would be a safe glycemic goal for improving the outcome measures of DPN in patients with poorly controlled type 2 diabetes with mild to moderate neuropathy. The near-normoglycemia is effective for preventing the development of neuropathy and nephropathy, but not retinopathy. However, achieving this is not feasible in many patients.Written informed consent was obtained from all subjects based on the Declaration of Helsinki. The ethics committee of Ishibashi Clinic approved the protocol of this study.FI designed the study, researched data, and wrote the entire manuscript. MT advised on the statistical analysis, interpreted the results, contributed to writing, and reviewed and revised the whole manuscript. FI and MT are the guarantors of this work and, as such, had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis and interpretation.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.


Pseudomonas aeruginosa at 5 weeks after cardiac transplantation, on a background of prior bridging therapy with left ventricular assistant device (LVAD). The patient was successfully treated with the newer cephalosporin, Ceftolozane/Tazobactam, in combination with surgery. This is the first reported case of mycotic pseudoaneurysm infected with MDR Pseudomonas. This case also highlights the importance of high vigilance and timely multimodality treatment in the diagnosis and management of mycotic pseudoaneurysm following cardiac transplant, especially in patients who had LVAD.Mycotic pseudoaneurysm of aorta following cardiac surgery is rare but is highly fatal if it is unrecognized and untreated. Here, we report a case of a 45-year-old male patient who presented with rapidly progressive multiple pseudoaneurysms of the ascending aorta infected with multidrug resistant (MDR) Mycotic aneurysm is defined as any extra or intracardiac aneurysm of infectious etiology excluding syphilis. It involves aorta after open-heart surgeries such as cardiac transplant or coronary artery bypass, mostly at the anastomotic site and aortic suture lines . The risA 45-year-old man presented with 3-day history of fever and thoracic back pain at 5 weeks after receiving an orthotopic heart transplant for idiopathic dilated cardiomyopathy. The patient was on a left ventricular assistant device (HeartWare LVAD) as the bridging therapy for decompensated heart failure for 6 months prior to the cardiac transplant. There was no prior history of infective endocarditis or LVAD related infections such as pump, pocket, or drivelines infections. Immediate posttransplant period was unremarkable and he was discharged on standard immunosuppressive therapy with Prednisolone, Cyclosporine, and Mycophenolate.On examination, he was alert, febrile at 38.7°C, and haemodynamically stable with blood pressure of 110/65 mmHg and pulse rate of 120 bpm. There was no spinal tenderness or other localizing sign. His sternotomy wound and the previous LVAD exit site had healed well without evidence of cellulitis. His heart sounds were dual and lungs were clear with good air entry on auscultation. There was no sign of cardiac failure. He did not have any focal neurological deficit.9/L (ref: 4.0–11.0), predominantly neutrophils (12.66 × 109/L) (ref: 2.0–8.0), low hemoglobin of 83 g/L (ref: 135–180), normal platelet count of 175 × 109/L (ref: 140–400), urea of 14.9 mmol/L (ref: 2.1–7.1), creatinine of 119 umol/L (ref: 60–110), normal liver function test, and coagulation profile. Chest X-ray showed mild cardiomegaly but no consolidation or effusion. Urinalysis was unremarkable with negative blood and white cells.Investigations showed elevated serum C-reactive protein (CRP) of 163 mg/L (ref: <5.0), elevated white cell count of 13.0 × 10 Pseudomonas aeruginosa, which was sensitive to Gentamicin, Amikacin, and Colistin by disc testing but resistant to Meropenem, Piperacillin-Tazobactam, Ceftazidime, Cefepime, and Ciprofloxacin. Further minimum inhibitory concentration (MIC) showed MIC = 12 for Meropenem and MIC = 2 for Ciprofloxacin. The same Pseudomonas species was previously isolated from a routine superficial wound swab from the LVAD exit site 4 weeks prior to the current presentation. The patient was afebrile without any obvious evidence of cellulitis at that time, but one week of IV Gentamicin was administered with subsequent good healing of the exit site.He was initially managed with empirical intravenous (IV) Gentamicin and Piperacillin-Tazobactam. Blood cultures subsequently grew multidrug resistant18F-fluorodeoxyglucose (FDG) tracer showed moderate FDG uptake in the aortic outpouchings and the diagnosis of mycotic pseudoaneurysm was made  and CT chest/abdomen/pelvic were performed. TOE excluded infective endocarditis but revealed 2 small outpouchings, measuring 2.5 × 2.4 cm and 0.45 × 0.6 cm, in the ascending aorta, which were also seen on CT chest . PET scawas made .24)/MIC ratio of 70–100 and Meropenem was administered as a continuous intravenous infusion of 6 grams over 24 hours, targeting a steady-state unbound plasma concentration above the MIC.The case was discussed with the cardiothoracic surgical team, and a trial of conservative management with IV Gentamicin and Meropenem was instituted. Gentamicin was administered once daily, targeting an area under the concentration-time curve from 0 to 24 hours of concentration  of brain also showed small areas of restricted diffusion in frontal and parietal lobes bilaterally suggestive of acute embolic infarcts although the patient did not have any focal neurological deficits. Pseudomonas and additional sensitivity test showed that it was sensitive to Ceftolozane/Tazobactam (MIC = 4). Postoperatively, antibiotic therapy was continued with IV Ceftolozane/Tazobactam for 8 weeks followed by oral Ciprofloxacin 750 mg twice daily.The antibiotic therapy was changed to IV Ceftolozane/Tazobactam 1000 mg/500 mg every 8 hours and the patient proceeded to have redo-sternotomy, total excision of pseudoaneurysm, and associated ascending aorta and aortic allograft implantation. Cultures of the resected specimen grew the same MDRThe patient is now 6 months after discharge and remains well without any evidence of recurrence. The plan is to continue oral Ciprofloxacin as lifelong suppressant therapy.Mycotic aneurysm is defined as any extra or intracardiac aneurysm of infectious etiology excluding syphilis. It involves aorta after open-heart surgeries such as cardiac transplant or coronary artery bypass, mostly at the anastomotic site and aortic suture lines  The risk Pseudomonas was isolated.The incidence seems to have increased over the last two decades. In 2011, Tang et al. reported a series of 8 cases from their single institution and 12 other cases that were published between 1999 and 2011. Five out of the eight cases had LVAD as bridging therapy, and the authors suggested that the increasing use of bridging LVAD might lead to an increase prevalence of mycotic pseudoaneurysm following cardiac transplantation . LVADs aPresentation with mycotic pseudoaneurysm is usually delayed after the transplant procedure, from a couple of months to years . Most ofMycotic aneurysm postcardiac transplant poses very high mortality, up to 50% –8 despit Staphylococcus aureus, Candida species, and Pseudomonas aeruginosa but infection with Coagulase negative Staphylococcus, Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococci, multidrug resistant Enterobacter cloacae, and Aspergillus has also been reported [ Pseudomonas aeruginosa, which has not previously been reported in the literature to our knowledge.Common isolated causative microorganisms includereported –4, 7, 9.T>MIC) and to allow administration of a higher dose without peak concentrations that might result in neurotoxicity. While studies investigating clinical outcomes of administering beta-lactams by continuous infusion or extended infusion versus standard dosing have had mixed clinical outcomes [T>MIC. Using a continuous infusion, we achieved a free fraction steady-state Meropenem concentration of 44 mg/L which was approximately 3.5 times the MIC and was tolerated without any apparent neurotoxicity. On the other hand, therapeutic drug monitoring of Gentamicin was performed by calculating an AUC as described by Begg et al. [Cmax: MIC of >10 and an AUC0–48 h: MIC of 140–200 mg·h/L by dosing every 48 hours.In choosing an appropriate antibiotic regime in our patient, microbial sensitivity (MIC), renal function, drug interactions in context of nephrotoxic immunosuppressant therapy, and practicality of therapeutic drug monitoring were taken into consideration. Meropenem was administered by continuous infusion in an attempt to optimize time above MIC , chromosomal AmpC, and upregulation of efflux pumps . The use of Ceftolozane/Tazobactam has been well described in the treatment of complicated intra-abdominal infections in combination with Metronidazole and in complicated urinary tract infections including pyelonephritis [Despite this, medical treatment appeared to fail and the Gentamicin was substituted with IV Ceftolozane/Tazobactam. Ceftolozane is a novel bactericidal antipseudomonal cephalosporin, which exerts bactericidal activity by inhibition of bactericidal cell-wall synthesis. Tazobactam is a beta lactamase inhibitor by inhibiting certain class A and class C beta lactamase. The combination is stable against most commonephritis , 15. To  Pseudomonas aeruginosa postcardiac transplant, in a patient who had bridging therapy with LVAD. The case displayed rapid progression but was successfully managed by timely surgery in conjunction with appropriate concurrent antibiotic therapy. The case highlights the importance of close follow-up, low index of suspicion for vascular imaging, early diagnosis, and multimodality intervention in cardiac transplant patients bridged by VAD.In summary, this is the first reported case of mycotic pseudoaneurysms of aorta with MDR


The lymph node (LN) status is very important for the survival in pancreatic neuroendocrine tumors (PNETs). Therefore, the investigation of factors related to LN metastases has a great clinical significance. The aim of this study was to evaluate the predictive value of the preoperative neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and possible clinical parameters on the LN metastases in nonfunctional PNETs (NF-PNETs). A retrospective review of 101 NF-PNET patients following curative resection and lymphadenectomy was conducted. The associations between clinicopathological factors and LN metastases and prognosis were determined. Twenty-seven (26.7%) patients had LN metastases. LN metastases was independently associated with disease-free survival (P = 0.009). Ideal cutoff values for predicting LN metastases were 1.80 for NLR, 168.25 for PLR and 2.5 cm for tumor size according to the receiver operating characteristic curve. On multivariable analysis, NLR (P = 0.017), symptomatic diagnosis (P = 0.028) and tumor size (P = 0.020) were associated with LN metastases. These results indicate that preoperative NLR ≥ 1.80, tumor size ≥2.5 cm and symptomatic diagnosis are independently associated with LN metastases for patients undergoing resection of NF-PNETs. It is anticipated that these findings are useful for further planning of lymphadenectomy before surgery. The annual incidence of PNETs in the United States is estimated to range between 2 and 5 cases per one million individuals but appears to be rising, due to the application of imaging and endoscopic ultrasound2. PNETs can be classified as either functional or nonfunctional, while nonfunctional PNETs (NF-PNETs) account for 60% to 90% of all PNETs. Unlike functional PNETs with the typical clinical manifestations of hormone overproduction, NF-PNETs often had grown to an advanced stage with a large mass, local invasion and distant metastasis, because of the nonspecific symptoms in the early days, such as abdominal pain and distension, nausea and vomiting, abdominal mass, and others3. Complete surgical resection of a NF-PNET has been suggested to be the only potentially curative treatment for the disease, similar to pancreatic adenocarcinoma. The 5-year survival rate is about 40 to 60% with a median survival of 38 to 104 months5.Pancreatic neuroendocrine tumors (PNETs) are a heterogeneous group of neoplasms, accounting for approximately 1–2% of all pancreatic neoplasms and 7.0% of all neuroendocrine tumors8. In addition, more and more evidence demonstrated that LN metastasis was an independent prognostic factor for PNETs10. Therefore, the investigation of factors related to LN metastases has a great clinical significance. However, preoperative factors predictive of LN metastases are not well defined in NF-PNETs. In recent years, markers of systemic inflammation, such as the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR), have been identified as prognostic factors. An elevated NLR and PLR have been shown to be correlated with advanced stages and poor prognosis in a variety of human tumors13. Tao L et al. reported that preoperative NLR, CA125 and CA19-9 are useful biomarkers for the prediction of LN metastasis in pancreatic ductal adenocarcinoma14. Whether preoperative NLR and PLR can predict the LN metastases of NF-PNETs remains unknown. Thus, we performed a retrospective analysis of predictor value of NLR and PLR and possible clinical parameters on the LN metastases of NF-PNETs before operation.The current American Joint Committee on Cancer Staging (AJCC) and European Neuroendocrine Tumor Society (ENETS) considers tumor size, lymph node (LN) metastasis, and presence of distant metastasis in its staging criteriaA total of 101 patients with primary NF-PNET who underwent curative resection and lymphadenectomy were enrolled, including 53 males and 48 females. In this cohort, described in Table Twenty-seven (26.7%) patients were discovered with LN metastases in the pathology. It demonstrated that both PLR and NLR were significantly higher in those patients with LN metastases, while lymphocyte-to-monocyte ratio (LMR) was significantly lower in those patients with LN metastases  Fig. . WhereasROC curve analysis showed that the AUCs of the NLR, PLR, LMR, and tumor size were 0.734, 0.565, 0.656, 0.647, respectively, and that the best cut-off values for the above parameters were 1.80, 168.25, 3.92, and 2.5, respectively, as these values were both the most sensitive and the most specific with respect to predicting LN metastases , symptomatic diagnosis  and tumor size   months. Thirty-nine patients (38.6%) had tumor recurrence during the follow-up. The results of the univariate disease-free survival (DFS) analysis for each of the clinicopathologic variables were shown in Table 10. Interestingly, our study also showed that preoperative NLR ≥ 1.80, tumor size ≥ 2.5 cm and symptomatic diagnosis were independently associated with LN metastases for patients undergoing resection of NF-PNETs.In the current study, we showed that 26.7% (27/101) NF-PNETs patients had LN metastases when diagnosed. Furthermore, LN metastases was associated with decreased DFS, which was similar to previous studies16, lung cancer17, colorectal cancer18, hepatocellular carcinoma12 and breast cancer13. Interesting, Lee et al. investigated the clinical impact of NLR as a prognostic factor in malignant pleural effusion and a new scoring system that use NLRs in the serum and malignant pleural effusion (smNLR score) in lung cancer patients. As a result, ECOG-PS (p < 0.001), histology (p = 0.001), and smNLR score (p < 0.012) were independent predictors of overall survival17. Regarding NETs, the report by Salman T et al. revealed that an elevated NLR and PLR were associated with a high tumor grade and an advanced tumor stage for NET. The study also verified that NLR and PLR were simple laboratory parameters that could be used to identify NETs with worse outcomes19. Recently, a retrospective analysis of 165 PNETs from Luo G et al. revealed that NLR was an independent predictor of overall survival for patients with PNETs. However, NLR > 2.4 was not significantly associated with positive lymph status (P = 0.067)15. In a retrospective study of 95 patients  undergoing resection for PNET, Tong Z et al. demonstrated that 15.8% (15/95) patients were histologically confirmed to have LN metastasis. The preoperative NLR was a potential independent predictor for LN metastasis and recurrence-free survival. The nomogram, including NLR, T stage, and grade, achieved a more optional performance in predicting LN metastasis, especially during the initial diagnosis for resectable PNET16. Both of the articles included functional PNETs or NF-PNETs, radical resection or non-radical resection. Since various studies have reported that function status and resection status are the independent predictors in patients with PNET, our study included 101 patients with NF-PNET who underwent curative resection to exclude these factors. Compared to functional PNET, NF-PNETs show a worse outcome in part due to the delay of diagnosis and higher malignant potential. Moreover, previous reports have demonstrated that only 30–40% of patients with NF-PNETs present with LN metastases at diagnosis, suggesting that most patients could be spared a lypmphadenectomy. In the present study, 26.7% (27/101) NF-PNETs patients had LN metastases when diagnosed. Addtionally, NLR ≥ 1.80 was associated with presence of LN positivity in 38.7% of patients, yet for NLR < 1.80, the rate of LN positivity was 7.7%. Further study revealed that NLR was an independent prognostic factor associated with LN metastases in patients with NF-PNET, which was similar with the results by Tong Z16. However, Arima et al. reported that NLR ≥ 2.4 predicted postoperative liver, but not lymph node, metastasis in PNET20. Heterogeneity across Arima and our studies may be attributed to several factors, such as differences in NLR cutoff values, differences in inclusion and exclusion criteria and differences in period of LN metastasis (LN metastasis during the surgery in our study and postoperative LN metastasis during the follow-up in Arima’s study). The best cut-off value for the NLR was based on the prediction of LN metastases in our study, while that in the research of Arima was based on the prediction of recurrence.NLR, which can comprehensively reflect inflammatory and immune status in patients with cancer, has been a reliable marker for predicting the survival of patients with different types of tumor, such as PNET23. In addition, neutrophils, major part of WBCs, have a crucial role in tumor metastasis25. The study of Zhang J indicates that the abundance of circulating tumor-associated neutrophils in advanced cancer patients contributes to the tumor metastasis by inhibiting the activation of the peripheral leukocytes26. Other studies have indicated that tumor-associated neutrophils promote tumor proliferation, facilitate metastasis by releasing pro-angiogenic mediators (VEGF) and lead to more aggressive tumors27. It has also been pointed out that through interaction with neutrophils, tumor cells could be brought to the endothelium, which is an essential step in LN metastases. Wculek and Malanchi identify neutrophils as the main component and driver of metastatic establishment within the (pre-)metastatic lung microenvironment in mouse breast cancer models28. Furthermore, neutrophils can promote the adhesion of tumor cells to the lymphatic endothelium, which would bind to an endothelial cell if the endothelial is also sufficiently activated30. Therefore, neutrophils might be an important driver in LN metastasis.Increasing amounts of evidence have confirmed that tumor development is associated with inflammation and immunity. Inflammatory cells including leukocytes and lymphocytes play an important role in controlling proliferation, survival, and migration of tumor cells through apoptosis and angiogenesis pathways34. Tsutsumi K et al. reported increased prevalence of LN metastases in gastrinoma patients and non gastrinoma patients with tumor size ≥1.5 cm 32. They also found that 2 (8%) patients with gastrinoma out of 26 patients with tumor <1.5 cm had lymph nodal metastases32. Postlewait LM et al. also reported that tumor size ≥2 cm , male gender  and head/uncinate location  were associated with nodal-positivity. In addition, ROC analysis revealed that tumor size ≥ 2 cm was associated with nodal-involvement 35. In contrast, Joyce Wong et al. reported that tumor size did not predict LN metastases. Furthermore, LN metastases did not impact OS or DFS, while tumor differentiation appears to be more important in determining prognosis36. In the current study, tumor size of ≥2.5 cm was associated with presence of LN positivity in 32.5% of patients, yet for tumors <2.5 cm, all the tumors had negative LN. In addition, we also found that patients who were symptomatic at diagnosis were more likely to have LN metastases, compared to incidentally diagnosed NF-PNETs (P = 0.003). However, there were still 3 cases out of 35 incidentally diagnosed patients (8.6%) had LN metastases. Our data suggested that tumor size was more useful to predict LN metastases, while NLR and symptomatic diagnosis could not reliably predict LN metastases.Tumor size and symptomatic diagnosis, reliably available to a surgeon preoperatively, are also identified as predictors of LN metastases. A large number of researches have explored and reported on the LN positivity rates or progression rates at distinct size intervals: <1 cm 15%, <1.5 cm 13%, <2 cm 8–12%, <2.5 cm 8%, <3 cm 37%Our study had several limitations that must be considered. First, given its retrospective design, the current study was subject to possible selection bias, as well as diagnostic bias. Second, the NLR and PLR, a marker of systemic inflammation, may be affected by many conditions, including chemotherapy toxicity, chronic inflammatory diseases, granulocyte colony-stimulating factor administration, pathogenic inflammation and other diseases. Therefore, these conditions must be accounted for in clinical practice. Finally, the present study was conducted at a single institution. The performance of multicentre studies of the markers used herein would strengthen our conclusions.In conclusion, this study highlights that NLR ≥ 1.80, tumor size ≥ 2.5 cm and symptomatic diagnosis are independently associated with LN metastases for patients undergoing resection of NF-PNETs. It is anticipated that these findings are useful for further planning of lymphadenectomy before surgery.Patients who underwent surgical resection and lymphadenectomy for NF-PNETs from November 2003 to August 2016 at the First Affiliated Hospital, Zhejiang University School of Medicine, were retrospectively reviewed. The diagnosis of NF-PNET was made based on standard histologic criteria. The TNM stage of each PNET was determined based on the American Joint Committee on Cancer TNM Classification, while the grade of each PNET was determined according to the 2010 WHO classification of NETs of the GEP system. Patients who showed clinical evidence of infection or evidence of hyperpyrexia at the time of diagnosis  were excluded from the study (n = 8), as were patients who received preoperative radiochemotherapy (n = 2) and who had a history of cancer of any type (n = 3). We included only those patients who had survived for at least 60 days after surgery in the study to exclude perioperative mortality-related bias. Finally, 101 patients undergoing curative resection and lymphadenectomy were enrolled.The radiological examination before operation included ultrasonography, abdominal computed tomography and magnetic resonance imaging. Since 2012, the endoscopic ultrasonography or endoscopic ultrasonography guided fine needle aspiration biopsy has been performed in some patients, whose diagnosis was indistinct. Radical resection was considered the first-choice treatment for patients with PNET. For the nonmetastatic PNET patients undergoing radical resection, no postoperative somatostatin analogue therapy, targeted therapy or systematic chemotherapy was carried out. In patients presenting with metastatic PNET, multiple treatment modalities were used after operation, including somatostatin analogue therapy and systematic chemotherapy. Laboratory tests including blood routine, tumor markers and liver function were routinely performed within 7 days before the surgical resection. The NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. The PLR was calculated by dividing the absolute platelet count by the absolute lymphocyte count, while LMR was calculated by dividing the absolute lymphocyte count by the absolute monocyte count on preoperative routine blood tests. Meanwhile, we defined normal values of CA199, CEA, AFP, and CA125 as 0–37 U/ml, 0–5 ng/ml, 0–20 ng/ml, and 0–35 U/ml, respectively. ECOG-PS is an ordinal scale with scores from 0 to 5: 0, normal activity; 1, symptomatic but ambulatory; 2, symptomatic-confined to bed/chair < 50% of waking hours; 3, symptomatic-confined to bed/chair > 50% of waking hours; 4, 100% bedridden; and 5, dead. The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants before the commencement of the study. All methods and research activities were performed in accordance with the guidelines and regulations.Patient follow-up was performed by reviewing hospital records or contacting patient family members. Overall survival (OS) was defined as the time span extending from the date of initial diagnosis until the date of death from any cause or the date of last known contact. Disease-free survival (DFS) was calculated from the day of surgery until the time of recurrence. Our department follows up with patients every 6 months for the first 5 years after surgery and then yearly thereafter. The following postoperative follow-up data were collected for each patient: clinical symptoms and signs, laboratory test results and radiological examination results. Once recurrence was confirmed, patients were treated by repeat tumor resection, radiofrequency ablation (RFA), transarterial chemoembolization (TACE), systematic chemotherapy and somatostatin analogue therapy, according to the sizes, numbers and locations of their recurrent tumors. And no patients received the targeted therapy including everolimus or sunitinib.All statistical analyses were performed using SPSS 16.0 software  for Windows. Differences in the NLR and PLR and other clinicopathologic features between positive LN and negative LN were evaluated by t tests in the case of normally distributed variables or by the Mann-Whitney U test in the case of abnormally distributed variables. Area under the curve (AUC) values obtained from receiver operating characteristic (ROC) curve analysis were used to compare the predictive efficacies of NLR and PLR. The associations between the clinical and histopathological parameters with LN metastases were evaluated by both univariate analysis and multivariate logistic regression analysis. The Kaplan-Meier method and the log-rank test were used to calculate DFS. Prognostic analysis was performed using univariate and multivariate Cox regressions models. A P value < 0.05 was considered statistically significant. All data generated or analysed during this study are included in this published article.