Elevated serum triglyceride concentration  or in other words hypertriglyceridemia (HTG) is common in the populations of developed countries. This condition is accompanied by an increased risk for various diseases, such as acute pancreatitis (AP). It has been proposed that HTG could also worsen the course of AP. Therefore, in this meta-analysis, we aimed to compare the effects of various seTGs on the severity, mortality, local and systemic complications of AP, and on intensive care unit admission. 16 eligible studies, including 11,965 patients were retrieved from PubMed and Embase. The results showed that HTG significantly elevated the odds ratio (OR = 1.72) for severe AP when compared to patients with normal seTG (<1.7 mM). Furthermore, a significantly higher occurrence of pancreatic necrosis, persistent organ failure and renal failure was observed in groups with HTG. The rates of complications and mortality for AP were significantly increased in patients with seTG >5.6 mM or >11.3 mM versus <5.6 mM or <11.3 mM, respectively. We conclude that the presence of HTG worsens the course and outcome of AP, but we found no significant difference in AP severity based on the extent of HTG. In the disease of hypertriglyceridemia (HTG), the fasting seTG exceeds 1.7 mM. The aetiology of HTG may be primary (caused by genetic mutations), but it is most commonly secondary2. The causes of secondary HTG include obesity, unhealthy diet or lifestyle , pregnancy, hypothyroidism, hepatic steatosis, nephrotic syndrome, type-2 diabetes mellitus, and some drugs 3. The extent of HTG has been classified by the Endocrine Society into the following groups based on fasting seTG: mild (1.7 to 2.3 mM), moderate (2.3 to 11.2 mM), severe (11.3 to 22.4 mM) and very severe HTG (>22.4 mM)4. However, the classification of HTG based on fasting seTG was somewhat different according to the National Cholesterol Education Program Adult Treatment Panel III guideline5. They specified borderline-high seTG from 1.7 to 2.2 mM, high seTG between 2.2 and 5.6 mM, and very high seTG above 5.6 mM. HTG is associated with different comorbidities. In the general population, people with moderate or severe non-fasting seTG compared with individuals with non-fasting seTG in the normal range have a significantly higher risk for ischemic heart disease, ischemic stroke and mortality1. HTG is also an important risk factor for the development of acute pancreatitis (AP)6.Triglyceride is a component of lipoproteins, and its reference range in the blood serum is below 1.7 mM (150 mg/dL). Nowadays, a high-fat diet and resultant obesity are common; therefore, the prevalence of elevated serum triglyceride concentration (seTG) is high, affecting approximately 27% of all adults6. The severity of the disease can be classified into three groups based on the modified Atlanta criteria: mild, moderately severe and severe7. Moderately severe and severe forms of AP are associated with various local and systemic complications which also influence the rate of mortality. Necrosis, infections, abscesses and/or pseudocysts can occur in the pancreas. AP may also cause systemic complications, such as sepsis, systemic inflammatory response syndrome, and transient or persistent organ failure9. Organ failure most commonly affects the lungs, kidneys and cardiovascular system. About 60–80% of AP cases are due to massive alcohol consumption and gallstone disease, whereas 1–9% of the cases are HTG-induced10. In pregnancy, up to 56% of AP cases are HTG-related2. It is widely accepted that severe HTG markedly increases the risk for AP11. However, some authors define HTG-induced AP (HTG-AP) when seTG is >5.6 mM10. There is no significant evidence for HTG-AP at <5.6 mM seTG12. Beyond the increased risk for AP in severe HTG, previous publications have indicated that there is a relationship between seTG and the severity of AP14, even in the case of mild or moderate HTG18. Nawaz et al.15 reported that HTG independently and proportionally correlates with persistent organ failure regardless of AP aetiology. Zeng et al.16 showed that seTG >2.26 mM increases the risk for systemic and local complications in acute biliary pancreatitis. However, some studies have shown no relationship between seTG and the severity of AP20. To clarify this discrepancy, earlier efforts have aimed to analyse the effect of seTG on the severity of AP through meta-analyses14. Only one meta-analysis has been published in which the authors investigated the effect of seTG on the outcomes of AP13. However, the groups of patients classified by seTG overlapped, which is a significant limitation of that study.AP is a sudden inflammation of the pancreas that lasts for a short period (from days to some weeks)To the best of our knowledge, there is no meta-analysis where the effect of several different and well-defined seTGs have been investigated on the course of AP. Therefore, our aim was to evaluate and compare the effects of normal seTG with the effect of mild, moderate and severe HTG on the severity, mortality and other complications of AP. In addition, various ranges within the HTG were compared.https://www.crd.york.ac.uk/PROSPERO/, ID: CRD42017071264). The methodology for this analysis followed recommendations by Stroup et al.21 and the guidelines for the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols  model22. The problem was AP. The intervention was HTG with various groups formed for the analysis: >1.7, 1.7–5.6, 1.7–11.3, >5.6 and >11.3 mM seTG. The comparison interventions were normal (<1.7), <5.6, 1.7–5.6, 1.7–11.3 and <11.3 mM seTG. Different outcomes were investigated: AP severity, mortality, pancreatic necrosis, persistent organ failure (POF) and multi-organ failure (MOF), pulmonary and renal failure, and admission to an intensive care unit (ICU).This systematic review and meta-analysis was conducted according to the protocol previously registered in the PROSPERO database  and PubMed Library (published from 1961 to July 2017). Furthermore, ClinicalTrials.gov was also screened for additional unpublished data. The search contained the following terms for Embase: pancreatitis AND (‘triglyceride’/exp OR triglyceride OR hypertriglyceridemia OR ‘hyperlipidemia’/exp OR ‘hyperlipidemia’) AND [english]/lim AND (‘human’/de OR patient OR patients) NOT (‘conference abstract’/it OR ‘review’/it OR ‘case report’/de OR ‘nonhuman’/de OR ‘practice guideline’/de). The following terms were used for PubMed: pancreatitis AND (hyperlipidaemia OR hyperlipidemia OR triglycerides OR triglyceride OR hypertriglyceridaemia OR hypertriglyceridemia) AND (human OR patient OR patients) AND English NOT “case reports”[Publication Type]. The search terms for Clinicaltrials.gov were pancreatitis and hypertriglyceridemia.Case control or cohort studies.Studies involving AP patients.HTG (>1.7 mM) was present in at least one of the groups under investigationSeTGs were defined.Outcome data were provided for at least one of the following: severity of AP according to the revised Atlanta Classification, mortality, pancreatic necrosis, POF, MOF, pulmonary failure, renal failure and intensive care unit (ICU) admissionWritten in English.Articles were included if they fulfilled the following criteria:The seTG in different groups used as controls was below 1.7, 5.6 or 11.3 mM as well as within the 1.7–5.6 and 1.7–11.3 mM ranges. Systematic reviews, meta-analyses, reviews, conference abstracts, letters, replies, reports, commentaries, notes, case studies, animal studies, practice guidelines and non-English-language  articles were excluded from the analysis.24. Since seTG decreases rapidly when food intake is restricted25, the NOS was supplemented with another scoring system in which the articles were also evaluated based on the timing of the seTG measurement . The investigators read and selected the articles to be included in the statistical analysis and extracted the data. The Newcastle–Ottawa Scale (NOS) was used . The numbers of patients with regard to AP severity, mortality, pancreatic necrosis, POF, MOF, pulmonary failure, renal failure and ICU admission were used to calculate odds ratios (OR) to compare these outcomes in different seTG groups. ORs were pooled using the random effects model with the DerSimonien–Laird estimation and displayed on forest plots. Summary OR estimation, p value and 95% Confidence Interval (CI) were calculated. ORs with corresponding CIs and p values are indicated in the text according to the following order: OR . P < 0.05 was considered a significant difference from summary OR = 1. The different ORs were compared using the analysis of variance with random effect weights.2 statistic and the chi-square test to acquire probability values; p < 0.05 was defined to indicate significant heterogeneity. The small-study effect was visually investigated on funnel plots.Statistical heterogeneity was analysed using the IThe search for articles in three databases resulted in 2261 records  and multicentre (3) cohort studies were included. Population sizes ranged from 43 to 3203, and six trials involved over 300 patients. The aetiology of AP was noted in all the studies, eight comprised HTG-AP (>11.3 mM seTG), and twelve contained alcoholic and biliary pancreatitis patients. Other aetiologies, such as post-ERCP, idiopathic, mixed and drug-induced AP, were also included in some articles. The studies were performed in the following countries: China (nine cohorts), the USA (three cohorts), Hungary (two cohorts), the UK (one cohort) and Spain (one cohort). Table 3 and mulDifferent groups were created based on the extent of HTG, and the outcomes for AP were compared with those in the normal (<1.7 mM) seTG group. Figure The small-study effect was checked by placing the data acquired from articles on funnel plots  resulted in a significant elevation of ORs  for severity when all the groups were compared with the effect of normal seTG on AP severity. These data suggest that HTG aggravates the course of AP. Our findings are in line with earlier animal studies, in which hyperlipidaemia increased the severity of AP32. These NEFA are toxic and cause MOF to worsen the outcome of AP36. The administration of NEFA induced sustained elevation of [Ca2+] in pancreatic acinar cells and inhibited mitochondrial function and ATP production38. Consequently, NEFAs cause damage to acinar and vascular endothelial cells, thus leading to inflammation39. Our earlier studies have also indicated that fatty acids inhibit CFTR activity and decrease the HCO3− and fluid secretion of pancreatic ductal cells41. In addition, in the case of HTG, the concentration of chylomicrons is elevated. This increases blood viscosity, which impairs blood flow and results in pancreatic ischemia and acidosis32. Since AP is not induced in all patients with HTG10, we assume that other factors also contribute to the development of AP.The underlying mechanism by which HTG exacerbates the severity of AP is unknown. One of the possible processes is that pancreatic lipases metabolize seTG to non-esterified fatty acids (NEFA)Interestingly, the mortality of patients did not show statistically significant differences between groups  group and the non-TG-related AP (NTGAP) group. The patients categorized as TGAP had seTG > 11.3 mM or >5.6mM with a previous history of HTG. Otherwise, patients were categorized into the NTGAP group. However, we are unsure how this categorization relates to their statistical analysis because in some of the articles involved the seTGs were classified as <2.26 or <1.7 mM and used for the NTGAP group, and the TGAP group contained patients with seTG > 2.26 or >1.7 mM. For example, Nawaz et al.15 had 115 patients with normal (<1.7 mM) seTG who were included in the NTGAP group, while the 86 patients with HTG (seTG > 1.7 mM) were listed in the TGAP group. Another example of this discrepancy could be found in Fig. 13, where Zeng et al. (2014) involved groups with seTG < 1.7 (NTGAP) and ≥5.65 mM (TGAP), Yin et al. (2015) consisted of groups with seTG < 5.6 (NTGAP) and >5.6 mM (TGAP), Tariq et al. (2016) contained groups with seTG < 2.27 (NTGAP) and ≥2.27 mM (TGAP), and Jiang et al. (2005) comprised groups with seTG < 1.7 (NTGAP) and >1.7 mM (TGAP). However, all these different elevations of seTG were used within one analysis. Furthermore, some errors could also be identified for the patient numbers used for the analysis  should be 4/828 instead of 4/1459). Thus, Wang et al.13 compared two groups of patients, one with seTG > 1.7 mM and one with seTG < 5.65 mM, where the two groups overlap between 1.7 and 5.65 mM seTG. The authors proved that the AP group with higher seTG had an increased risk for systemic complications and an elevated mortality rate compared to patients with lower seTG. Our results partly confirm the findings from Wang et al.13.Most of the earlier attempts to investigate the effect of HTG on the severity of AP via meta-analysis were unsuccessful due to the small number of available observational studies47 were published in which the effect of HTG on the severity of AP was investigated, and this allowed us to prepare a meta-analysis by combining those cohorts with those in earlier papers. However, our study has several limitations: (1) Although the literature is more extensive nowadays, most of our analyses contained a small number of articles . (2) The different populations (China or the USA) and the various baseline data  could represent a bias. (3) Aetiologies  for AP differ. (4) Only English-language articles were included in this study, which can affect the results. (5) There were no statistical significances for all the investigations even if the ORs were high . A further increase in the number of articles and patients could clarify any discrepancies related to ORs or significance. (6) Meng et al.48 observed significant differences (more than ±10%) between the seTG values obtained using various commercially available kits. (6) Significant heterogeneities were detected for some analyses. (7) Notably, seTG changes dynamically, which relates to food intake and fasting. Current treatment protocols for most AP patients include fasting at the beginning of hospitalization, except for suspected severe AP cases where early enteral feeding (within 48 hours) is recommended50. Fasting results in a rapid (within 48 hours) drop of seTG10 and measuring seTG 48 or 72 hours after the admission might underrepresent levels at the onset of AP. Dominguez-Munoz et al.25 demonstrated a dramatic decrease in seTG during fasting: seTG falls from approx. 30 mM to 5 mM within three days. Other authors also confirmed this phenomenon51. To take this bias into account, we scored the articles based on the timing of the seTG measurement. Having high scores for NOS and seTG measurement timing represents good quality for the selection of articles for this meta-analysis.In 2016 and 2017, eight well-written articles52. If patients have HTG on admission for AP, then lipid-lowering therapy (such as plasmapheresis) is recommended according to guidelines. The use of these therapies would also improve further studies. Due to large differences between the kits used for seTG determination, standardizing the method for seTG measurement is also recommended. Based on basic discoveries, cohort analyses, clinical studies and meta-analyses, early intervention (e.g. heparin and/or insulin therapies and plasmapheresis) to normalise HTG may be beneficial for patients, and this should be investigated in randomised controlled trials.To improve the design of further retrospective or prospective cohorts related to HTG-AP, we would suggest some recommendations. It is advisable to keep the time interval for the seTG measurement consistent. Preferably, it should be performed within 48 hours after the onset of the first symptoms and repeated regularly. Publishing the medical history of patients with respect to seTG-lowering therapies would also be advantageous, e.g. describing the regular use of statins or fibrates, which could decrease the incidence of AP53.In conclusion, HTG worsens the severity of AP and increases the risk for local and systemic complications Fig. . HoweverSupplementary Information


The efficacy of thulium laser prostate enucleation (ThuLEP) for large prostates is unclear. This study aimed to explore the expanded utility of 150–200-W ThuLEP by studying patients with a large prostate (> 80 mL).t-test, the z-test, and logistic regression analysis.We retrospectively reviewed records of 125 patients with large prostate glands (> 80 mL) who underwent ThuLEP performed by a single surgeon from June 2012 to June 2016. The ThuLEP data from our previous pilot study were used as the control. Operative variables, patient profiles, preoperative and postoperative urine flow rates, prostate volume, and the international prostate symptom score (IPSS) were recorded and analyzed using Student’s P = 0.68, 0.18, 0.98, respectively). Preoperative and postoperative IPSSs were 27.09 and 7.35, respectively. The postoperative prostate-specific antigen was reduced by 85.59% in comparison to the preoperative level. The estimated prostate size was reduced by 74.17% postoperatively. The modified Clavien-Dindo classification system was used to identify the overall rate of complications, which was approximately 22.4%. The mortality rate was 0.8%.Of 366 patients who underwent ThuLEP, 125 (34.15%) were enrolled. The ages and estimated prostate volumes were compared with those of the control. Overall, 39.2% underwent Foley placement and 4% received an anticoagulant agent preoperatively. Maximum urinary flow rates before and 3 and 12 months postoperatively were 9.93, 23.20, and 19.00 mL/s, respectively, which were generally equal to those of the control groups (High-power ThuLEP is safe and effective for patients with large prostate glands (> 80 mL). Prostate enucleation using a high-power thulium laser is feasible for patients who exhibit contraindications for surgery. Open prostatectomy (OP) offers the highest probability of symptomatic improvement and the lowest failure rate of all treatment modalities for bladder outlet obstruction (BOO) due to benign prostatic hyperplasia (BPH) . DespiteHolmium laser enucleation of the prostate (HoLEP) has been adopted by many urologists worldwide because of the increased commercial availability (beginning in 1994) of high-powered holmium:yttrium-aluminum-garnet lasers. Since then, numerous single-center and multicenter large-scale series and randomized clinical trials have demonstrated its effectiveness and safety when used to treat BOO , 7. HoLEWith the evolution of technology, thulium laser prostate enucleation (ThuLEP) has become a novel treatment option for BOO . In 2005The power of an advanced thulium device is 150–200 W. In our pilot study published in 2015, a high-power thulium laser was used to perform ThuLEP in patients from the general or unselected public. One year of follow-up showed that ThuLEP and traditional transurethral resection of the prostate (TURP) effectively alleviated subjective and objective voiding symptoms with a low rate of complications , 17. TheLimited number of randomized controlled trials with long-term follow-up support the efficacy of ThuLEP, there is a need for ongoing investigation of the technique . FurtherTo confirm ThuLEP as an endourological alternative to OP and HoLEP for numerous specific conditions, the application of ThuLEP for treating large prostates should be evaluated. Therefore, in this study, we assessed the outcomes  of ThuLEP for patients with a large prostate (> 80 mL).We retrospectively reviewed data from 125 patients who underwent ThuLEP performed by a single surgeon (JYH) between June 2012 and June 2016. Approval was received from the institutional review board (VGHIRB 2016–03-007 AC), along with a waiver for informed consent.All patients, regardless of the presence of malignancy, had a prostate gland that was larger than 80 mL in volume, as measured using transabdominal ultrasonography and the prolate ellipsoid volume formula. No exclusion criteria were applied during chart review. The Cyber 150 device  was used with 150–200 W of energy in continuous mode . For all statistical comparisons, significance was assumed when P <  0.05.The database from published articles were introduced as the the OP, HoLEP, and ThuLEP control group , 20, 21.P = 0.02) and had a higher estimated prostate volume  [Of 366 patients  who underwent ThuLEP during a 4-year period, 125  were enrolled. Fifteen patients exhibited malignant pathological findings    reflects this trend. Certain patients who were unhappy with the outcomes often visited the clinic more frequently and for a longer period. This may have affected our measurements of surgical outcomes  is safe and effective for patients with large prostates (> 80 mL).Therefore, promising outcomes associated with ThuLEP suggested that enucleation using a high-power laser is feasible for patients with large prostate > 80 mL.


Lassa virus, the causative agent of Lassa fever, is a zoonotic pathogen causing annual outbreaks in West African countries. Human patients can develop lethal hemorrhagic fever in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few available countermeasures, such as antiviral drugs or vaccines. Moreover, the fact that animal models are not readily accessible and the fact that mostly laboratory viruses, which have been passaged many times after isolation, are used for studies further limits the successful development of countermeasures. In this study, we demonstrate that a human isolate of Lassa virus causes lethal infection uniformly in Hartley guinea pigs. This novel animal model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics. Arenaviridae, is the causative agent of Lassa fever. Lassa virus is endemic in West African countries, such as Nigeria, Guinea, Liberia, and Sierra Leone, and causes outbreaks annually. Lassa fever onset begins with “flu-like” symptoms and may develop into lethal hemorrhagic disease in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few licensed vaccines or therapeutics against Lassa fever. The fact that animal models are limited and the fact that mostly laboratory-derived viruses are used for studies limit the successful development of countermeasures. In this study, we demonstrated that the LASV isolate LF2384-NS-DIA-1 (LF2384), which was directly isolated from a serum sample from a fatal human Lassa fever case in the 2012 Sierra Leone outbreak, causes uniformly lethal infection in outbred Hartley guinea pigs without virus-host adaptation. This is the first report of a clinically isolated strain of LASV causing lethal infection in outbred guinea pigs. This novel guinea pig model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics.Lassa virus (LASV), a member of the family IMPORTANCE Lassa virus, the causative agent of Lassa fever, is a zoonotic pathogen causing annual outbreaks in West African countries. Human patients can develop lethal hemorrhagic fever in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few available countermeasures, such as antiviral drugs or vaccines. Moreover, the fact that animal models are not readily accessible and the fact that mostly laboratory viruses, which have been passaged many times after isolation, are used for studies further limits the successful development of countermeasures. In this study, we demonstrate that a human isolate of Lassa virus causes lethal infection uniformly in Hartley guinea pigs. This novel animal model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics. Arenaviridae, is the causative agent of Lassa fever (LF). LASV is a zoonotic pathogen maintained in the rodent Mastomys natalensis as its natural reservoir (Lassa virus (LASV), a member of the family eservoir . Humans eservoir . Additioeservoir . LASV iseservoir . An estieservoir . LASV mueservoir , 7. Howeeservoir , 8. Guineservoir , 10. Outeservoir . Animal Here we show that the LASV isolate LF2384-NS-DIA-1 (LF2384), which was directly isolated from a serum sample of a fatal human LF case in the 2012 Sierra Leone outbreak and which has been reported to cause lethal infection in STAT1 knockout mice  and two of five guinea pigs inoculated with 10 PFU of LASV LF2384 succumbed to disease at 13 to 22 days postinoculation (dpi). The mean survival times with standard errors for the groups inoculated with 104, 103, and 102 PFU are 17.6 ± 1.0, 16.4 ± 1.5, and 17.0 ± 0.3 dpi, respectively (50) is <14.7 PFU. Animals inoculated with the virus started losing weight and developed a fever, a body temperature greater than 40°C, at 10 dpi  , lymphocytes (LYM), monocytes, neutrophils, red blood cells, platelets (PLT), hemoglobin level, and hematocrit were determined. WBC, LYM, and PLT from guinea pigs that succumbed to disease were abnormally low and significantly lower than those of surviving guinea pigs ( < 0.01)  to D. Th10.1128/mSphere.00428-19.1TABLE S1n = 13) or at the end of the study (n = 3), blood samples were collected for hematology. Uninfected guinea pig blood was used for negative control (n = 8). Whole-blood samples collected in EDTA tubes were used for standard hematologic analysis using a VETSCAN HM5 (ABAXIS) according to the manufacturer’s instructions. Means and standard deviations are shown. Download Table S1, DOCX file, 0.02 MB.Hematology data. At the time of euthanasia , 1% penicillin-streptomycin, and 4 to 10 PFU) in 100 μl of phosphate-buffered saline (PBS) and monitored daily for 28 days after inoculation. Animals were humanely euthanized once they showed neurological symptoms such as paralysis or were unable to access their food or water and/or lost more than 20% of their body weight based on their baseline measurement at the time of inoculation. Blood and organs  were collected at the end of the study.Five- to 7-week-old Hartley guinea pigs were purchased from Charles River. All animals were housed in animal biosafety level 2 (ABSL2) and ABSL4 facilities in the GNL at UTMB. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at UTMB and were conducted according to the National Institutes of Health guidelines. Body temperature measurements were performed using subcutaneously implanted BMDS IPTT-300 transponders and a DAS-6007 transponder reader (Bio Medic Data Systems). Body weights were measured by using a Sartorius AY1501 digital scale. Guinea pigs were intraperitoneally inoculated with diluted LASV strain LF2384  according to the manufacturer’s instructions.2. After the inoculum was removed and the wells were washed, each well was overlaid with minimum essential medium containing 2% FBS, 1% penicillin-streptomycin, and 0.6% tragacanth (Sigma). Following a 5-day incubation, cells were fixed with 10% formalin and stained with crystal violet. Viral titers were represented as PFU.Virus titers were determined by plaque assay. Briefly, confluent Vero cells in 12-well plates were inoculated with virus diluted 10-fold and incubated for 1 h at 37°C with 5% COMN275172 and MN275173.Sequences have been deposited in GenBank under accession no.


Sodium borohydride (NaBH4) was used as the reducing agent. The properties of AgNPs in Cts, Cts/gelatin and gelatin bionanocomposites (BNCs) were studied in terms of their surface plasmon resonance, crystalline structure, average diameter size, particle distributions, surface topography and functional groups. All the samples were characterized by UV-visible spectroscopy, powder X-ray diffraction, transmission electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy. In this research, silver nanoparticles (AgNPs) were synthesized in chitosan (Cts), Cts/gelatin and gelatin suspensions using a chemical reducing agent. Cts and gelatin were used as natural stabilizers and solid support, whereas AgNO The field of nanotechnology has emerged as one of the most active areas of research in materials science over the last decades. The fact that nanoparticles (NPs) exhibit quite a number of interesting and unique properties based on their specific characteristics, such as size, distribution and morphology leads itself to various applications , for insetc. This accounts for the different properties of composites that are synthesized, which allow them to serve different purposes in our daily life.During the preparation of silver nanoparticles (AgNPs), stabilizers are one kind of agent that should be present and that play an important role in controlling the formation of nanoparticles, as well as their dispersion stability. Polymers are often used as particle stabilizers due to the fact that they are effective in preventing agglomeration and precipitation of the particles. This is important in synthesizing nanoparticles with homogenous distributions. Various methods have been introduced to synthesize Ag nanocomposites, including photochemical ,6,7, cheN-acetyl-D-glucosamine (acetylated unit). Cts has been investigated as a natural cationic biopolymer because of its known excellent biocompatibility, biodegradability, nontoxicity, bioactivity, and multifunctional groups from years of research. It is also extensively being studied for food packaging film, bone substitutes, artificial skin, biomedical applications and pH sensitive drug delivery among others due to a number of great properties it possesses [Chitosan (Cts) is a polysaccharide that occurs naturally. Its units are composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and ossesses .Gelatin is made up of a mixture of peptides and proteins produced by partial hydrolysis of naturally occurring collagen. The carboxyl groups on its chain backbones are one of its amazing features that enables the possibility to form hydrogen bonds with chitosan for a well-mixed hybrid . Besides4. Cts, Cts/gelatin and gelatin suspension were used with the original intention to prevent aggregation of AgNPs. In addition, these matrixes were found to be functioning as stabilizer also in later stage of experiment. The size of the silver nanoparticles synthesized is less than 15 nm.Here, we report synthesis of AgNPs by chemical reduction method using NaBH3 using NaBH4 as strong reducing agent. As a result, AgNO3 was successfully reduced by NaBH4 in the presence of Cts/gelatin or either one, resulting in the formation of AgNPs according to the following equations (1–3) [In this research, Cts, Cts/gelatin and gelatin suspension were used as the stabilizers for reducing AgNOns (1–3) :Ag+/Cts 3/Cts/gelatin suspensions (S0) were colorless. After the addition of the reducing agent to the suspensions, they turned dark brown (S1-S3) indicating the formation of AgNPs in the Cts, Cts/gelatin and gelatin suspensions. The formation of AgNPs was also followed by measuring the surface plasmon resonance bands of the AgNO3/Cts/gelatin (S0), Ag/Cts (S1), Ag/Cts/gelatin (S2) and Ag/gelatin (S3) suspensions at wavelengths within the range of 300–700 nm , 3085 cm−1 for alkenyl C–H stretch, 2956 cm−1 for CH2 asymmetrical stretching, 1631 cm−1 for C=O stretch/HB coupled with COO−, band at 1533 cm−1 for N–H bend coupled with CN stretch, 1444 cm−1 for CH2 bend, 1240 cm−1 for NH bend, and 1078 cm−1 for C–O stretch [−1 to 1634 and 1537 cm−1 in Cts/gelatin composite, corresponding to the bend vibration of amine chitosan. The pure gelatin was characterized by an amino band at 1533 cm−1 and a carbonyl peak at 1631 cm−1 shifted to 1634 and 1537 cm−1 as well. The shifting of both amino and carbonyl bands in the spectra of Cts/gelatin composite showed the formation of complex involving HB in between chitosan and gelatin [−1 to 1635 and 1544 cm−1, respectively, and also displayed a high intensity peak at 1544 cm−1. The vibration band of Ag/Cts at 1397 cm−1 could indicate the interaction between AgNPs with Cts. The spectrum of Ag/Cts/gelatin BNCs  was used as the silver precursor, and was obtained from Merck . Meanwhile, NaBH4, the reducing agent, was obtained from Sigma–Aldrich . Low molecular weight chitosan and glacial acetic acid (99%) were also obtained from Sigma–Aldrich. The gelatin was obtained from HiMedia . All the aqueous solutions were prepared with double-distilled water. All reagents in this work were of analytical grade and were used as received without further purification. AgNO3  was added immediately into the suspension under constant stirring for 2.0 hours for preparation of the AgNO3 in chitosan suspension. NaBH4  was added to the suspension of AgNO3/Cts and an immediate color change from pale yellow to brown indicating the formation of AgNPs was noted. This suspension was stirred for 1.0 hour more, then the obtained Ag/Cts BNCs were finally made into thin films for further characterization. The chitosan suspension was prepared by solubilizing chitosan (1.0 g) in acetic acid  solution. Then, AgNO3  was added directly into the suspensions. The AgNO3/Cts/gelatin suspension was stirred for 2.0 hours. The NaBH4  was added immediately to the suspension of AgNO3/Cts/gelatin. A color change from colorless to dark brown was observed. The suspension was stirred for another 1.0 hour, then the obtained Ag/Cts/gelatin BNCs were made into thin films for characterization. Chitosan (0.5 g) was dissolved in acetic acid  solution. On the other hand, gelatin (0.5 g) was dissolved in warm (40 °C) distilled water (25 mL). Next, both suspensions were mixed together and AgNO3  was mixed into the suspension and stirring continued for 2.0 hours. NaBH4  was introduced immediately to the suspension of AgNO3/gelatin. An immediate color change from colorless to light brown was observed. This suspension was stirred for 1.0 hour. The product was finally prepared in thin films for further characterization. For preparation of gelatin suspension, gelatin (1.0 g) was solubilized in warm (40 °C) distilled water (50 mL). After that, AgNO−1 using a Series 100 Perkin Elmer FT-IR 1650 spectrophotometer. The AFM image was obtained on an Ambios Q-scope  (SPM) machine.The prepared Ag BNCs were characterized by UV-visible spectroscopy (UV-visible), powder X-ray diffraction (PXRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR). The UV-visible spectra were detected over the range of 300–700 nm using a Shimadzu H.UV.1650 PC UV-visible spectrophotometer. Crystalline structures of the synthesized Ag were examined using Philips X’pert Pro Panalytical PW3040MPD X-ray diffraction. TEM image observations were carried out on Hitachi H–7100 electron microscope and the particle size distributions were determined using the UTHSCSA Image Tool program . FTIR spectra were recorded over the range of 300–4000 cmAgNPs were successfully introduced into Cts, Cts/gelatin, and gelatin natural polymers using a chemical reduction method. This was confirmed by the maximum surface plasmon resonance peak at 408–414 nm for each sample indicating the formation of Ag NPs. The crystalline structures of AgNPs were found to be face centered cubic type. The average diameters of the AgNPs were revealed to be 10.50, 3.37 and 4.38 nm for Ag/Cts, Ag/Cts/gelatin and Ag/gelatin, respectively. The best distribution of AgNPs was found to be in Cts/gelatin suspension and their average diameters were found to be the smallest from TEM results. Hence, we conclude that Cts/gelatin works better as stabilizer and size control compared to Cts and gelatin alone.


About 10% latent tuberculosis infections (LTBI) would progress to active tuberculosis (TB), if left prophylactic therapy. Tuberculin skin test (TST) is the most widely used method for LTBI screening in the school of China. However, for college students, the association between TST reaction size and active TB risk was unclear.We conducted a retrospective study to assess whether the TST reaction size would predict active TB during the next two years after TST screening for college students. Multivariable Cox regression was performed to identify the size of TST reaction and other factors associated with active TB risk.A total of 67292 college students in Beijing, China were included in this study; 8021 (11.92%) individuals were TST positive (≥10 mm), and 3879 (5.76%) of them were strong TST positive (≥15 mm). During the two years of follow-up, 26 active TB cases were reported in 134575 person-years with an incidence rate of 19.32 (95% CI: 12.61–28.32) per 100000 person-years. The adjusted hazard ratios (HR) (95% CI) were 1.094 (0.247~4.846), 3.644 (1.188~11.179), 6.832 (2.436~19.163) and 9.768 (2.203~43.315) of cohorts with the TST reaction size intervals 5~9, 10~14, 15~20 and ≥ 20 mm, respectively, compared to cohort with interval 0~4 mm. Besides, the adjusted HR (95% CI) was 3.593 (1.354~9.537) of males compared to females.This study indicated that the risk of active TB increased in college students when the TST reaction size was ≥10 mm, and males had a higher risk compared to females.The online version of this article (10.1186/s12879-019-4238-2) contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis (Mtb). In 2017, about 10 million people suffered from tuberculosis, and 12–14% of them died. Wherein China has the second largest number of TB patients  in the world [Worldwide, it is estimated that one-third of the world’s population is infected with Mtb infection screening in China, because of its handleability and inexpensive price.School is a people-intensive place, in which students have been in prolonged face to face contact with each other. It is one of the most common places that reported for the community-based outbreak of TB in China , 5. It iSeveral studies have been performed among primary school students to estimate the risk of TB disease according to TST reaction size , 7. HoweWe performed a retrospective cohort study of the college freshmen TB infection investigation of 20 colleges, located in Changping district, Beijing, China. The baseline examination took place in 2013–2016. Follow-up for all individuals started at the time they received TST, mainly in mid-September each year. We ended follow-up for each individual with 2 years follow-up. The data were excluded if any of the following conditions exist: active TB diagnosis in freshmen TB screening, having active TB history or missing TST results.Beijing Changping institute for tuberculosis prevention and treatment (BCITPT) is the public health institution mainly responsible for TB prevention and control in Changping district. Raw records of colleges’ freshmen TB screening of 2013–2016, which contained the age, sex, ethnicity, region, TST reaction size, and diagnosis result, were obtained from BCITPT. Data on reported TB cases of the colleges in Changping district were retrieved from national TB reporting system from 2013 to 2018. Besides, the incidence time of each TB case was collected for further analyses.Mtb culture of sputum and microscopy examination of sputum smears and so on. The individuals with TST reaction size ≥20 mm (886 ones in this study) were advised to receive preventive therapy. However, it was non-obligatory, a few students received preventive therapy, and because it needs three months of treatment, few ones successfully complete treatment. In this study, no one was excluded from the analysis due to receiving LTBI treatment. In the follow-up years, the student who was suspected to be TB case would be taken to BCITPT by the head teachers of classes of colleges in the first time, and all the suspected active TB cases in Changping district were confirmed and treated in BCITPT. They all accepted X-ray examination, microscopy examination of sputum smears and Mtb culture of sputum. The diagnosis and classification of TB were based on the national guidelines for diagnosis for pulmonary tuberculosis (WS 288—2008) and Classification of tuberculosis (WS 196—2001) [According to the medical records of BCITPT, TST was conducted with 2 units of tuberculin  on the left forearm using the intradermal technique for each college freshman. And results were measured and recorded after 72 h of TST. For students with the strong positive reaction of TST (induration size ≥15 mm or the presence of blister or necrosis), the subsequent medical diagnosis was performed using X-ray, osis WS 16—2001 [996—2001) .P value of less than 0.05 was used to identify the statistical significance for all tests. Separately, cumulative incidence curves for different categories of sex and TST reaction size levels were plotted by using the Kaplan-Meier method, and the log-rank test was used to compare the curves. Subsequently, to identify the potential factors related to TB incidence, regression analysis based on Cox’s proportional hazards model was applied to estimate the unadjusted and adjusted hazard ratio (HR) and 95% CI. The univariates with P values less than 0.10 were then candidates included in multivariate Cox’s regression analysis. Besides, HR analyses of subgroups of males and females were performed with the same methods. The absolute risk of active TB (1/100000) of different TST reaction sizes was calculated and plotted.All analyses were performed using R 3.5.2 in this study. Pearson’s Chi-square test or Fisher’ exact test, where appropriate, was used to compare the frequency of categorical variables between the cohorts. The incidence rate (IR) and 95% confidence interval (CI) for each cohort were calculated. A 2-tailed As shown in Table There were 8021 (11.92%) TST positive individuals (induration size ≥10 mm or the presence of blister or necrosis), and about half of them  were strong TST positive (induration size ≥15 mm or presence of blister or necrosis)  per 100000 person-years. And for two-year follow up, 26 active TB cases were recorded in 134575 person-years with an incidence rate of 19.32 (95% CI: 12.61–28.32) per 100000 person-years. However, no significant difference in incidence rate was observed between the one-year and two-year follow up. Compared with the incidence rate of female , male individuals  had a higher incidence rate (P = 0.01). The comparison among subgroups who came from different regions of China indicated that those from western China had the highest incidence rate, which might be due to the higher rate of LTBI in those regions at baseline. But no significant difference in the incidence rate was observed among students from different regions of China, as well as Han and non-Han ethnicity (Table P < 0.001).There were 26 active TB cases during the 2 years of follow-up. Three of them were diagnosed by microscopy examination of sputum smears; three of them were diagnosed by P = 0.91). However, when the induration size was greater than 10 mm , the adjusted HR (95% CI) showed an upward trend  (Table P < 0.001) compared to those with reaction size < 10 mm for males, and 5.08  for females. This suggested that in males, the risk of active TB was higher in college students with the TST positive results, but the difference was not significant for females.Factors that significantly related to active TB incidence were sex and TST response. As shown in Fig. In this retrospective study, we found that TST reaction size ≥10 mm in college freshmen predicted an increased risk of active TB in the subsequent 2 years of TST, compared with those with a reaction size <10 mm. Specifically, the active TB risk increased about 3 times per 5 mm increasing of the interval of TST reaction size compared to reaction size ≤5 mm, up to 9.768 times with reaction size ≥20 mm. And the distribution of the absolute risk of active TB of different induration sizes had similar features. Some previous studies also indicated that a person with a TST reaction size ≥15 mm had a high risk of active TB . HoweverMtb infection, vaccine coverage rate for bacillus Calmette-Gue’rin (BCG) has been around 97% since 1999 in China [As the most effective and economical means for prevention from in China . Howeverin China , which min China , 14, 15,in China , 16, 17,Mtb is a person-to-person transmitted pathogen mainly according to the droplet, but behavior-related exposure is usually higher among males, especially after puberty [Mtb exposure may lead to the different risk levels of incidence of active TB.Furthermore, the males of students predicted a higher risk of active TB compared to females, which was consistent with the results of previous studies –20. Adol puberty . Thus, tMtb infection [There were a few limitations in this study. At first, the absence information of some other influencing factors, such as BMI, smoking and alcohol drinking history, close contact history, comorbidity and so forth, in the raw records might lead to ignoring some risk factors and influencing the accuracy of results. Secondly, because of the missing information of students received LTBI treatment, the risk of active TB for those with TST reaction size ≥20 mm might be underestimated in this study. Thirdly. follow up time was not long enough. It was reported that TB risk decreased rapidly with time after nfection , 24. LonIn summary, the intensity of TST reaction could reflect the active TB risk to some extent for college students. And males had a higher risk compared to females. This may allow us to do a better job in the prevention of pulmonary tuberculosis of college students in the future.Additional file 1:Table S1. The age distribution of the follow-up cohort. Table S2. The TST reaction size distribution of follow-up cohort. Table S3. The characteristics of individuals with TST positive and TST negative. (DOCX 21 kb)


To characterize the clinico-pathological features including estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu (HER2) expression in breast cancers in Botswana, and to compare them by HIV status.This was a retrospective study using data from the National Health Laboratory and Diagnofirm Medical Laboratory in Gaborone from January 1, 2011 to December 31, 2015. Clinico-pathological details of patients were abstracted from electronic medical records.p < 0.001). The majority of patients  presented with stage III at diagnosis. Stage IV disease was not presented because of the lack of data in pathology records surveyed, and additionally these patients may not present to clinic if the disease is advanced. Overall, 68.9% (151/219) of tumors were ER+ or PR+ and 16.0% (35/219) were HER2+. ER+ or PR+ or both, and HER2- was the most prevalent profile , followed by triple negative , ER+ or PR+ or both, and HER2+,  and ER−/PR−/HER2+ . There was no significant difference in receptor status noted between HIV-infected and HIV-uninfected individuals.A total of 384 unique breast cancer reports met our inclusion criteria. Of the patients with known HIV status, 42.7% (50/117) were HIV-infected. Median age at the time of breast cancer diagnosis was 54 years (IQR 44–66 years). HIV-infected individuals were more likely to be diagnosed before age 50 years compared to HIV-uninfected individuals  at diagnosis and hormone receptor positive disease. HIV-infected breast cancer patients tended to present at a younger age compared to HIV-uninfected patients. HIV status does not appear to be associated with the distribution of receptor status in breast cancers in Botswana. Breast cancer is the most commonly diagnosed cancer among women worldwide. Mortality due to breast cancer disproportionately affects women in low- and middle-income countries (LMIC) . PreviouBotswana is an upper-middle-income country in SSA with a population of 2.2 million, and the third highest prevalence of HIV in the world, 17.6% among the general population –12. BreaThe aim of this retrospective review was to characterize the clinico-pathological features, including age at diagnosis, stage at presentation, estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal Growth Factor Receptor2 (Her-2/neu) of breast cancer pathology specimens reviewed at the National Health Laboratory (NHL) and Diagnofirm Medical Laboratory (DML) in Gaborone, Botswana.The NHL is located in Gaborone and currently serves as a referral laboratory for the southern region of the country providing services to 14 district hospitals, including Princess Marina Hospital, a tertiary referral center. DML is an independent laboratory in Botswana with twelve locations throughout the country. While NHL sees about 85% of the breast cancer patients in Botswana, DML sees about 15% . ArchivaWe conducted a retrospective analysis using archival data from NHL and DML in Gaborone for breast cancer pathology specimens analyzed and reported from January 1, 2011 to December 31, 2015. The data includes all patients who underwent a biopsy, wide local excision and mastectomy for breast cancer. We manually abstracted available data from the pathology report: age, gender, tumor laterality, tumor grade, tumor size and nodal status; and demographic data and information on HIV status from the electronic medical record. Because information was ascertained from pathology records and breast and lymph node procedures only, data on metastatic sites were not available. Staging data was computed according to AJCC v7, using tumor size and lymph node status from breast surgical specimens with the assumption that patients who underwent definitive surgery had been ruled out for metastatic disease. AJCC v7 vs v8 was utilized as we used retrospective staging records and did not actively stage tumors. Stage IV patients were not presented in our data. Pathology records reported breast an axillary lymph node staging only, while most stage IV disease is diagnosed both radiographically and clinically and available in individual patient notes, we were unable to account for these in the pathology records.ER, PR, and HER2 immunostaining was performed in an automated stainer at NHL and manually at the DML at the time of diagnosis as part of routine care. All staining was done using a standardized protocol under optimum physical conditions as prescribed by the manufacturer. In brief, formalin fixed, paraffin-embedded breast tumor blocks were obtained and 4-mm tissues were sectioned, deparaffinized, and rehydrated. Antigen retrieval was performed by using a microwave in Tris/EDTA buffer, pH 9.0 (for anti - ER and PR) and Citrate buffer, pH 6.1 (for anti – HER2/neu) at 97 °C for 20 min and blocked with Phosphate buffer containing hydrogen peroxide, 15 mmol/L NaN3 and detergent (Dako EnVision™ FLEX Peroxidase-Blocking Reagent). Tissue sections were then incubated for 30 mins with primary antibodies at room temperature: anti-ER , anti-PR , and anti-HER2/neu , followed by goat secondary antibody molecules against rabbit and mouse immunoglobulins . Staining was visualized by using diaminobenzidine  and counterstained with hematoxylin. IHC manufacturers and sources are Dako Products, Agilent Technologies, USA.Immunohistochemical stains were evaluated using standardized controls provided by the manufacturer. ER, PR and HER 2/neu status were assessed semi-quantitatively using Standard Allred scoring system. ER and PR were considered positive if > 1% nuclei of tumor cells stained . HER2 waData from all pathology reports were extracted and recorded in a Research Electronic Data Capture (RedCap) database. Descriptive statistics was used to quantify mean, median, range and respective proportions. We also performed a chi squared test to compare the equality of proportions of age > 50 , receptor status, and grade at initial breast cancer diagnosis between HIV-infected and HIV-uninfected breast cancer patients. All analyses were performed in STATA.This study was reviewed and approved by the Institutional Review Board of the University of Botswana, Health Research and Development Committee of Ministry of Health and Wellness and the Institutional Review Board of DML.There were 409 breast pathology reports analyzed from January 2011 to December 2015. Seventeen were excluded because biopsies were taken before 2011 or because they were not breast/axillary lymph node biopsies, and 8 were excluded because of insufficient residual tumor for analysis. Using one sample per patient, a final total of 384 reports of pathologically confirmed breast cancer cases were included in our analysis. Six of these patients were male.Age was reported on 330 (85.9%) reports Table . Median Left breast was involved in 42.0% (161/384) of reports and right breast in 40.0% (153/384) of reports. Bilateral breast cancers were reported in 3 cases. There was no mention about the laterality in 17.4% (67/384) cases. There was no predilection for the laterality as no significant difference was noted between the proportions of left vs. right breast cancers.The most common histologic subtype was invasive ductal carcinoma . Second most common type was mucinous carcinoma  followed by medullary  and invasive papillary . Invasive lobular carcinoma constituted 1.53% (6/384) of all breast cancers. Presence of ductal carcinoma in-situ (DCIS) was not specified in 90% (344/384) cases, but in those where it was specified, DCIS was present in 90% (36/40) of cases along with invasive carcinoma. Of the six male breast cancers, four were invasive ductal carcinomas.n = 2), 27.4% presented with Stage II (n = 14), and 68.6% (n = 35) presented with stage III breast cancer . Mean tumor size was 4.3 cm (range 0.8–16 cm). Four percent of patients presented with Stage I disease  of the tumors were of histologic grade 2, with a quarter  presenting with features of grade 3. Eighty-nine reports either had grade noted as “not specified” or were missing grade information. Ki-67 status was available for only 12/211 tumor samples. This is a common data challenge for pathology samples in Botswana.Immunohistochemistry was completed on 219 of the samples at the time of their removal. From retrospective review, eight of the samples had at least one receptor type noted as “not specified,” allowing for complete IHC profile to be completed on 211 samples. Overall, 69.2% (148/214) of tumors were ER+, 52.3% (112/213) of tumors were PR+, and 16.4% (35/214) were HER2+ overexpressed. 62.6% of samples included a profile of ER+ or PR+ or both, and HER2- , 21.3% were triple negative , 9.0% were ER+ or PR+, or both, and HER2+  and 7.1% were ER−/PR−/HER2+ (15/211). ER−/PR−/HER2+ cancers were more likely to be seen in patients ≥50 years of age (58.3% vs. 41.7%) (Table 3 (IQR 209 cells/mm3–525.6 cells/mm3). Antiretroviral treatment (ARV) coverage was not available for this population, however ARV coverage in Botswana is well-documented to be 84% among adults [p < 0.0001). HIV-uninfected individuals had a slightly higher prevalence of ER+ disease than HIV-infected individuals (82.9% vs 70.0%), but this was not statistically significant. This trend was not seen for HER2+ and TNBC; however, our sample size for these groups were small  of patients had known HIV status; of those patients, 43.0% (49/114) were HIV-infected and 57.0% (65/114) were HIV-uninfected. The median CD4 count among HIV-infected patients with breast cancer was 396 cells/mmg adults . Table 384 of patTo our knowledge, this study analysing data on 384 patients with breast cancer is the first large pathology-based study describing clinical and pathological features of breast cancer in Botswana. We noted that breast cancer in Botswana was largely hormone receptor positive and tended to present more frequently at stage III than stages I and II. Median age at diagnosis for our cohort was 54 years, seven years younger than the median age (61 years) of both African-American and Caucasian women in the United States (U.S.), with 40% of women diagnosed before 50 years of age. Comparatively, median age of breast cancer diagnosis in the U.S. is 58 years for African-American women and 62 years for white women . A previIn our study, 67.6% of breast cancer cases were ER+, similar to data published in SSA and parts of East Africa, but different from West Africa where studies have reported a low rate of ER+ cases (35%) and high triple negative subtypes . The preThe prevalence of TNBC in Botswana (21.3%) was higher than those documented in white populations (10–16%) , 41 but Breast cancers in Africa are often characterized by a higher proportion of advanced disease, including large median tumor diameter and advanced stage. On average, 60% of black Africans in SSA present with advanced stage disease compared to 27% among white women and 32% among African-American women . SeventyThis may be a reflection of delayed recognition of symptoms, diagnosis, or treatment or an indication of more aggressive tumor biology . Delay fHIV prevalence among women in Botswana is 9.5%, and total HIV prevalence among adults aged > 15 years old is 24% , 52. TheMajor strengths of this study include documentation and characterization of over 200 breast cancer specimens in a five-year period in Botswana, where little data exists about the characteristics of breast cancer. Diagnostic information on the cancer was acquired from histopathological assessment as opposed to clinical chart review, assuring accuracy of diagnostic information. This data provides much-needed information for further studies looking to analyze the treatment and survival of breast cancer in Botswana, especially for younger and HIV-infected patients. It also establishes a baseline to understand the current practice of pathology and how it contributes to cancer care.Limitations of this study include small sample size and retrospective nature of the study.We do not assume a selection bias as both pathology centres serve the surrounding population. We are limited by the data found in medical records. Since staging data was pulled from pathological records, we do not have information on metastasis limiting our ability to characterize stage IV disease. There are challenges present in acquiring staging information, especially M stage with limited imaging resources and lack of trained staff to stage appropriately. Two retrospective reviews have evaluated completeness of breast cancer pathology reports across sub-Saharan Africa, both which identified substantial gaps documentation of basic pathology information , 54. It Despite limited staging info, our data provides important data on HIV, receptor status and age of breast cancer patients in Botswana and contributes to literature on this topic from SSA. This will help guide future studies on breast cancer patients with HIV as well as breast cancer screening and treatment strategies.Breast cancer patients in Botswana present at a younger age compared to their Western counterparts, but at a similar age compared to other SSA countries. Additionally, we found a higher prevalence of HIV among our population of breast cancer patients compared to the general Botswana population. HIV-infected individuals tended to present at a younger age at initial diagnosis compared to HIV uninfected individuals. Further investigations into the link between HIV and breast cancer, and understanding the impact on treatment are needed. Immunohistochemistry (IHC) of breast tumors seen is Botswana is similar to other countries in Southern and East Africa, with a fifth of cancers expressing triple negative type. There is no difference in receptor status by HIV-status. To our knowledge, this is the first attempt to characterize the clinico-pathological and immunohistochemical characteristics of breast cancer in Botswana, providing essential data to a growing body of knowledge of tumor biology in SSA.


Endometrial cancer (EC) is the most common malignancy of the female reproductive tract. Despite years of research, the accurate screening strategy is still not available in this disease and it is usually diagnosed only after the clinical signs are present. The recent technological advances in analytical methodologies enabled detection of multiple molecules in one, small sample of biological materials. Such approach was undertaken in the presented study.Concentrations of aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), carbonic anhydrase IX (CA9), CD44, epithelial cell adhesion molecule (EpCAM), hepsin, kallikrein-6, mesothelin, midkine, neural cell adhesion molecule L1 (L1CAM), and transglutaminase 2 (TGM2) were measured using MAGPIX®System in plasma samples of 45 EC, 20 healthy controls and 11 patients with endometriosis.p <  0.001), EpCAM (p = 0.033) and TGM2 (p <  0.001). EpCAM and mesothelin concentrations differed based on FIGO stages. Regression analysis revealed marker panels with high accuracy in detection of EC. The highest AUC 0.937 was attributed to the 3-marker panel of CD44/TGM2/EpCAM , FIGO IA samples were discriminated from more advanced stages of EC with the mesothelin/grade 1 model featuring AUC of 0.911 .Significantly increased concentration in EC as compared to healthy controls were found in case of CD44  contains supplementary material, which is available to authorized users. Multiple serum and plasma circulating molecules have been suggested as possible biomarkers in endometrial cancer (EC) detection . HoweverIn previous years the search for possible markers in cancer detection was based on studying single analytes in one sample. That strategy was laborious, expensive and required relatively large sample volumes. The recent technological advances in analytical methodologies enabled detection of multiple molecules in one, small sample of biological material, and offered the opportunity of developing biomarker panels, which could increase accuracy in detection of certain diseases including endometrial cancer . With reThe authors of the study aimed to determine markers discriminating: 1) EC and the pooled control and endometriosis samples; 2) EC and the healthy controls; 3) different FIGO stages of EC.Median = 58, 95% CI 54.83–61.35; p = 0.011), but not from FIGO IA patients. There were no significant differences in patient age between subgroups distinguished based on FIGO stage, histological grade and myometrial invasion.Seventy-six females, patients of the III Department of Gynecology, Medical University of Lublin, were included in the study . Patients in the control group did not have history of malignancy, reproductive tract pathology or symptoms of endometriosis. None of the included patients had a history of any serious illness e.g. diabetes, autoimmunological, liver or kidney disease. Patients in EC and endometriosis groups did not have history of other malignancies or were submitted to neo-adjuvant therapy. The EC patients underwent total hysterectomy with bilateral salpingoophorectomy due to EC diagnosed with endometrial biopsy prior to operation. All cases were endometrioid endometrial carcinomas. After the operations patients were submitted to radiotherapy and/or chemotherapy according to International Federation of Gynecology and Obstetrics (FIGO) guidelines. Clinical stage of the disease was determined according to 2010 FIGO classification. Median age of the control samples was 51 (95% CI 46.53–56.81) and significantly differed from all EC patients  and was stored for maximum one hour in 4 °C until it was further processed. Blood was centrifuged for 15 min  and plasma was collected. Plasma samples were aliquoted to 300 μL and were stored in − 80 °C until further analyses.Methodology used in the study was based on the Luminex® xMAP® technology capable of performing immunoassays on the surface of fluorescent-coded magnetic beads coated with specific capture antibodies. After an analyte from a test sample is captured by the bead, a biotinylated detection antibody is introduced. The reaction mixture is then incubated with Streptavidin-PE conjugate, the reporter molecule, to complete the reaction on the surface of each microsphere. Each individual microsphere is identified and the result of its bioassay is quantified based on fluorescent reporter signals. The study was performed with commercially available MILLIPLEX® MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 4 . The analytes were simultaneously detected in 25 μL samples according to manufacturer’s protocol using MAGPIX®System  and mean fluorescence intensities were calculated with the xPonent 4.2 software, using five-parameter logistic curve fitting to derive the analyte concentrations in each sample.p-value less than 0.05 was considered statistically significant. All tests were two-sided. Data were analyzed with MedCalc  version 12.2.1.Mean or median with 95% IC were used to present the results depending on the data distribution, which was evaluated with Shapiro-Wilk test. Comparison of variables between two groups were performed using the t-student test or Mann-Whitney U test depending on Shapiro-Wilk test results. Comparisons of variables between three groups were performed with Kruskal-Wallis test and pairwise comparison of subgroups according to Conover was used for post-hoc testing. Correlations between variables were estimated with Spearman test. The diagnostic accuracy of single biomarkers was evaluated using logistic regression. For the selected markers, the receiver operating characteristic (ROC) curves were obtained and the area under curve (AUC) was calculated with 95% confidence intervals according to the nonparametric method. ROC curves AUCs were compared using the method of Delong et al.  with 2-sp = 0.028) and TGM2 (p = 0.004), were significantly increased in plasma of patients with EC as compared to non-EC samples , but the subsequent subgroup analysis revealed that it was due to highly increased midkine level in endometriosis samples (S2-S3).Concentrations of EpCAM , EpCAM (p = 0.033) and TGM2 (p < 0.001)  , CA9 (p = 0.005), CD44 (p < 0.001), hepsin (p = 0.008), midkine (p < 0.001), and TGM2 (p < 0.001), whereas concentration of a single marker – kallikrein-6, was found to be decreased (p = 0.006) .Seven analytes differentiated endometriosis and control samples. Six analytes were increased in patients with endometriosis: ALDH1A (p = 0.019), CA9 (p = 0.031), CD44 (p = 0.010), hepsin (p = 0.007), midkine (p < 0.001)  Figs. .Fig. 2CoDescriptive statistics of markers’ concentrations and inferential tests’ results for all comparisons were provided in the Additional file For the purpose of the analysis EC patients were divided into three subgroups based on FIGO classification: FIGO IA, FIGO IB and FIGO III. Patients with higher stage were combined into one group due to small numbers of those patients. The group consisting of patients presenting with FIGO IA stage and histological grade 1 was also distinguished (FIGO IA-G1), which represented the earliest phase of disease progression.p < 0.001, post-hoc p < 0.05, and TGM2: p < 0.001, post-hoc p < 0.05). No significant differences were however encountered between individual FIGO stages in regards to CD44 and TGM2 concentrations.Significant differences were encountered in CD 44 and TGM2 concentrations between control samples and each of the FIGO stage Fig. a, d. Thep = 0.025), and in FIGO1B samples was significantly higher comparing to controls (p = 0.003).EpCAM concentration was higher in FIGO IA and IB stages as compared to FIGO III group Fig. b. Medianp = 0.011), and between FIGO I and FIGO III samples (p = 0.038). It was also found that concentration of mesothelin in FIGO IB – III samples was significantly lower comparing to control samples (p = 0.018)  were correlated in control group. No correlations were found between studied analytes and patients’ age (S4-S6).The associations between altered levels of circulating CD44, TGM2 and EpCAM and hematologic prognostic parameters reflecting a systemic immune and inflammatory response, such as neutrophil/lymphocyte ratio (NLR), monocyte count, and platelet/lymphocyte ratio (PLR) were also evaluated in patients with endometriosis and with endometrial cancer and are presented in the Additional file SE 0.04, 95% CI 0.79–0.96, p < 0.001) and 0.945   were calculated and compared. The highest AUC value was calculated for TGM2 and that result was significantly higher in comparison to CD44 and EpCAM Table , Fig. 4bbackward method retrieved model consisting of EpCAM  and TGM2  as most suitable for discrimination between EC and control samples. Subsequently, models were created using logistic regression enter method. Out of the four models obtained in that analysis, the 3-marker model was characterized by the highest AUC of 0.937 . Comparison of the AUCs revealed no significant difference between CD44/TGM2/EpCAM model, the TGM2 and all three 2-marker models  and CD44 , which distinguished FIGO IA samples from the controls with the 69% sensitivity and 94% specificity and AUC of 0.847   biomarkers. The logistic regression b = − 0.57, SE 0.21, p = 0.006, OR 0.57) and histological grade 1 , as factors predicting the lower progression of EC. The ROC curve plotted based on that model retrieved AUC of 0.911 , 95% sensitivity, and 78% specificity , however this result needs to be considered as preliminary due to the small number of endometriosis samples.It was also found that a regression model consisting of CD44 and TGM2 discriminated endometriosis from control samples with high accuracy  or gene expression studies –25. The Accurate tool in endometrial cancer diagnosis needs to be sensitive but also specific, as this carcinoma rarely occurs as a lone disorder. It is often associated with obesity, hypertension and diabetes, but can be also accompanied by endometriosis . In lineAnalysis of data revealed that concentrations of CD44, EpCAM, and TGM2 were significantly increased in EC comparing to control samples. It was also found that concentrations of the two stem-cell markers, CD44 and TGM2 were significantly correlated in EC samples. These results correspond to results of increased CD44 expression in EC tissues reported by Elbasateeny et al. and Wojciechowski et al., as well as upregulation of EpCAM in serous uterine cancers presented by El-Sahwi et al. , 9, 11. High tissue expression of mesothelin has been linked to aggressive tumor behavior and worse prognosis in ovarian and pancreatic cancers as well as in mesothelioma . The diaWhen concentrations of other analytes were compared according to clinicopathological characteristics the most important finding regarded EpCAM, which concentration was higher in FIGO IA as compared to more advanced stages (>FIGO IA). No differences in concentrations of other markers were found in regards to histological grading, myometrial invasion and FIGO staging. The lack of correlation of CD44 level with clinicopathological features despite its highly significant increase in EC samples corresponds to the results obtained by other authors , 8.Further analyses including the subset of endometriosis samples revealed interesting findings. Concentration of seven analytes  differed in endometriosis samples as compared to control samples and levels of five markers  were different between endometriosis and EC samples. We acknowledge that those results can only be regarded as preliminary findings, due to a small number of patients with endometriosis. However, they seem to explain the observation, that only three analytes were different  in EC in comparison to non-EC group.backward analysis method retrieved the model consisting of EpCAM and TGM2 as most suitable model for discrimination between EC and controls, which yielded AUC of 0.909 with 84% sensitivity and 94% specificity. Subsequently, regression models were created using the enter method and different combinations of CD44, TGM2, and EpCAM. Out of the four models obtained in that analysis, the 3-marker model was characterized by the highest AUC of 0.937 with sensitivity of 84 and 100% specificity. Comparison of ROCs revealed that the AUC for CD44/TGM2/EpCAM model did not differ significantly from the AUC of TGM2 and all three 2-marker models. Analysis aiming at discrimination between EC samples and non-EC group, which included endometriosis samples, required input of least five markers to obtain a satisfactory AUC of 0.895, and the utilization of eight markers was necessary to increase the AUC to 0.945.Regression analysis revealed panels of analytes characterized by high diagnostic accuracy in discriminating EC samples. Firstly, TGM2 was found to be a highly accurate, single marker able to differentiate between EC and healthy controls with the AUC of 0.901 . Secondly, multivariable logistic regression models were constructed to evaluate, if the input of several biomarkers would improve the diagnostic accuracy. The Comparing to the work of other authors, marker panels discovered in this study seem to offer similar or better accuracy. Yurkovetsky et al. reported prolactin as the strongest single biomarker for EC with 98.3% sensitivity and 98.0% specificity and the 5-marker panel , which yielded high sensitivity and specificity in discrimination between EC, ovarian and breast cancers . AnotherEC has a very good prognosis providing it is diagnosed at the early stage of clinical progression. Nowadays most EC are discovered only after the clinical signs are already present, and the screening method is still not available . One of Accurate preoperative diagnosis of the disease progression has an important clinical significance in endometrial cancer management, as early EC does not require lymphadenectomy and radical surgery. A method with reasonable accuracy in depicting tumors at the earliest stage of progression could decrease morbidity and complications connected with unnecessary lymphadenectomies and extensive resection of pelvic tissues. In line with that notion a multivariable regression analysis was performed to discriminate between FIGO IA samples and more advanced stages of EC. That analysis incorporated histological grading as one of the variables and retrieved model consisting of mesothelin concentration and histological grade 1, as factors predicting early clinical stage of EC. The ROC curve plotted based on that model retrieved AUC of 0.911, with 95% sensitivity and 78% specificity.These results present similar accuracy as compared to blood derived markers reported by other research groups , 35, 41.There are some limitations to our study that need to be addressed. Firstly, although we investigated multiple markers the number of patients included in the study was only moderate. Therefore, we consider obtained results as preliminary, which need to be repeated in larger population, preferably in the multicenter setting. Secondly, we acknowledge that the research would benefit from tissue expression analysis. We plan to address these issues in the future studies.In conclusion, in this study we report novel, potential biomarkers that could offer a good diagnostic accuracy in diagnosis of EC, with TGM2 being reported for the first time as plasma marker. In addition, we point to the fact, that endometriosis might share similarities in the pattern of markers alterations characteristic for EC, what leads to the conclusion that its undiagnosed presence could alter results of EC studies and need to be taken into consideration during the research design.Additional file 1:The dataset: clinicopathological characteristics of the endometrial cancer patients. (XLSX 10 kb)Additional file 2:Table S1. Descriptive statistics of the studied analytes’ plasma concentrations. (DOCX 19 kb)Additional file 3:Table S2. Statistical tests’ results for the comparisons between groups: Mann-Whitney (M-W) or t-test were used based on Shapiro-Wilk test results. (DOCX 19 kb)Additional file 4:Table S3. Results of the correlation analysis for the analytes in the EC group. (DOCX 26 kb)Additional file 5:Table S4. Results of the correlation analysis for the analytes in the control group. (DOCX 27 kb)Additional file 6:Table S5. Results of the correlation analysis for the analytes in the endometriosis group. (DOCX 27 kb)Additional file 7:Table S6. Correlation analysis results of CD44, TGM2 and EpCAM plasma levels and neutrophil/lymphocyte ratio (NLR), monocyte count, and platelet/lymphocyte ratio (PLR). (DOCX 14 kb)Additional file 8:The dataset supporting the conclusions of this article. (XLSX 17 kb)


Nano-emulsions are promising carriers for antigen delivery. Here, we evaluated the efficacy of a water-oil nano-emulsion containing concentrated, inactivated Clostridium novyi (C. novyi) type B supernatant culture (nano-iCnB) in protecting Swiss mice against a lethal dose of alpha toxin concentrated extract. Proteins were confirmed in the nano-iCnB and their stabilities were determined according physical parameters such as Zeta Potential (ZP). Biochemical, hematological parameters and morphological appearance of liver, spleen and thigh muscle alterations were examined to determine the safety of the compound. Partial protection against lethal doses was achieved in immunized mice despite low IgG titers. These data suggest that our nano-emulsion is a simple and efficient method of promoting antigen delivery for toxin-related diseases. C. novyi type B is a strict anaerobic, endospore-forming, gram-positive bacterium. Its major virulence factor is an alpha toxin that causes necrotic hepatitis, known as black disease. Black disease symptoms include hemoglobinuria, reduced appetite, fever, lethargy, decreased milk production and blood-stained feces, all of which reduce productivity in sheep, horses, pigs and cattle2. Pathogenesis begins with spore germination in the liver, followed by development of vegetative cells and production of alpha toxin. Liver infection causes necrotic infarction with thick areas of hyperemia4. The acute disease is rapidly progressive and associated with a high mortality rate6.8. Aqueous adjuvant induces short-term responses, besides the fact that costs with handling and revaccination make them more expensive10. Thus, alternatives to reduce animal handling as well as elimination of interactions between antigens are necessary for vaccine development10. Antigens in nano-emulsions are promising alternatives to improve vaccine efficacy because they delay antigen delivery, generating good cellular and humoral responses for long periods11. Here, we demonstrated that our formulation is a substantial improvement and advance in terms of adsorption of antigens for control of clostridiosis.Current vaccines are composed of multiple antigens adsorbed on aluminum hydroxide; nevertheless, their efficacies remain unsatisfyingC. novyi cause gas gangrene, characterized by hemorrhage and muscle and soft tissue. After alpha toxin production, mice were inoculated to determine the lethal dose (LD50/mL) up to 72 hours , while the LT50s of nano-milliQW and nano-iCnB were 30.0 (19.9–40.1) and 56.0 (40.8–71.2), respectively. The log rank statistic for the survival curves showed a significant difference between survival curves (p = 0.002). The survival curves were compared to the curves of the three groups  simultaneously, using TL50 as the comparison unit. Only animals in the nano iCnB group showed significant differences.Kaplan-Meier survival curves showed that mice immunized with nano-iCnB had greater survival when challenged with a lethal dose of 157 LDice Fig. . The letC. novyi type B has substantial economic importance because of the damage it generates in livestock production chains. Here, we evaluated a nano-iCnB containing alpha toxin with 2178 amino acid residues, molecular weight ranging from 260 to 280 kDa13. We demonstrated partial protection in immunized Swiss mice using nano-iCnB, indicating its promising action as carriers for antigen delivery.Alpha toxin-producing 14. New controlled release systems have been proposed as antigen delivery vehicles because of their utility as immunological adjuvants, with absence of toxicity, biodegradability, and slow release of encapsulated antigens14. Here, we observed that the nano-iCnB showed a high volume with 66.5% of the sample, suggesting improved encapsulation and controlled release of antigen , infectious bovine rhinotracheitis (IBR), and inactivated C. botulinum toxin with protection and safety being similar to that of the conventional vaccine17. As a proof of concept in this murine model, we consider it to be a relevant technological contribution of the use of nano-iCnB for immunization of Swiss mice. It represents an improvement in production of multiple antigens vaccines, specifically for the use of alpha toxin of C. novyi for control of hepatic necrosis.The present study represents an innovative and simple technological strategy for the emulsification of inactivated toxins as immunogen18. Here, the nano-milliQW consisted of droplets of three diameters: 896.5, 618.5 and 1280 nm, suggesting that the system containing only the adjuvant did not produce adequate size control. However, the nano-iCnB showed only two bands: 509.4 and 882.4 nm with high volume, suggesting that the antigen helps to control droplet size in the nano-emulsion, giving rise to a fine emulsion20. The surface area to the volume in nanoemulsion can be affected by properties of the particles and their interaction with other systems. Small size particles show higher barrier properties for evaporation and increase the occlusion. These data suggest that the effect is affected by applied volume, particle size and crystallinity21.Size, intensity and ZP are parameters used for analysis of interactions, transport of molecules and the availability of antigen at the immunization site22. In nano-iCnB, the ZP was lower than that of nano-milliQW, demonstrating desirable physical characteristics for a W/O type emulsion vaccine formulation20. This suggests product stability, controlled release and stimulation of the immune response23.ZP values are directly related to the stability of products, being obtained by the ratio of repulsion forces between charged particles that avoids the natural tendency to aggregate14. Significant reduction of viremia (BVDV), even with a lower viral antigen concentration, was observed with use of vaccines formulated with ISA 61 VG. Here, for the first time, we used Montanide ISA 61 VG adjuvant together with bacterial antigen in nanoemulsion, and detected partial protection of Swiss mice after dose challenge (150 LD50g−1) with alpha toxin from C novyi tipe B. Immunized animals had later manifestations of the disease in some individuals, with a survival rate of 40%. Hepatocellular lesions and hepatocyte permeability may be indicated by changes in AST, ALT and cholestatic disorders caused by obstruction of the bile ducts, suggesting high metabolic activity in serum, as occurs in necrosis or disease in target tissues25. In contrast, nano-iCnB mice showed normal biochemical and hematological parameters. Furthermore, these animals did show signals of hypertrophy and congestion in liver. No edema in the right thigh nor with body or liver weight after 7 days of the final immunization indicated its safety. These data suggest that our nano-emulsion is a simple and efficient method that can be used to promote antigen delivery for toxin-related diseases.Montanide ISA 61 VG is a new ready-to-use mineral oil-based adjuvant developed by SEPPIC Inc.  being employed in the improvement of vaccines against FMD and BVDV virusThis study had technical limitations as to improve the concentration of antigen present in nanoemulsion, as well to explore nano emulsion containing multiples antigen clostridial understanding your protective effect.C. novyi type B on a nanoemulsion. Nano-iCnB displayed safe and effective for stimulating immune responses in Swiss mice challenged with lethal doses of toxin.In conclusion, we proposed a simple and straightforward method of determining the effect of inactivated alpha toxin from Mus musculus) weighing between 17 and 23 g were purchased from the Goiânia Central Goat Hospital - GO of the Federal University of Goiás (UFG). The animals were kept under conditions controlled for temperature, luminosity and odor. They were given feed (Biobase® - Brazil) and purified water ad libitum. The animals were maintained for a quarantine period of 7 days before the start of the experiments. The study was authorized by the Ethics and Research Committee of the Federal University of Tocantins (UFT) under number 23.101.006.832/2017-53 according to international recommendations for ethics in animal experimentation and by guidelines for animal use and care were based on the standards established by The Brazilian College of Animal Experimentation (COBEA).Female Swiss mice  and xylazine 22 mg/Kg  intraperitoneally and were euthanized by cervical dislocation to achieve rapid and painless death when mice showed intense lethargy, according to ethical protocolsC. novyi type B, virulent strain used for alpha toxin production and vaccinal antigen purchased from the NCTC  was grown and stored in a culture bank with storage at the Laboratory of Biomolecules and Vaccines of the Federal University of Tocantins (LaBVac/UFT). Alpha toxin production was started in culture of C. novyi type B in Tarozzi culture medium and incubated at 37 ± 2 °C in BOD for 24 hours. Cultures were spun at 3000 g for 15 minutes to collect the supernatants. Vivaspin 6 MWCO 30000 membranes were used to concentrate inactivated alpha toxin used in W/O 40/60 nano-emulsions.C. novyi type B was determined in Swiss mice by diluting the toxin from 1:4–1:16. From each dilution, 200 μL was administered intraperitoneally to four mice27. Determination of lethal dose (LD50/mL) was performed by observing the effect of the alpha toxin in the inoculated animals for 72 hours and quantifying by mathematical interpolation28. A lethal dose of 157DL50.g−1 was employed for challenge test  to a final concentration of 0.16 mg/ml in the 40% aqueous phase and the 60% ISA adjuvant (oily phase). Nano-iCnB and nano-milliQW were prepared by passing the volume between sterile syringes connected by a silicone hose.In addition to LDThe physical characteristics of nano-iCnB were evaluated in the presence (culture supernatant) and water (nano-milliQW) of inactivated alpha toxin22. The diameter measurements of the emulsion droplets and zeta potential were performed using a Zetasizer Nano ZS90 from Malvern Instruments at 25 °C. Samples were diluted using ISA adjuvant at a ratio of 1:10.The Dynamic Light Scattering (DLS) technique was used to determine the diameter of the droplets. In this technique, a beam of light is focused on the sample and droplet size is estimated from fluctuations in the intensity of light scattered at a given angle. The zeta potential is a measure of the magnitude of the static charges present at the interface between the droplets and the solvent. This parameter is important because it affects the stability of the droplets.Characterization of the inactivated alpha toxin was performed using SDS/PAGE and antigenicity was evaluated by immunoblotting. We used 11% SDS/PAGE gels with buffer containing 1.5 M Tris-HCl pH 8.8 and 0.4% SDS. Electrophoresis buffer contained 25 mM Tris-HCl pH 8.3, 200 mM glycine and 1% SDS. The samples were heated to 100 °C for 5 minutes and were run at 70 Volts. The gels were stained with Coomassie blue for 12 h, and the protein bands were transferred to polyvinyl difluoride (PVDF) membranes using Trans-Blot® Turbo ™ Transfer System from Bio-Rad Lab and buffer with 5.82 g of tris base, 2.83 g glycine, 3.75 mL 10% SDS and 20% methanol, pH 9.2 for 60 minutes.Electrotransfer was performed at 15 volts for 30 minutes. After transfer, the membranes were immersed in blocking solution with 1x PBS buffer + 3% milk powder, left overnight, and were subsequently washed in PBS 0.05%-Tween three times. Sera from healthy and diseased animals were diluted 1:2000 in 1x PBS + 0.5% BSA buffer and were applied to the membranes with gentle agitation for 60 minutes. Then, membranes were washed again for addition of alkaline phosphatase-conjugated anti-mouse at a dilution of 1:10000 under gentle agitation for 60 minutes. Membranes were again washed with PBS 0.05% Tween three times and once with alkaline phosphatase buffer under gentle agitation for 5 minutes. Membranes were submerged in NBT/BCIP substrate and the reaction was subsequently stopped. The membranes were washed with distilled water and dried in an oven.50.g−1 after 7 days of the final immunization. Sick animals were anesthetized and euthanized and Kaplan-Meier survival curves were determined.Female Swiss mice were separated into three groups  with 10 animals per group. In the immunized group, 100 µl of the nano-iCnB supernatant were administered at 0, 21, 42 days intramuscularly injected into the right thigh. Animals in the nano-emulsion and non-immunized groups were given 100 µl of PBS and nano-milliQW, respectively. The three experimental groups were monitored daily with weights measured and challenged with 157 LDThree animals were evaluated 7 days after the final immunization (day 42) to evaluate changes in liver, spleen and right thigh morphology. The degree of lesions at the site of administration was evaluated in terms of variation of organ weight, swelling, and redness as follows: 0) alterations absent; 1) discrete changes up to 25%; 2) moderate changes greater than 25%, but less than 50%; and 3) severe changes greater than 50%.50.g−1, followed by blood collection in the first 24 hours. The biological material was analyzed by the Veterinary Pet Shop Dog Center (Gurupi/TO - Brazil). Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), leukograms, hemoglobin and hematocrit were determined. A group of three non-immunized animals were challenged with a lethal dose of 157 LD50.g−1 for the collection of sera from animals infected with active alpha toxin.Seven days after the final immunization (day 42), experimental groups were challenged with a lethal dose of 157 LDThe Kruskal-Wallis ANOVA test was used to verify the average and zeta-specific (ZS) sizes of the nano-iCnB and nano-milliQW. Morphological differences in liver, spleen, as well as liver and hematological data were compared between the immunized, nano-emulsion, non-immunized and sick animal groups (positive control) using data from three animals in each group. Significant differences were defined as p < 0.05 with 95% confidence intervals. Kaplan-Meier survival curves were used to display vaccine efficacy.All experiments were performed in accordance with the ethical guidelines for experiments with mice, and the protocols were approved by the National Council for Control of Animal Experimentation, Federal University of Tocantins Animal Experimentation Committee (CEUA n° 23101.006.832/2017-53). The guidelines for animal use and care were based on the standards established by The Brazilian College of Animal Experimentation (COBEA).